engineering complex orthopaedic tissues via strategic...

Engineering Complex Orthopaedic Tissues Via Strategic Biomimicry

DOVINA QU, CHRISTOPHER Z. MOSHER, MARGARET K. BOUSHELL, and HELEN H. LU

Biomaterials and Interface Tissue Engineering Laboratory, Department of Biomedical Engineering, Columbia University, 1210Amsterdam Avenue, 351 Engineering Terrace, MC 8904, New York, NY 10027, USA

(Received 3 July 2014; accepted 13 November 2014; published online 3 December 2014)

Associate Editor Fei Wang oversaw the review of this article.

Abstract—The primary current challenge in regenerativeengineering resides in the simultaneous formation of morethan one type of tissue, as well as their functional assemblyinto complex tissues or organ systems. Tissue–tissue syn-chrony is especially important in the musculoskeletal system,wherein overall organ function is enabled by the seamlessintegration of bone with soft tissues such as ligament,tendon, or cartilage, as well as the integration of musclewith tendon. Therefore, in lieu of a traditional single-tissuesystem (e.g., bone, ligament), composite tissue scaffolddesigns for the regeneration of functional connective tissueunits (e.g., bone–ligament–bone) are being actively investi-gated. Closely related is the effort to re-establish tissue–tissueinterfaces, which is essential for joining these tissue buildingblocks and facilitating host integration. Much of the researchat the forefront of the field has centered on bioinspiredstratified or gradient scaffold designs which aim torecapitulate the structural and compositional inhomoge-neity inherent across distinct tissue regions. As such, giventhe complexity of these musculoskeletal tissue units, thekey question is how to identify the most relevantparameters for recapitulating the native structure–functionrelationships in the scaffold design. Therefore, the focus ofthis review, in addition to presenting the state-of-the-art incomplex scaffold design, is to explore how strategicbiomimicry can be applied in engineering tissue connec-tivity. The objective of strategic biomimicry is to avoidover-engineering by establishing what needs to be learnedfrom nature and defining the essential matrix character-istics that must be reproduced in scaffold design. Appli-cation of this engineering strategy for the regeneration ofthe most common musculoskeletal tissue units (e.g., bone–ligament–bone, muscle–tendon–bone, cartilage–bone) will bediscussed in this review. It is anticipated that theseexciting efforts will enable integrative and functional

repair of soft tissue injuries, and moreover, lay thefoundation for the development of composite tissuesystems and ultimately, total limb or joint regeneration.

Keywords—Scaffolds, Interface, Tissue engineering, Com-

plex tissues, Strategic biomimicry.

INTRODUCTION

The prevalence of trauma and disease resulting inthe loss or failure of tissue and organ function hasengendered an unmet clinical need for the developmentof strategies to repair and regenerate damaged tissues.Combining biomaterials, cells, as well as chemical andphysical cues, tissue engineering principles59,107 havebeen successfully applied in recent years to form avariety of single-tissue systems both in vitro and in vivo.From a structure–function perspective, the organs ofthe body are comprised of diverse tissue types whichinterface with each other and operate in synchrony toenable complex functions. Therefore, to recapitulatenative biological function, the next horizon in the fieldof tissue engineering resides in how to assemble thesesingle-tissue systems into multi-tissue units or facilitatethe integration of these composite tissue grafts in vivo.

Synchronized tissue units are especially important inthe musculoskeletal system, wherein physiologic mo-tion is orchestrated through well-organized and con-certed actions of bone in conjunction with a variety ofsoft tissues, including, ligaments, which connect boneto bone, tendons, which join muscle to bone, and car-tilage, which lines the surface of articulating joints. Thetissue–tissue junctions through which they integratewith each other are characterized by multiple tissueregions that exhibit well-defined, spatial changes in cellphenotype, matrix composition and organization, aswell as region-specific mechanical properties (Fig. 1).

Address correspondence to Helen H. Lu, Biomaterials and

Interface Tissue Engineering Laboratory, Department of Biomedical

Engineering, Columbia University, 1210 Amsterdam Avenue, 351

Engineering Terrace, MC 8904, New York, NY 10027, USA. Elec-

tronic mail: [email protected]

Annals of Biomedical Engineering, Vol. 43, No. 3, March 2015 (� 2014) pp. 697–717

DOI: 10.1007/s10439-014-1190-6

0090-6964/15/0300-0697/0 � 2014 Biomedical Engineering Society

697

Unfortunately, these connective junctions are alsoprone to injury and degeneration, and are not rees-tablished via standard surgical repair methods. Forexample, in the majority of anterior cruciate ligament(ACL) injuries, a relatively homogenous tendon graftis used to replace the damaged ligament and is fixedwithin the bone tunnel with pins or screws. However,this single-tissue oriented strategy does not facilitateregeneration of the functional soft-hard tissue unit ofthe native ACL. Instead disorganized fibrovasculartissue is formed within the bone tunnel,93 resulting in astructurally weak link between graft and bone that isoften susceptible to failure, leading to poor long-termoutcomes and reduced mobility for the patient.35

Similarly, repair methods for rotator cuff tears can beassociated with post-surgical failure rates as high as94%.31 The prevalence of osteoarthritis, especiallyamong the aging population, has further increased thedemand for soft tissue repair therapies as current car-tilage treatment options are similarly limited by poorintegration of grafted tissue with the underlying boneand host cartilage.44 It is clear that there exists anunmet clinical need for functional and integrativetreatment strategies for the repair of orthopaedic softtissue injuries.

While a number of approaches to musculoskeletalsoft tissue regeneration have been explored withpromising results,46,61,120,125 successful clinical trans-lation of these grafts will depend largely on how best toachieve functional and extended integration of theengineered tissue units with each other and the sur-rounding host tissue. The inherent complexity andstructural intricacy of the native junction between softand hard tissues underscore the difficulties in engi-neering tissue–tissue interfaces. Each tissue phase ischaracterized as having distinct cellular populationsand unique matrix composition and organization, yetmust operate in unison with adjoining tissues to facil-itate physiologic function and maintain tissue homeo-stasis. Inspired by these multi-tissue structures, avariety of complex scaffold designs seeking to reca-pitulate the native spatial and compositional inhomo-geneity have been developed.4,28,77 This review willdiscuss current regenerative engineering efforts in lig-ament-bone3,11,19,21,54,62,68,71,72,86,87,96–98,110,111,113–115

tendon–one,24,78,79,134 muscle–tendon,57,58,60,117,118 andcartilage–bone integration1,5,13,15–18,25–27,30,32,33,36,37,39,41,47,49,52,53,55,56,69,74,92,95,100–104,106,119,128,133,135–137 focusing onbiomaterial- and cell-based strategies for engineering bio-mimetic, functional spatial variations in composition andmechanical properties. In light of the complexity of multi-tissue regeneration, the application of strategic biomimicryacross tissue–tissue junctions, or prioritizing what needs tobe recapitulated from native tissues and identifying themost crucial parameters for complex scaffold design, is

essential for avoiding over-engineering the scaffold system.This review will highlight these strategic design approachesand conclude with a summary and reflections on futuredirections in complex tissue engineering.

COMPLEX SCAFFOLD DESIGN FOR

INTEGRATIVE LIGAMENT TISSUE

ENGINEERING

Anatomically, ligaments are anchored to bone ei-ther through fibrous insertions, wherein aligned col-lagen fibers connect the soft tissue to theperiosteum,90 or through direct insertions via a two-region fibrocartilaginous interface.130 In other words,the functional ligament is a multi-tissue unit whichconsists of several compositionally distinct andstructurally continuous regions: bone–interface–liga-ment–interface–bone. For example, the ACL, which isthe primary stabilizer of the knee joint, spans fromfemur to tibia and joins to each bony end via afibrocartilaginous insertion (Fig. 1). The fibrocartilageinterface is further subdivided into mineralized andnon-mineralized regions, with an exponential increasein mineral content observed within the calcifiedregion.109 A structurally and/or compositionally het-erogeneous scaffold design is therefore necessary torecapitulate the composite tissue structure across theligament-bone junction.

Ideally, such a scaffold should exhibit phase-specificmechanical properties which increase from the liga-ment to bone phase and exhibit mechanical compe-tence under physiologic tension and torsion.Additionally the scaffold must support the growth anddifferentiation of relevant cell populations, facilitatetheir interactions, promote and maintain controlledmatrix heterogeneity, and be biodegradable in order tobe gradually replaced by newly generated tissue. Spe-cifically, the neoligament tissue should exhibit orga-nized matrix comprised predominantly of collagen Iand III, while fibrocartilaginous tissue is characterizedby a matrix consisting of glycosaminoglycans (GAG)in collagens I and II, and bone exhibits a mineralized,collagen I matrix. Complex scaffolds for multi-tissueregeneration must not only facilitate regeneration ofeach discrete tissue type of interest, but also achieveintegration of these multiple tissue phases. To this end,interconnectivity and integration of adjacent phases, aswell as adaptability and compatibility with currentsurgical repair methods, are associated requirements inscaffold design and fabrication.

While traditional efforts to develop tissue-engi-neered ACL grafts have predominantly focused on theligament proper,125,129 there has been a recent shifttowards forming multi-tissue units consisting of inte-

QU et al.698

grated bone–ligament–bone, ligament–bone or liga-ment–interface–bone regions (Table 1). For bone–liga-ment–bone designs, Bourke et al. first reported ascaffold consisting of polydesamino tyrosyl-tyrosineethyl ester carbonate or polylactide (PLA) fibersembedded in polymethyl methacrylate plugs.11

Improving upon this design concept, Cooper et al.

developed a continuous multi-phased synthetic ACLgraft, which consisted of braided polylactide-co-gly-colide (PLGA) microfibers arranged to form a liga-ment with two denser fiber regions at either end tofacilitate bone formation.19,21,68 In vitro19,68 andin vivo21 evaluation of this design in a rabbit ACLreconstruction model demonstrated biocompatibility

FIGURE 1. Common orthopaedic tissue–tissue interfaces. Ligaments, such as the anterior cruciate ligament (ACL) in the knee(Modified Goldner’s Masson Trichrome),126 and tendons, such as the supraspinatus tendon in the shoulder (Toluidine blue),70

connect to bone via a fibrocartilaginous (FC) transition, which can be further subdivided into non-mineralized (NFC) and miner-alized (MFC) regions (Von Kossa). The muscle–tendon junction (Modified Goldner’s Masson Trichrome) consists of an interdigi-tating band of connective tissue.60 Articular cartilage (AC), which can be subdivided into surface (SZC), middle (MZC), and deep(DZC) zones (Modified Goldner’s Masson Trichrome), connects to subchondral bone via a transitional calcified cartilage (CC)region (Von Kossa).

Engineering Complex Orthopaedic Tissues 699

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QU et al.700

and extensive collagenous tissue infiltration by12 weeks. More recently, Altman et al. developed asimilar multi-region silk bone–ligament–bone graftconsisting of silk yarns connecting more densely knitregions at either end for bony attachment.3,42 After12 months of implantation in a caprine ACL recon-struction model, a collagenous ligament-like structurewith aligned cells and crimped matrix was observed.To harness the potential use of growth factors to en-hance graft-bone integration, Kimura et al. developeda multi-phased system comprised of a braided PLA-collagen scaffold with basic fibroblast growth factor-releasing gelatin hydrogels at either end to be placedwithin the bone tunnels.54 This design supported theformation of ligament and bone regions, and resultedin enhanced tensile mechanical properties compared tosingle-phased controls. Similarly, Paxton et al. inves-tigated the incorporation of hydroxyapatite (HA) andthe RGD cell-adhesion peptide into a polyethyleneglycol (PEG) hydrogel for engineering ligament-boneattachments and observed that the incorporation ofHA improved interface formation.86 The bioresorbablecalcium phosphate brushite replaced HA in subsequentgraft anchor designs,87 with the two ends embedded infibrin gel to form a bone–ligament–bone construct.Using a cell-based approach, Ma et al. formed bone–ligament–bone constructs through co-culture of mes-enchymal stem cell (MSC)-derived bone constructswith a MSC-derived ligament monolayer rolledbetween.71 In vivo evaluation in an ovine modelshowed graft integration with the native bone and theformation of an interface between the ligament andbone phases which structurally resembles fibrocarti-

lage.72 Overall, these novel bone–ligament–bone designsrepresent significant advances in forming multi-tissueunits, and, from a strategic biomimicry perspective,elegantly demonstrate the importance of multi-tissueover single-tissue design, especially in terms of biomi-metic tissue regeneration and resultant functionality.

One of the challenges in the implementation of thebone–ligament–bone design is that the fibrocartilagi-nous interface between the ligament and bone regionsis not consistently or uniformly regenerated. Struc-ture–function studies have revealed that the ligament-bone insertion is optimized to withstand the uniquecombination of tensile and compressive loading sus-tained at this interface,7,20,75,112 and is therefore criticalfor mediating load transfer and minimizing stressconcentrations between soft tissue and bone.7,20,80 Asthe elastic modulus of bone is more than an order ofmagnitude greater than that of the ligament, thegradual transition in mechanical properties facilitatedby the intermediate fibrocartilage regions protects thesoft tissue from contact deformation and damage athigh strains.8,40 Therefore, incorporating interfaceregeneration into graft design will be essential forachieving physiological joint function after ligamentreconstruction. To this end, Spalazzi et al. reported onthe design and optimization of a stratified ligament–interface–bone scaffold (Fig. 2).110,111 The ligamentphase was comprised of a PLGA mesh, the interfacephase was comprised of sintered PLGA microspheres,and the bone phase consisted of sintered PLGA and45S5 bioactive glass (BG) microspheres. Phases werejoined together via solid state sintering. In vitro andin vivo tri-culture of fibroblasts, chondrocytes, and

FIGURE 2. Scaffold design for ligament-interface-bone regeneration. Mimicking the stratified structure (Modified Goldner’sMasson Trichrome)126 and composition (FTIR-I: Fourier Transform infrared spectroscopy)89 of the native insertion, a tri-phasicscaffold (Phase A: PLGA mesh, Phase B: PLGA microspheres, Phase C: PLGA-BG microspheres) was designed for ACL-boneinterface regeneration.110,111 This design allowed for spatial control over cell distribution (Fb: fibroblasts on Phase A, Ob: oste-oblasts on Phase C, along with chondrocytes in a hydrogel in Phase B) enabled the formation of compositionally distinct yetstructurally continuous tissue regions in vivo (Modified Goldner’s Masson Trichrome).


osteoblasts on the tri-phasic scaffold led to the for-mation of interconnected ligament-, fibrocartilage- andbone-like matrix in each respective region (Fig. 2).110

In addition to supporting multi-tissue formation, thestratified design exhibited graded mechanical proper-ties across the scaffold, with the highest elastic mod-ulus and yield strength in the bone phase, mimickingthe mechanical transition of the native ligament–interface–bone junction. These studies demonstrate theutility of stratified design in engineering complex tis-sues as well as the importance of exercising spatialcontrol of cell distribution in multi-tissue formation.

More recently, building upon these findings, Su-bramony et al. developed a five-phased, nanofiber-based scaffold for ACL tissue engineering.115 In thisdesign, a poly-e-caprolactone (PCL)-based bone–inter-face–ligament–interface–bone scaffold was fabricated,and mechanoactive collars were applied at either liga-ment-bone junction to form a continuous, five-phasedconstruct. In vitro evaluation of stem cell-seeded con-structs showed upregulation of fibroblast, fibrochon-drocyte, and osteoblast-specific markers on theligament, interface, and bone phases, respectively. Invivo implantation of these scaffolds resulted in accel-erated formation of mineralized tissue within the bonetunnels, as well as superior mechanical propertiescompared to single-phased controls. Together, thesepromising results emphasize that biomimetic, stratifiedscaffold design can allow for spatial control over thedistribution of interface-relevant cell populations,facilitate formation of phase-specific matrix heteroge-neity, and enable regeneration of the fibrocartilaginoustransition.

Taking into consideration the mineral distributionacross the calcified fibrocartilage interface,109 Samav-edi et al. employed offset co-electrospinning methodsto fabricate fibrous scaffolds with a continuous gra-dation in polymer composition and mineral contentwhich in turn guided osteogenic differentiation of stemcells in a gradient-dependent manner.96,97 Thesemethods have most recently been applied to formbone–ligament–bone scaffolds consisting of a region ofaligned PCL fibers for ligament regeneration contigu-ous with regions of unaligned PLGA nanofibers ateither end for bone regeneration.98 Meanwhile, otherdesigns have used a gradient of chemical cues, such asgrowth factors, to induce the formation of a gradedcalcified matrix. Phillips et al. fabricated collagenscaffolds with a compositional gradient of retroviralcoating for the osteogenic transcription factorRUNX2, which induced seeded fibroblasts to producea gradient of mineralized matrix both in vitro andin vivo.88 These studies demonstrate that gradientscaffold design is a promising approach for interface

tissue engineering, with the potential to fully recaptureand pre-design the micro- and nano-scale organizationof the native tissue transitions. Unlike stratified scaf-folds, gradient designs exhibit more gradual, continu-ous transitions in composition and mechanicalproperties. On the other hand, however, the stepwiseincrease in mineral content between phases of stratifiedscaffolds better approximates the exponential increasein mineral content across the interface regions.109

Moreover, gradient scaffolds are relatively challengingto fabricate at physiologically relevant scales comparedto stratified scaffolds. Therefore from a strategic bio-mimicry standpoint, it will be important to systemati-cally investigate and compare gradient scaffolds withstratified designs and determine whether either or bothare optimal for multi-tissue formation. It is alsoemphasized that cells and the host environment alsoplay a role here. Namely, as the stratified or gradient-based scaffold system degrades and is remodeled bycells, a cell-engineered biomimetic gradient of compo-sition will emerge and give rise to the physiologicallyrelevant structure–function profile.

It is also noted that one of the challenges in stratifiedscaffold design is achieving functional integration ofcompositionally or mechanically distinct layers. Whileadhesives have beenwidely used to join scaffold or tissuephases, the addition of such materials may restrict thediffusion of nutrients, metabolites, or cytokines whichare crucial for cell function and tissue development.38

Moreover, a sharp transition between dissimilar mate-rials or tissues is inherently weaker than a gradualinterface consisting of interdigitated phases.63,73 Onestrategy to circumvent this limitation is to ensure that allphases of the stratified scaffold are predominatelycomprised of the same type of biomaterial, such asPLGAwhich was used by Spalazzi et al. in the ligament–interface–bone graft.110,111 Specifically, by sintering thephases beyond the glass transition temperature ofPLGA, they can be joined seamlessly and functionallyto prevent delamination and ensure structural conti-nuity. More recently, Harley et al. demonstrated thatinterdigitated stratified scaffolds can be fabricated byallowing interdiffusion of liquid collagen-GAG sus-pensions followed by lyophilization.38

In summary, the extensive body of work on liga-ment-bone engineering reaffirms that integration ofsoft tissue to the native bone remains a primarychallenge in functional ligament tissue engineering.One of the advantages of the stratified and gradientdesigns is that pre-engineering the ligament-boneinterface via biomimetic scaffold designs ex vivo,allows for subsequent focus in vivo to be on therelatively easier task of facilitating bone–bone inte-gration.

QU et al.702


INTEGRATIVE TENDON TISSUE ENGINEERING

Similar to the ligament, the functional tendon,which joins muscle to bone, is a multi-tissue structureconsisting of structurally contiguous yet composition-ally distinct regions: muscle–interface–tendon–inter-face–bone. Like the ACL, major tendons, such as thoseof the rotator cuff, insert into subchondral bonethrough a graded fibrocartilage transition, divided intonon-mineralized and mineralized regions.7,10,20 Anumber of studies have shown that the region betweentendon and bone is relatively compliant, with a tensilemodulus that is approximately half that of the ten-don,43,99,121 which minimizes the formation of stressconcentrations at the interface.66 As the tendon-boneand ligament-bone interfaces are similar in structure,function, and composition, many of the multi-phasicand gradient scaffold design strategies discussed above,as well as criteria for mechanical and compositionalevaluation, can also be applied for functional andintegrative tendon tissue engineering. The ideal tendonscaffold should similarly incorporate structural andcompositional heterogeneity which enable well-de-fined, phase-specific changes in mechanical propertiesand support interactions amongst heterotypic cellpopulations. Currently, in addition to the tendonproper, many studies have focused on engineering ei-ther the tendon–interface–bone (Table 2) or muscle–interface–tendon (Table 3) unit. It is noted that despitesimilarities between tendon and ligament, the scaffolddesign must also be adapted to account for the tendon-specific loading environment. While both of theseconnective tissues withstand high tensile loads, tendonstypically experience loading in a uniaxial direction, incontrast to ligaments which are also loaded in tor-sion.94 Furthermore, current surgical repair methodsunique to the anatomic location and disease conditionof the joint must also be considered.

Rotator cuff tears represent one of the most com-mon tendon injuries that necessitate clinical interven-tion. However, unlike treatment strategies for ligamentinjuries, which are dominated by reconstructions, ten-don injuries are managed clinically via repair wherebythe tendon is reattached to bone by mechanical means.However, restoration of the native tendon-boneinsertion is challenging and tendon detachment re-mains the primary cause of surgical failure. To im-prove fixation, Chang et al. and Sundar et al. haveshown that surgically interposing periosteum tissue14

or demineralized bone matrix116 between the nativetendon and bone helps to facilitate the development ofa fibrocartilage-like matrix and improve mechanicalfunction. However, the clinical relevance of suchmethods is limited by the challenges associated with

harvesting and preparing these additional tissues dur-ing repair procedures. Focusing on the fibrocartilagi-nous tendon–bone interface and taking intoconsideration current cuff repair surgical techniques,Moffat et al. designed a biphasic scaffold consisting ofcontiguous layers of aligned PLGA and PLGA-HAnanofibers joined via electrospinning, intended to mi-mic the non-calcified and calcified fibrocartilageregions, respectively (Fig. 3).78 In vivo evaluation inboth rodent78 and ovine134 rotator cuff repair modelsusing this scaffold as an inlay between tendon andbone resulted in the formation of a fibrocartilage-likematrix in both scaffold phases (Fig. 3). Mineral dis-tribution was maintained, in which calcified fibrocar-tilage formed only on the HA-containing phase. Pre-seeding the biphasic scaffold with bone mar-row-derived cells also promoted fibrocartilage matrixmaturation and enhanced collagen organization at thetendon-bone junction. From a biomimetic scaffolddesign perspective, it was found that regeneration of anorganized interface was only evident with the biphasicdesign, but not when the tendon was repaired witheither single-phased PLGA or PLGA-HA only scaf-folds (Fig. 3). These observations suggest that themineral-free top layer of the biphasic scaffold guidestendon and fibrocartilage formation, with nanofiberalignment promoting integration with tendon, whilethe controlled spatial distribution of mineral content inthe bottom layer of the scaffold supports calcifiedfibrocartilage formation and osteointegration.

A number of gradient scaffold designs have alsobeen explored for tendon–one integration, seeking topre-engineer the mineral gradient34 at the tendon-bonejunction by employing various methods of calciumphosphate incorporation. For example, Li et al.developed a simple and elegant coating method togenerate a linear gradient of calcium phosphate onpolymeric nanofiber scaffolds by varying the incuba-tion time of these scaffolds in a concentrated simulatedbody fluid.64 It was reported that the mineral distri-bution imparted a gradation in mechanical propertiesalong the length of the scaffold. The system was thenfurther optimized by increasing the concentration ofbicarbonate ions in the soaking solution, whichresulted in denser mineral coating on the fibers andfurther improved mechanical properties.67 This gradedscaffold was shown to exhibit spatial control overosteogenesis of adipose-derived MSCs, with moreextensive staining of osteogenic markers observed onareas of higher scaffold mineral content.65 Anothernovel gradient scaffold design focused on protein dis-tribution, whereby a fibronectin concentration gradi-ent was generated on polymer nanofiber scaffolds andwas shown to exhibit control over density and mor-phology of cultured fibroblasts.105 Also using con-


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QU et al.704

trolled soaking methods, Dickerson et al. recentlydeveloped a regionally demineralized bone-basedscaffold for tendon-bone integration.24 Evaluation ofthe scaffold in an ovine rotator cuff repair modelshowed that the scaffold also facilitated formation offibrocartilaginous tissue at the tendon-bone junction,albeit the neo-fibrocartilage was thicker than that ofthe native insertion. An alternative method for con-trolling nucleation and growth of HA crystals onnanofibers was reported by Cui et al., whereby PLAfibers were functionalized with carboxyl, hydroxyl, andamino groups, which served as induction sites forin situ HA formation.22,23 This method was applied tofabricate fibrous scaffolds with graded HA contentwhich supported spatial control over MC3T3 celldensity, osteogenesis, and collagen deposition.139

These studies demonstrate the potential of gradient-based scaffolds for composite tissue formation. Thenext frontier in the design of these novel scaffolds is toachieve physiologically relevant gradient profiles withmicro- and nano-scale compositional changes acrossthe scaffold length.

While the majority of tendon injuries occur in eitherthe tendon proper or the tendon-bone insertion, mus-cle atrophy and detachment from tendon have alsobeen associated with tendon degeneration. The nativemuscle–tendon junction serves to distribute mechanicalloads between skeletal muscle and bone132 and conse-quently the reestablishment of this interface is alsoimportant for full restoration of musculoskeletalfunction following injury. Paralleling the previouslydiscussed tissue interfaces, the muscle–tendon interfacerepresents a junction between tissues with vastly dif-ferent mechanical properties. In particular, the tendonis significantly stiffer than muscle, with reported elasticmodulus values as high as three orders of magnitudegreater than those of muscle.83,131 The native interface

between muscle and tendon consists of an interdigi-tating band of fibroblast-laden tissue which connectselastic muscle fibers to dense tendon collagen fibers(Fig. 1).123 This inter-digitation results in an approxi-mate ten-fold increase in muscle–tendon contact sur-face area and serves to distribute stresses induced bymuscle contraction over a wide area, thereby mini-mizing the risk of tearing122–124 Thus, an ideal scaffoldfor muscle–tendon interface repair should also bemulti-phasic in design, mimicking the differentmechanical profiles of the adjacent muscle and tendonregions. Structurally and compositionally, muscle andtendon are also distinct, and engineered muscle shouldbe distinguishable from tendon by the presence ofmulti-nucleated myotubes.

Early efforts to engineer the muscle–tendon inter-face involved culturing myoblasts in collagen gelsin vitro, which resulted in the formation of contractilemuscle constructs with fibril terminals similar to thosefound at the native junction.117,118 Using a cell-basedapproach, Larkin et al. co-cultured skeletal muscle andengineered tendon constructs in vitro to form a muscle–interface–tendon construct.60 The neo-interface regionexhibited upregulated expression of muscle–tendonjunction-specific paxillin, and was able to sustain ten-sile loading at super-physiologic strain rates. Further-more, microscopic evaluation of the constructsrevealed the termination sites of the engineered myof-ibers also resembled the structures found at the fetalmuscle–tendon junction.57 More recently, Ladd et al.developed a tri-phasic scaffold for engineering a mus-cle–interface–tendon unit by co-electrospinning PCL-collagen and PLA-collagen onto opposite ends of asingle mandrel.58 The resulting scaffolds exhibited asimilar spatial strain distribution as the native muscle–tendon interface and facilitated both myoblast andfibroblast attachment.

TABLE 3. Complex scaffold designs for integrative tendon tissue engineering (muscle–tendon).

Study

Material and scaffold

design Induction agents Cell source Animal model Tissues formed*

Swasdison

et al.117,118Collagen I gel (muscle) – Quail pectoral

myoblasts,117 Chick

tendon fibroblasts118

– Muscle

Tendon

Ladd et al.58 PCL-collagen I fibers

(muscle) with gradient to

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myoblasts, Murine

NIH3T3 fibroblasts

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Larkin et al.60 and

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et al.57

Scaffold-less, cell-based

muscle–tendon

construct

– Rat soleus myocytes,

Rat tendon fibro-

blasts

– Muscle

Interface

Tendon

*Note Tissue formation was determined by staining, immunohistochemistry, or gene expression for pertinent matrix components (muscle:

myosin; interface: paxillin; tendon: collagen, collagen I, and/or collagen III). PCL: poly-e-caprolactone; PLA: polylactide.


In summary, current scaffold design approaches inintegrative tendon repair are a reflection of the prev-alence of tendon injuries and current clinical practice,and complex scaffold designs in the form of stratifiedor gradient design are promising approaches forfunctional and integrative tendon tissue engineering.


INTEGRATIVE CARTILAGE TISSUE

ENGINEERING

Similar to the other soft connective tissues discussedhere, articular cartilage health and function is inti-mately tied to the subchondral bone.138 Structurally,the two tissue types are connected via the osteochon-dral interface, which consists of a calcified cartilagebarrier that is instrumental for load bearing and force

distribution across these tissues.12,91,108 Specifically,the mineral is localized to the calcified cartilage andbone regions (Fig. 4), with an exponential increase inmineral content between non-calcified and calcifiedregions that persists with age.51 The modulus of thecalcified cartilage layer is intermediate between that ofarticular cartilage and bone,76 resulting in a gradationin mechanical properties which enables force trans-mission across the system while reducing the formationof stress concentrations at the soft tissue-bone inter-face.84,85 Thus, in addition to meeting the complexmechanical demands of articulation, the ideal cartilagescaffold must also enable cartilage–bone integration byconnecting these two tissues through a stable andphysiologically relevant calcified cartilage layer. Inother words, consideration of multi-tissue regeneration(i.e., cartilage–bone, cartilage–interface, cartilage–interface–bone) is also functionally relevant for inte-

FIGURE 3. Scaffold design for tendon-interface-bone regeneration.78 A biphasic scaffold comprised of layered aligned PLGA andPLGA-HA nanofibers was fabricated by electrospinning, which led to phase-specific mineral deposition in vivo (Von Kossa,subcutaneous athymic rat model). The bilayer scaffold was subsequently tested in a rat rotator cuff repair model, and disorganizedscar tissue was observed in the single-phased controls (PLGA, PLGA-HA only). Interestingly, tendon-bone integration via anorganized bilayer fibrocartilage interface was only observed with the biphasic design (Picrosirius red, Alcian blue).

QU et al.706

grative cartilage repair (Table 4). The mechanicalfunctionality of regenerated tissue should therefore beevaluated by compressive mechanical testing whilematrix composition should be evaluated by histologyand immunohistochemistry. Cartilage tissue is com-prised of a GAG and collagen II-rich matrix whilecalcified cartilage is characterized by the additionalpresence of collagen X and mineral.

A number of stratified cartilage–one designs havebeen developed and evaluated for osteochondral tissueengineering. As they have been thoroughly reviewedelsewhere,46,61,120 only a brief summary is providedbelow. Early designs consisted predominantly of fusingdistinct cartilage and bone regions together throughthe use of sutures or glue. For example, Schaefer et al.developed a biphasic scaffold by suturing chondrocyte-seeded PLGA meshes with periosteal cell-seededPLGA/PEG foams.100 Early cell–cell interactionsproved vital to osteochondral interface formation, asintegration was enhanced when the constructs weresutured together 1 week post-seeding as opposed to4 weeks.100 Later, Gao et al. joined a stem cell-seededhyaluronan sponge with a porous stem cell-seededcalcium phosphate scaffold using fibrin glue andreported the formation of continuous collagen fibersbetween the two phases in a subcutaneous rat model.33

Similarly, Alhadlaq and Mao developed a bi-layered,PEG-based hydrogel osteochondral construct bysequential photo-polymerization, where the top andbottom hydrogel layers contained stem cell-derivedchondrocytes and osteoblasts, respectively.1 Distinctbut histologically integrated cartilaginous and osseous

regions developed after subcutaneous implantation inathymic mice, albeit it is unclear whether consistent oruniform osteochondral interface formation wasachieved. Comparable results were obtained fromother stem cell-seeded biphasic scaffolddesigns.16,32,37,47,103,119 Collectively, these studiesdemonstrate the feasibility of engineering both carti-lage- and bone-like tissues. However, similar to othersoft tissue-bone designs discussed above, these biphasicscaffolds are for engineering cartilage and bone, whilethe osteochondral interface between the two tissues hasbeen underemphasized in these designs. The signifi-cance of the interface as a structural barrier protectingthe healing cartilage from vascular invasion was dem-onstrated by Hunziker et al. using a full-thicknesscartilage defect model and a Gore-Tex� membranewith a 0.2 lm pore diameter.45 Placing the membranebetween cartilage and bone compartments preservedthe integrity of newly formed cartilage, limiting vas-cular ingrowth from the subchondral bed, and pre-venting ectopic mineralization. Taking theosteochondral interface into consideration, Aydinet al. designed a biphasic scaffold whereby a PLA-PCLbone region with vertical channels was combined witha PGA cartilage region using a pigmented polymericblend that allowed for visualization of the interfaceregion. Scanning electron microscopy and micro-CTimaging showed the establishment of cartilage- andbone-like regions, as well as an interface region, al-though biochemical composition of the regeneratedtissues was not determined. To more closely mimic thenative osteochondral junction, Jiang et al. developed

FIGURE 4. Hydrogel-ceramic composite scaffold for osteochondral interface regeneration. Articular cartilage (AC) connects tosubchondral bone via the osteochondral interface, which consists of calcified cartilage (CC, Modified Goldner’s Masson Tri-chrome). Analysis via Fourier Transform infrared spectroscopy (FTIR-I) reveals an exponential increase in mineral content betweenthe articular cartilage and calcified cartilage regions.51 The hydrogel-HA composite design52 guided chondrocyte-mediateddeposition of a mineralized matrix (Von Kossa) that is positive for collagen X (immunohistochemistry) and can be used inconjunction with cartilage grafts.


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a stratified cartilage–interface–bone scaffold consistingof a hydrogel-based region for cartilage regeneration, ahybrid hydrogel and polymer-ceramic compositemicrosphere region for interface regeneration, and apolymer-ceramic composite microsphere region forbone regeneration.49,69 Chondrocyte and osteoblastco-culture on this scaffold system resulted in the for-mation of distinct yet continuous cartilaginous andosseous matrices, as well as a calcified interfacialregion largely due to the pre-engineered mineralizedscaffold phase.

Cell-based approaches for cartilage–bone integra-tion were pioneered by Kandel et al., and have laid thefoundation for current approaches to osteochondralinterface regeneration. For example, Kandel et al.seeded deep zone chondrocytes (DZC) on collagen IIpre-coated filter inserts and observed the formation ofmineralizing matrix in the region directly adjacent tothe inserts.50 In a follow-on study, Allan et al. seededDZCs at high density on porous calcium polyphos-phate scaffolds in media containing b-glycerophos-phate (b-GP). A semi-crystalline calcium phosphate-containing matrix formed adjacent to the scaffold,suggesting that DZCs are a promising cell populationfor calcified cartilage formation.2 Building on thiswork and focusing specifically on the osteochondralinterface, Khanarian et al. evaluated both degradable(alginate)53 and non-degradable (agarose)52 hydrogel–mineral composite scaffolds for calcified cartilage for-mation (Fig. 4). Both scaffold systems were found topromote the deposition of a collagen II and proteo-glycan-rich matrix by DZCs, resulting in enhancedcompressive and shear mechanical properties, as wellas chondrocyte hypertrophy and collagen X deposition(Fig. 4). Most importantly, the scaffolds supportedcalcified cartilage formation. The advantage of thescaffold-based approach is that fewer cells are requiredto achieve superior functional mechanical properties.Moreover, scaffolds can be modified prior to implan-tation to further enhance mechanical performance and,clinically, they may be used in conjunction with acartilage graft for repairing full-thickness defects.

Given the mineral transition which inherently oc-curs across the osteochondral interface, gradient scaf-folds have also been investigated for integrativecartilage repair. As heterogeneous mechanical andchemical properties must be established and main-tained across the cartilage-bone interface, composi-tional and/or chemical gradients can also allow forregional control of cell and tissue phenotypes. Sher-wood et al. established one of the earliest gradientosteochondral scaffold designs by using 3D printingtechnology to fabricate an osteochondral graft withgraded variation in porosity and ceramic composi-tion.104 Chondrocytes were found to preferentially

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QU et al.710

attach to the cartilaginous phase and the tensilestrength of the bone region was comparable to that ofnative tissue. More recently, Singh et al. developed aPLGA microsphere scaffold which exhibited continu-ous gradients in compressive mechanical properties,achieved by encapsulating a higher stiffness nano-phase material into portions of the microspheres.106

Another interesting system developed by Aviv-Gavrielet al. consists of a gelatin membrane with a ceramicgradient formed by exposing one side of the calcium orphosphate ion-containing gels to a solution of thecomplementary ion.5 This resulted in the formation ofa partially calcified hydrogel membrane which can beadapted for integration of cartilage grafts with bone.Combining elements of both stratified and gradientdesigns, Harley et al. fabricated layered collagen-GAGscaffolds consisting of distinct cartilage and boneregions connected by a continuous interface via liquid-phase co-synthesis.39 This unique method of fabrica-tion resulted in small gradients of dissimilar materialsextending across a soft interface. In vivo evaluation ofthe acellular scaffold in a caprine model revealed thatthis design supported significant formation of bothcartilaginous and osseous tissue on the respectivephases.36 Using a novel electrospinning method, Eris-ken et al. fabricated PCL nanofiber scaffolds contain-ing a linear tricalcium phosphate (TCP) gradient,29

and the graded calcified matrix was maintained bycultured MC3T3 cells. It is emphasized that in thisstudy, the scaffold exhibited a compositional gradientthat is relevant at the physiological scale (~100 lm).

In order to engineer a growth factor gradient, Wanget al. developed a silk-based microsphere scaffoldsystem capable of generating opposing insulin-likegrowth factor-1 and bone morphogenetic protein(BMP)-2 gradients via spatially- and temporally-con-trolled delivery.128 Human MSCs seeded on the scaf-fold underwent chondrogenic and osteogenicdifferentiation along the growth factor concentrationgradients. Similarly, Dormer et al. generated opposing,spatially defined mixtures of transforming growthfactor-b1 and BMP-2 on a PLGA microsphere-basedscaffold, which led to the formation of both cartilage-like and bone-like matrix in a rabbit mandibular con-dyle defect model.26 While scaffolds with dual growthfactor gradients did not out-perform sham defects inthe mandibular model,27 bone regeneration was en-hanced in a rabbit knee model when both growthfactor and HA gradients were simultaneously incor-porated into the scaffold design.81 These studies andothers82 collectively demonstrate the promise of cou-pling growth factor and compositional gradients forenhancing composite tissue formation, and the impactof non-growth factor inductive agents (e.g., HA) fordirecting tissue regeneration.

In summary, composite scaffold designs in bothstratified and gradient form can be used to engineercartilage-interface or cartilage-interface-bone grafts.The success of these tissue regeneration approacheswill depend on defect site and animal model used forevaluation, and both compositional and growth factorgradients may be required for functional multi-tissueformation.

SUMMARY AND FUTURE DIRECTIONS

An overview of current concepts in engineeringcomplex tissues for musculoskeletal soft tissue repair,with an emphasis on scaffold design strategies thatenable physiologic tissue connectivity and multi-tissueregeneration has been presented here. These biomi-metic scaffold designs seek to recapitulate the spatialdistribution in compositional, structural, andmechanical properties inherent between bone and softtissues, especially across the native tissue–tissue junc-tions. These studies have collectively delineated severalstrategic biomimicry design principles for multi-tissueregeneration. First, one-tissue centric, single-phasedscaffold systems are insufficient for recapitulating softtissue functionality, especially when it comes toachieving graft integration with host tissues. Next,incorporation of physiologically relevant interfaceregions or interface formation strategies on multi-tis-sue scaffold designs (bone–interface–ligament–inter-face–bone, tendon–interface–bone, muscle–interface–tendon and cartilage–interface–bone) is a prerequisitefor achieving graft integration and physiologic func-tion. In addition, regional scaffold cues and cellularinteractions can be used to direct cell fate in the ab-sence of differentiation media either in vitro or in vivo.Specifically, spatial patterning of relevant key factorshave been shown to exercise spatial control in stem celldifferentiation on stratified and gradient scaffolds inwhich all regions are bathed in a commonmedia.15,16,30,65,92,139 Furthermore, heterotypic cellularinteractions have been reported to ensure phenotypicmaintenance and matrix integrity between zonalchondrocytes.48 It is likely that spatial control of celldistribution and relevant inductive agents on thestratified or gradient scaffold is required to control thefate of each cell population and direct region-specificmatrix elaboration. Moreover, it is noted that designrequirements vary by tissue type and must take intoconsideration existing surgical practices for soft tissuerepair. For example, while multi-tissue units are nec-essary for ligament reconstruction, scaffolds that cansupport non-calcified and calcified interface formationare sufficient for achieving functional tendon-boneintegration. In contrast, calcified cartilage formation


alone is not sufficient for functional repair of a carti-lage defect. Most importantly, for multi-tissue forma-tion, the complex scaffold must exert spatial controlover heterotypic cell interactions through incorpora-tion of spatial and/or temporal compositional andchemical gradients, as well as through the use of bio-chemical and biomechanical stimulation to support theformation of integrated composite-tissue systems.Furthermore, scaffolds which support in vivo cellularinfiltration and stem cell homing represent a promisingfuture direction in the field as they have the potentialto minimize the need to obtain, expand, and seed cellsonto the scaffold prior to implantation. This approachwill ultimately simplify the pathway to clinical trans-lation and improve accessibility. The studies high-lighted herein also demonstrate that functional andintegrative connective tissue repair may be achieved bycoupling both scaffold- and cell-based approaches.

Despite the exciting advancements in scaffold designand fabrication made in a relatively short period, thereremain a number of challenges in this fast-growingfield. One of the technical challenges in ex vivo engi-neering of complex tissues resides in how to devise anoptimal culturing media or loading regimen that en-sures the phenotypic maintenance of multiple cellpopulations and the elaboration of related matrix. Forexample, Wang et al. investigated the effects ofascorbic acid and b-GP dose on human osteoblastsand ligament fibroblasts, and devised a co-culturemedia which maintained osteoblast function withoutinducing unwanted mineralization by fibroblasts.127 Tothis end, the mechanistic effects of biological, chemical,and physical stimuli must be thoroughly evaluated toenable more refined and targeted scaffold design andgraft fixation. In addition to in vitro culture challenges,there remains a need for a greater understanding of thestructure–function relationships at the native tissue–tissue interfaces, as well as the mechanisms governinginterface development and healing at these junctions.Furthermore, physiologically relevant in vitro andin vivo models are needed to evaluate the clinicalpotential of these designs.

The strategic biomimcry approach emphasized here,where scaffolds can be designed to recapitulate the keycompositional and structural organization propertiesof the native interface, will be instrumental for rees-tablishment of integrated musculoskeletal tissue sys-tems and restoring physiologic function. It isanticipated that these efforts will lead to the develop-ment of the next generation of functional fixation de-vices for orthopaedic repair, as well as augment thepotential for clinical translation of tissue-engineeredorthopaedic grafts. Moreover, by bridging distincttypes of tissues, interface tissue engineering will be

instrumental towards engineering complex organs andtotal limb or joint regeneration.

ACKNOWLEDGMENTS

This work was supported by the National Institutesof Health (R21-AR056459, R01-AR055280, AR059038), and the New York State Stem Cell ESSC Board(NYSTEM C029551).

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