Native organization

Collagen is the main component of the enthesis. Three major types of collagen are found across the interface: type I, type II, and type X. Collagens III, V, and VI are also found in these interfaces at much lower concentrations.^[Reference Waggett, Ralphs, Kwan, Woodnutt and Benjamin^24] Types I and II collagen are fibrillar collagens, meaning they self-assemble into organized nanofibers, ~50–500 nm in diameter.^[Reference Fratzl and Weinkamer^25] Type I collagen is deposited in association with tensile forces and is found in bone and ligamentous tissue. Type II collagen is associated with compressive forces and is found primarily in fibrocartilaginous regions.^[Reference Gao^15, Reference Moffat, Sun, Pena, Chahine, Doty, Ateshian, Hung and Lu^26] The presence of compressive forces has been postulated to aid in the reduction of stress concentrators in the enthesis, possibly indicating the origin of type II collagen.^[Reference Liu, Thomopoulos, Birman, Li and Genin^27] These fibrillar collagens can further assemble into increasingly large fiber-like structures. This type of organization is found within the oriented soft tissue of the entheseal attachments and is typically associated with type I collagen. Collagen fibrils in this region can bundle together into structures called fascicles, ~50–300 µm in diameter, which are in turn bundled into large fibers, ~100–500 µm in diameter.^[Reference Fratzl and Weinkamer^25] Type X collagen is found in calcified fibrocartilage regions and is non-fibrillar.^[Reference Gao^15] Type X collagen is thought to play a role in ossification, but its exact function is unknown. Hypotheses include type X collagen acting to regulate mineralization at the ossifying front, and type X collagen functioning as a matrix protein and facilitating the ingrowth and mineralization of new bone.^[Reference Shen^28]

The enthesis contains proteoglycans that bind water, provide compressive strength, and contribute to collagen fiber formation. Proteoglycans consist of a central core protein and at least one covalently attached glycosaminoglycan (GAG) chain. GAGs are linear, highly charged polysaccharides that can contain sulfate and carboxylate groups.^[Reference Hardingham and Fosang^29] Aggrecan is the predominant proteoglycan in cartilage and fibrocartilage and is the largest of the proteoglycans. In tendon and ligament, aggrecan is most prevalent in regions of compression and in the fibrocartilage of the insertion site.^[Reference Waggett, Ralphs, Kwan, Woodnutt and Benjamin^24, Reference Benjamin and Ralphs^30] Similar to ligament and tendon, aggrecan in the meniscus is located in the inner portion of the meniscus, which is subject to more compressive loads, and the entheseal attachments.^[Reference Melrose, Smith, Cake, Read and Whitelock^31, Reference Rossetti, Kuntz, Kunold, Schock, Grabmayr, Sieber, Burgkart and Bausch^32] The large “bottle brush” structure and negative charge of aggrecan helps to bind and retain water, which contributes to the ability of the tissue to resist compression.^[Reference Tavakoli Nia, Han, Soltani Bozchalooi, Roughley, Youcef-Toumi, Grodzinsky and Ortiz^33] Small leucine-rich proteoglycans (SLRPs) such as fibromodulin, decorin, and biglycan are known to bind to collagen and help to regulate collagen fiber formation and maintenance.^[Reference Garg, Berg, Silver and Garg^34–Reference Vogel, Paulsson and Heinegård^36] SLRPs are prevalent in association with highly organized collagen bundles of tendon, ligament, and meniscus.^[Reference Rossetti, Kuntz, Kunold, Schock, Grabmayr, Sieber, Burgkart and Bausch^32, Reference Vanderploeg, Wilson, Imler, Ling and Levenston^37] Aggrecan is often found in regions of soft tissue-to-bone interfaces under compressive loading, while SLRPs are found in regions with large fibers experiencing tensile loads.^[Reference Rossetti, Kuntz, Kunold, Schock, Grabmayr, Sieber, Burgkart and Bausch^32]

Apatitic mineral is found within the subchondral bone and calcified fibrocartilage regions of the interfacial tissues. The mineral phase is primarily non-stoichiometric hydroxyapatite with carbonate substitutions, as opposed to geologic hydroxyapatite,^[Reference Wopenka and Pasteris^38] that is arranged into nanocrystalline platelets. In bone, these platelets are contained within collagen fibrils and are oriented with their c-axis parallel to the direction of the fibril. Non-collagenous proteins are thought to organize the apatite platelets into the intrafibrillar spacing of collagen, but the exact mechanism through which this hierarchical structuring occurs is unknown.^[Reference Nudelman, Lausch, Sommerdijk and Sone^39] Thorough reviews of bone structure are available.^[Reference Weiner and Wagner^40, Reference Reznikov, Shahar and Weiner^41] The mineral phase in calcified fibrocartilage is also mainly carbonate-substituted hydroxyapatite, but the organization of the crystals within the matrix is not as well understood.^[Reference Spalazzi, Boskey, Pleshko and Lu^12, Reference Schwartz, Pasteris, Genin, Daulton and Thomopoulos^42]

Aggregating the above information, subchondral bone consists of type I collagen fibrils infiltrated with nanocrystalline, carbonated apatite arranged circumferentially around pores, typically of the order of 1 mm in diameter.^[Reference Keaveny, Morgan, Niebur and Yeh^43] This structure transitions into non-porous calcified fibrocartilage, consisting of type II and type X collagen, apatitic mineral, and proteoglycans. Uncalcified fibrocartilage consists of splayed fibrils of type II collagen with proteoglycans. These splayed fibrils transition from the uncalcified fibrocartilage region into large type I collagen fibers that make up the oriented fiber region of the enthesis.^[Reference Rossetti, Kuntz, Kunold, Schock, Grabmayr, Sieber, Burgkart and Bausch^32, Reference Deymier-Black, Pasteris, Genin and Thomopoulos^44] Understanding native structure/composition should inform material selection for tissue engineering.

Materials selection

Appropriate materials for a tissue engineered scaffold must possess adequate mechanical properties, support cellular attachment and differentiation/proliferation, and potentiate cellular remodeling. Since the modulus ranges drastically across soft tissue-to-bone interfaces, a variety of materials have been used for tissue engineered constructs. To match the moduli of the stiff, mineralized regions of the enthesis, various calcium phosphate minerals, such as hydroxyapatite and tricalcium phosphate, and bioglass have been used.^[Reference Allan, Pilliar, Wang, Grynpas and Kandel^45–Reference Spalazzi, Doty, Moffat, Levine and Lu^51] The compliant portions of the enthesis have been constructed from polymers and copolymers consisting of poly(capralactone) (PCL), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and/or poly(ethylene glycol) diacrylate,^[Reference Nyberg, Rindone, Dorafshar and Grayson^49, Reference Spalazzi, Doty, Moffat, Levine and Lu^51–Reference Paxton, Donnelly, Keatch and Baar^56] as well as other biopolymers such as silk, agarose, gelatin, hyaluronic acid, and collagen.^[Reference Kim, Kim, Choi, Jeong, Jo and Cho^48, Reference Altman, Horan, Lu, Moreau, Martin, Richmond and Kaplan^57–Reference Wang, Shan, Choi, Oh, Kepler, Chen and Lu^62] Decellularized or demineralized native matrices have also been used to engineer the enthesis.^[Reference Nyberg, Rindone, Dorafshar and Grayson^49, Reference McCorry, Mansfield, Sha, Coppola, Lee and Bonassar^60, Reference Chang, Lin, Lin, Chou and Liu^61, Reference Xu, Kuntz, Foehr, Kuempel, Wagner, Tuebel, Deimling and Burgkart^63, Reference Sundar, Pendegrass and Blunn^64]

Materials-based design and fabrication

The complex structure of the enthesis is related to its function, providing continuity and integration between multiple tissues with differing properties. The necessity for this integration and continuity arises from the loading of such interfaces in tension;^[Reference Lu and Thomopoulos^8] hence, failure modes like delamination become relevant for poorly integrated constructs. To promote continuity, various materials processing techniques have been used. Many of these techniques revolve around the construction of gradients or the binding of multiple “compartmentalized” materials. Controlled crystal growth on electrospun poly(lactic-co-glycolic) acid (PLGA) resulted in a nanofiber scaffold with a mineral gradient.^[Reference Liu, Lipner, Xie, Manning, Thomopoulos and Xia^47] This gradient was formed through syringe pump-mediated injection of a calcium and phosphate salt-containing solution. Upon cell seeding, the activity of alkaline phosphatase (ALP), an enzyme associated with mineral deposition, the expression of RUNX2, an osteoblast-related transcription factor, and the expression of osteocalcin, an osteoblast-related protein, were correlated with mineral deposition. Cellular density and cell proliferation were negatively correlated with mineral deposition.^[Reference Liu, Lipner, Xie, Manning, Thomopoulos and Xia^47] These results indicate that proper processing and appropriate arrangement of enthesis-related materials result in spatially localized cellular responses that mimic those found in the body. However, implantation of this scaffold for the repair of the murine supraspinatus tendon resulted in scar formation, indicating that further processing is required for optimal enthesis reconstruction.^[Reference Lipner, Shen, Cavinatto, Liu, Havlioglu, Xia, Galatz and Thomopoulos^65]

Regional integration in scaffolds can also be induced through the production of microstructure gradients. For example, freeze-casting coupled with salt-leaching enabled the formation of a multi-region scaffold composed of silk fibroin [Fig. 6(a)].^[Reference Font Tellado, Bonani, Rosado Balmayor, Föhr, Motta, Migliaresi and van Griensven^58] The processing conditions were designed to form a continuous scaffold with a porous, trabecular bone-like structure that transitions into a fiber-like morphology. The porous region results from salt leaching, in which a solution of silk fibroin and NaCl is frozen and freeze-dried. The precipitated salt crystals are subsequently dissolved, creating pores within the silk matrix. This region transitions to a fiber-like morphology, created using freeze casting, a process in which directional freezing is used to grow elongated ice crystals within a polymer solution. During solidification, the polymer, i.e., silk fibroin, is concentrated into the boundaries between the crystals.^[Reference Wegst, Bai, Saiz, Tomsia and Ritchie^66] Upon sublimation of the ice crystals, oriented fiber-like structures composed of silk fibroin remain. These processing methods result in a continuous silk structure that mimics the morphology of collagen in the native enthesis.^[Reference Font Tellado, Bonani, Rosado Balmayor, Föhr, Motta, Migliaresi and van Griensven^58] Similar scaffolds have been produced for the osteochondral interface, demonstrating the customizability of these techniques.^[Reference Ding, Zhu, Xu, Zhang, Zhao, Ji, Wang, Wang, Li, Kong, Ma and Yang^67]

Figure 6. Examples of tissue engineered interface constructs for soft tissue to bone. (a) Biphasic anisotropic silk fibroin scaffold with integrated fiber/bone interface. Images from left-to-right: μCT of full construct, field emission scanning electron microscopy (FESEM) image of, anisotropic (fiber) region, fluorescence microscopy image of transition region, and FESEM image of porous (trabecular) region. Reprinted with permission from TISSUE ENGINEERING, Part A, Volume 23, Issue 15–16, published by Mary Ann Liebert, Inc., New Rochelle, NY^[Reference Font Tellado, Bonani, Rosado Balmayor, Föhr, Motta, Migliaresi and van Griensven^58] (b) Bone–ligament–bone ACL replacement generated using stem cell self-assembly and targeted differentiation. Images from left-to-right: full tissue engineered construct, immunostained for collagen (red) and DAPI-stained (nuclear stain) section showing bony region prior to implantation, immunostained for collagen (red) and DAPI-stained (nuclear stain) section showing ligament region prior to implantation, image of regenerated fibrocartilaginous region with aligned nuclei (arrow) after 2 months implantation. Reprinted with permission from TISSUE ENGINEERING, Part A, Volume 18, Issue 1–2, published by Mary Ann Liebert, Inc., New Rochelle, NY.^[Reference Ma, Smietana, Kostrominova, Wojtys, Larkin and Arruda^68] (c) Porous membrane with inverse gradients of PDGF and BMP-2 for tendon-bone repair. Reprinted from Acta Biomaterialia, Volume 10, Issue 3, Hyun Ki Min, Se Heang Oh, Jonh Min Lee, Gun Il Im, Jin Ho Lee, Porous membrane with reverse gradients of PDGF-BB and BMP-2 for tendon-to-bone repair: In vitro evaluation on adipose-derived stem cell differentiation, 1272–1279, Copyright © 2013, with permission from Elsevier^[Reference Min, Oh, Lee, Im and Lee^69]

Structural characteristics are important in that they not only affect the mechanical properties of the scaffold but also drive cellular differentiation. Fiber alignment drives matrix deposition rates in meniscal fibrochondrocytes and mesenchymal stem cells (MSCs) (the progenitor cells for bone, cartilage, etc.).^[Reference Baker and Mauck^70] Pore size affects the production of cartilage matrix proteins by cells. For example, smaller pore sizes cause cells to produce more GAGs.^[Reference Di Luca, Van Blitterswijk and Moroni^22] In addition to structurally derived cellular effects, the material composition of scaffolds also has an effect on differentiation. The addition of hydroxyapatite to scaffolds improves osteogenic properties and increases cellular attachment,^[Reference Allan, Pilliar, Wang, Grynpas and Kandel^45, Reference Paxton, Donnelly, Keatch and Baar^56] and the presence of GAGs in scaffolds drives cells toward a chondrogenic lineage.^[Reference Park, Yang, Woo, Yang, Na and Park^71, Reference Han, Heo, Driscoll, Delucca, McLeod, Smith, Duncan, Mauck and Elliott^72]

The use of materials to control cellular responses can also be achieved through biodegradability and remodeling. For example, a compartmentalized triphasic scaffold, created by sintering polyglactin mesh, PLGA microspheres, and bioglass and PLGA microspheres together, shows varying rates and types of tissue ingrowth into the scaffold following subcutaneous implantation.^[Reference Spalazzi, Doty, Moffat, Levine and Lu^51] All regions of the scaffold showed collagen deposition, and the bioglass and PLGA microsphere region allowed for mineral deposition. The polyglactin region degraded entirely, producing a fibrocartilage-like area between the polyglactin and PLGA microsphere region.^[Reference Spalazzi, Doty, Moffat, Levine and Lu^51, Reference Spalazzi, Dagher, Doty, Guo, Rodeo and Lu^73] In design of scaffolds that utilize multiple materials, structural considerations must be made for the types of mechanical forces to which the scaffold will be subjected. For example, fused microspheres are resistant to compression and polyglactin mesh is resistant to tension, but neither structure is designed to resist shear. Additionally, the differing structures could result in stress concentrations at the interface between the materials, arising from dissimilarities in their Poisson's ratios. Use of biodegradable materials can promote tissue infiltration, and particularly in this case, can be used to diminish the sharpness of the interface over time. The mechanism of biodegradation should also be considered for tissue engineering. Biodegradable polymers like PLA and PGA can be degraded through hydrolysis, opening up space for tissue to grow into, whereas biopolymers such as collagen must be enzymatically degraded and rearranged by cells, providing a template for remodeling. For example, cells rearranged collagen gels into large, oriented fibers during culture of a meniscus construct, indicating the value of incorporating cellular remodeling capability into a scaffold.^[Reference Puetzer, Koo and Bonassar^74] Implementation of continuity, integration of structure, and support for cellular remodeling in scaffolds allow for control of mechanical properties, strengthening of interfacial regions, and ability of the scaffold to integrate with native tissue upon in vivo implantation.

Native cell types

Soft tissue-to-bone interfaces span four regions with distinct cellular phenotypes (Figs. 3 and 4). Each cellular type found in the enthesis has a distinctive behavior, cell shape, matrix production profile, and genetic expression that define its phenotype. Osteoblasts, osteoclasts, and osteocytes are the three main cell types that reside in bone. Osteoblasts initiate new bone formation, while osteoclasts resorb and remodel bone. An osteocyte is a terminally differentiated osteoblast that resides in the mineralized matrix to maintain bone homeostasis [Fig. 4(a)]. Together, these cells regulate bone formation and maintenance. The bone phenotype is typically quantified by expression or accumulation of proteins, such as osteopontin and osteocalcin, type I collagen, markers of mineralization, such as ALP activity, and expression of transcription factors, such as RUNX2.^[Reference Lian and Stein^75] The transition from bone to the calcified fibrocartilage region is regulated by hypertrophic fibrochondrocytes [Fig. 4(b)]. Hypertrophy is the process in which chondrocytes increase in size and secrete type X collagen in a mineralized cartilaginous matrix.^[Reference Mackie, Ahmed, Tatarczuch, Chen and Mirams^76] In contrast, chondrocytes are found in cartilage and exhibit a rounded morphology. These cells reside in a proteoglycan-rich (mostly aggrecan) matrix composed of collagens I and II. Chondrocytes, found in the cartilaginous tissue, have upregulated levels of aggrecan, type II collagen, link protein, Sox9, and COMP genes.^[Reference Sandell and Aigner^77] Fibrochondrocytes that reside in the uncalcified fibrocartilage region of the enthesis are named as such because they exhibit phenotypic markers of both fibroblasts and chondrocytes [Fig. 4(c)].^[Reference Sanchez-Adams and Athanasiou^78] The native cell phenotypes serve as benchmarks for cell behavior and stem cell differentiation in tissue engineered constructs. Ligament cells and tenocytes (tendon cells) are often described as exhibiting a fibroblastic phenotype due to their spindle shape and production of type I collagen [Fig. 4(d)].^[Reference Benjamin and Ralphs^18] While these two cell types belong to distinctly different tissues, they share many common markers. Key positive markers for this phenotype are production of type I collagen, tenascin-C, scleraxis, tenomodulin, and proteoglycans such as decorin, biglycan, versican, lumican, and fibromodulin.^[Reference Spanoudes, Gaspar, Pandit and Zeugolis^79, Reference Hasegawa, Nakahara, Kinoshita, Asahara, Koziol and Lotz^80]

Tissue engineering with cells

A consistent challenge of producing these soft tissue-to-bone interfaces is incorporating cells into the materials processing methods of the scaffold. Cellularizing these constructs can be accomplished by either encapsulating cells directly into the scaffold or by seeding cells onto the material surface and directing migrating cells into the scaffold. Growth factors or other chemoattractants are useful to encourage cellular migration and infiltration into scaffolds.^[Reference Sundar, Pendegrass and Blunn^64, Reference Lee, Rodeo, Fortier, Lu, Erisken and Mao^81, Reference Monibi and Cook^82] Processing cells and materials together restricts the conditions in which a scaffold can be processed. Cells require a specific operational window of temperature, pH, pressure, strain, osmolarity, and ion and solute balance in order to maintain viability. Furthermore, the soft tissue-to-bone interface contains an array of cell types. The number of cell types and the cellular locations in the tissue engineered scaffold play a role in soft tissue-to-bone interface assembly, culture, and development.

Given the gradient of cell types present in the enthesis, co-culture is one method to generate a graded interface. Different cell types are constantly interacting during native development and have intertwined signaling feedback mechanisms that are critical to development.^[Reference Benjamin and Ralphs^18, Reference Mackie, Ahmed, Tatarczuch, Chen and Mirams^76, Reference Zelzer, Blitz, Killian and Thomopoulos^83] Ex vivo co-culture mediates cell–cell contact and paracrine interactions that have been shown to influence cellular phenotype. Co-culturing of chondrocytes and osteoblasts in direct contact using a high cell density 3D micromass reduced GAG deposition in chondrocytes and cell-mediated mineralization in osteoblasts.^[Reference Jiang, Nicoll and Lu^84] Furthermore, co-culture can be used as a tactic to guide stem cell differentiation.^[Reference McCorry, Puetzer and Bonassar^85–Reference Hoben, Willard and Athanasiou^89] One strategy to create an integrated gradient is to co-culture the desired cell types and generate cell gradients that utilize cell–cell interactions to mature the material into a graded interface. For ligament–bone interfaces, 2D co-culture of fibroblasts and osteoblasts, modeled using a fibroblast region, interface region, and osteoblast region, decreased cell proliferation and increased cell-mediated mineralization by fibroblasts. In addition, production of type II collagen and aggrecan increased, indicating that co-culture interactions can lead to cell trans-differentiation from one cell phenotype to another.^[Reference Wang, Shan, Choi, Oh, Kepler, Chen and Lu^62] Co-culturing of different cell types is a useful technique to integrate the different tissue regions of the soft tissue-to-bone interface.

An alternative to using multiple cell types is using MSCs, which can differentiate into multiple cell types. In vivo entheses begin as dense mesenchymal condensates and develop into complex interfaces.^[Reference Gardner and O'Rahilly^90–Reference Mérida-Velasco, Sánchez-Montesinos, Espín-Ferra, Rodríguez-Vázquez, Mérida-Velasco and Jiménez-Collado^92] Since MSCs have the capacity to differentiate into all the cell phenotypes of the enthesis, MSCs are a logical cell source to use for tissue engineering the enthesis.^[Reference Caplan and Dennis^93, Reference Mackay, Beck, Murphy, Barry, Chichester and Pittenger^94] MSCs from bone marrow (bmMSCs) are a popular source for tissue engineering, because they are easily obtained and expanded. Adipose-derived stem cells (ASCs) are another common stem cell source. Since ASCs are easily obtained from fat which is considered surgical debris, they are the most readily available stem cell source from human tissue. While ASCs do have multi-potent properties, they have been shown to be less effective than bmMSCs at differentiating into cartilage and bone.^[Reference Im, Shin and Lee^95] Stem cells seeded into tissue engineered enthesis constructs rely on mimicking the biomaterial and biochemical inputs that direct differentiation during development. For example, regional changes in pore alignment of a tendon/ligament enthesis silk fibroin scaffold resulted in corresponding ASC differentiation into cartilage and tendon lineages [Fig. 6(a)].^[Reference Font Tellado, Bonani, Rosado Balmayor, Föhr, Motta, Migliaresi and van Griensven^58] These examples emphasize the importance of scaffold material and biochemical design in order to inform cell behavior when generating enthesis constructs.

Construct geometry and chemistry have been shown in multiple systems to affect cellular phenotype through cell–material interactions.^[Reference Spalazzi and Lu^96, Reference Gunja and Athanasiou^97] Cellular phenotypes can be dictated by cell–material interactions. A material such as collagen has cellular adhesion sites, allowing cells to bind and spread, encouraging the cells to develop a fibroblastic morphology. Alternatively, a material lacking in cell adhesion sites, like alginate, prevents cells from binding, encouraging a chondrogenic morphology.^[Reference Zeltz and Gullberg^98, Reference Augst, Kong and Mooney^99] Cell morphology, such as a fibroblastic or chondrogenic morphology, has been shown to be an indicator for cell behavior and matrix production.^[Reference Baker, Nathan, Gee and Mauck^100] Scaffold mechanical properties are known to direct stem cell fate, where materials with stiffer structures promote osteogenic differentiation.^[Reference Tse and Engler^101, Reference Engler, Sen, Sweeney and Discher^102] Lastly, chemical composition dictates cellular response. Cells embedded in a proteoglycan-rich region experience increased strain shielding compared to cells embedded in collagen fibers when a tissue-level mechanical deformation is applied to the scaffold.^[Reference Han, Heo, Driscoll, Delucca, McLeod, Smith, Duncan, Mauck and Elliott^72] Furthermore, the arrangement and alignment of fibers in a 3D microenvironment affects cellular behavior and the distribution of applied loads. In an aligned poly(ethylene oxide) scaffold, cells attach and elongate in the direction of the fibers. Fiber alignment aided bmMSC differentiation into a fibrous phenotype compared with the same bmMSCs in pellet culture which developed a chondrogenic phenotype.^[Reference Baker, Nathan, Gee and Mauck^100] Furthermore, aligned fibers improved ligamentous phenotype, as cells develop characteristic spindle shapes and increase production of collagen when compared with random fiber alignment. The alignment of fibers also affects the cellular response to an applied strain, with longitudinal strains in the fiber direction inducing the largest increase in collagen production.^[Reference Lee, Shin, Cho, Kang, Kim, Park and Shin^103, Reference Subramony, Dargis, Castillo, Azeloglu, Tracey, Su and Lu^104] These studies emphasize the importance of material mechanical properties and structure when considering cellular interactions and response.

Another approach to tissue engineering soft tissue-to-bone interfaces shifts the focus from materials design to utilizing the cells themselves to create the scaffold. Cells grown in monolayers have the ability to proliferate to fill the surface area and then self-assemble to form their own matrix. A multi-component bone–ligament–bone tissue engineered anterior cruciate ligament (ACL) graft was created using confluent cell monolayers [Fig. 6(b)]. The bmMSCs were first pre-differentiated into ligamentous or bone pathways in monolayer and then assembled together into a 3D scaffold. The resulting scaffold showed sufficient properties for ACL replacement and performed well during long-term implantation in an ovine model.^[Reference Ma, Smietana, Kostrominova, Wojtys, Larkin and Arruda^68] Gene transfer is another strategy to spatially regulate genetic modification and differentiation of primary dermal fibroblasts. A retrovirus encoding the factors RUNX2 and cbfa1 was used to induce osteoblastic and fibroblastic differentiation, respectively.^[Reference Phillips, Burns, Le Doux, Guldberg and García^105] These techniques demonstrate that cells can be used to produce complex gradients for soft tissue-to-bone interfaces.

Native biochemical signaling

Soft tissue-to-bone interfaces have a wide array of growth factors that influence cellular activities such as differentiation, proliferation, apoptosis, and matrix production. Key growth factor contributors to bone growth, repair, and differentiation are bone morphogenic proteins (BMPs), transforming growth factor-βs (TGF-βs), and insulin-like growth factors (IGFs) (Table I).^[Reference Urist, DeLange and Finerman^106–Reference Lieberman, Daluiski and Einhorn^108] The growth plate contains similar regional zones to the enthesis since it is an interface between bone and cartilage. Growth factors secreted in the growth plate are essential to endochondral bone formation and include IGFs, Indian hedgehog (Ihh), parathyroid hormone-related peptide (PTHrP), BMPs, Wnts, fibroblast growth factors (FGFs), and TGF-βs.^[Reference Mackie, Ahmed, Tatarczuch, Chen and Mirams^76] Key players in bulk tendon and ligament development, healing, and remodeling are IGFs, growth and differentiation factors (GDFs), TGF-βs, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and FGFs.^[Reference Molloy, Wang and Murrell^109, Reference James, Kesturu, Balian and Chhabra^110] IGFs, FGFs, TGF-βs, and PDGF are the central growth factors that contribute to developing and maintaining organized collagen structures and high levels of proteoglycans in cartilage and meniscus.^[Reference Van der Kraan, Buma, Van Kuppevelt and Van Den Berg^111] While many of these growth factors are present in all of these tissues (Table I), the spatial and temporal expression of growth factors are important drivers of tissue development.

Table I. Growth factors commonly used in orthopedic tissue engineering applications. See the following reviews for more on specific growth factors.^[Reference Lieberman, Daluiski and Einhorn^108–Reference James, Kesturu, Balian and Chhabra^110, Reference Baylink, Finkelman and Mohan^112]

? indicates that no native study on these growth factors has been performed; however, TE applications have been performed using these growth factors and they are believed to play a role.

TGF-βs, GDFs, BMPs, and IGFs regulate bone and joint development by influencing stem cell differentiation, matrix synthesis and remodeling, and cellular migration and proliferation. The TGF-β superfamily is a group of structurally related proteins including TGF-βs, GDFs, and BMPs that influence a broad range of activities in musculoskeletal development. Members of the BMP family encourage stem cell proliferation and differentiation into osteoblasts.^[Reference Yoon, Pogue, Ovchinnikov, Yoshii, Mishina, Behringer and Lyons^134, Reference Minina, Kreschel, Naski, Ornitz and Vortkamp^135] Furthermore, BMPs are osteoinductive, encouraging bone formation and maintenance by recruiting bone-forming cells that result in the formation of mineralized bone. BMPs have successfully navigated the FDA approval process for bone healing applications, notably BMP-2 (Infuse^® Medtronic) and BMP-7 (also called OP-1 by Stryker).^[Reference Bessa, Casal and Reis^136] GDFs, specifically GDF-5, interact closely with BMPs and act as signaling molecules in the growth and differentiation of cartilage, tendon, and ligament.^[Reference Farng, Bs, Bs, Bs and Mcallister^128, Reference James, Kumbar, Laurencin, Balian and Chhabra^129, Reference Wolfman, Hattersley, Cox, Celeste, Nelson, Yamaji, Dube, Diblasio-smith, Nove, Song, Wozney, Rosen, Wolfman, Hattersley, Cox and Anthony^137–Reference Decker, Um, Dyment, Cottingham, Usami, Enomoto-Iwamoto, Kronenberg, Maye, Rowe, Koyama and Pacifici^139] TGF-βs are prevalent in all of these tissues and are known to play a role in proliferation and stem cell differentiation.^[Reference Heine, Munoz, Flanders, Ellingsworth, Lam, Thompson, Roberts and Sporn^140] In particular, TGF-β is heavily implicated for its role in chondrogenic differentiation and development.^[Reference Leonard, Fuld, Frenz, Downie, Massague and Newman^141, Reference Kulyk, Rodgers, Greer and Kosher^142] IGF is a regulator of longitudinal bone growth in that it stimulates osteoblast proliferation and differentiation as well as increasing general cell proliferation and ECM synthesis.^[Reference Isaksson, Jansson and Gause^143–Reference Abrahamsson^146] These growth factors are essential signaling contributors to stem cell differentiation and tissue development in native orthopedic tissues.

Several growth factors prevalent in the inflammatory and healing processes also aid in the production of tissue. Vascularization-related growth factors are important in soft tissue development and healing; common growth factors include PDGF and VEGF.^[Reference Dormer, Singh, Zhao, Mohan, Berkland and Detamore^54, Reference Lieberman, Daluiski and Einhorn^108, Reference James, Kesturu, Balian and Chhabra^110, Reference Thomopoulos, Harwood, Silva, Amiel and Gelberman^132, Reference Midy and Plouët^147–Reference Keck, Hauser, Krivi, Sanzo, Warren, Feder and Connolly^149] Basic fibroblastic growth factor (FGF-2) is known to contribute to cell proliferation and is expressed in the developmental and healing phases of these tissues.^[Reference Thomopoulos, Harwood, Silva, Amiel and Gelberman^132, Reference Minina, Kreschel, Naski, Ornitz and Vortkamp^135] Growth factors have been specifically targeted for tissue engineering applications, because they are exogenously secreted factors that can be dosed into a system with relative ease and can drive stem cell differentiation and tissue maintenance.

Biochemical applications in tissue engineering

Since growth factors are known to play influential roles in cell behavior, they have been frequently applied to tissue engineer bone, cartilage, meniscus, tendon, and ligament (Table I). BMPs are popular for tissue engineering applications in bone interfaces and have been shown to induce mineralization as well as to stimulate osteoblast proliferation in scaffolds.^[Reference Patel, Young, Tabata, Jansen, Wong and Mikos^115, Reference Augst, Marolt, Freed, Vepari, Meinel, Farley, Fajardo, Patel, Gray, Kaplan and Vunjak-Novakovic^124, Reference Bessa, Casal and Reis^136] Scaffolds doped with IGF-I increased cartilage regeneration in growth plate injuries in vivo and increased the collagen and GAG content in tissue engineered cartilage constructs in vitro.^[Reference Aguilar, Trippel, Shi and Bonassar^130, Reference Sundararaj, Cieply, Gupta, Milbrandt and Puleo^150, Reference Gooch, Blunk, Courter, Sieminski, Bursac, Vunjak-Novakovic and Freed^151] VEGF was incorporated into a mineralized degradable polymer scaffold to provide osteoconductive signals for bone growth and angiogenesis.^[Reference Murphy, Peters, Kohn and Mooney^152] GDFs increased type I collagen production in 3D tissue engineered scaffolds.^[Reference Farng, Bs, Bs, Bs and Mcallister^128, Reference James, Kumbar, Laurencin, Balian and Chhabra^129] TGF-βs increase GAG and collagen production which improves the quality of tissue engineered cartilage and meniscus.^[Reference Lee, Rodeo, Fortier, Lu, Erisken and Mao^81, Reference Macbarb, Makris, Hu and Athanasiou^121, Reference Park, Temenoff, Holland, Tabata and Mikos^125, Reference Steinert, Palmer, Capito, Hofstaetter, Pilapil, Ghivizzani, Spector and Evans^153] Growth factors provide signaling mechanisms to encourage cellular components to exhibit a specific phenotype.

Prominent growth factors in these developmental processes can also guide stem cell differentiation in tissue engineered constructs. BMP-2 combined with hydroxyapatite in a silk fibroin fiber scaffold supported MSC growth and differentiation toward an osteogenic phenotype, quantified by increased BMP-2 transcription levels and mineral deposition.^[Reference Li, Vepari, Jin, Kim and Kaplan^113] Delivering BMP-2 and BMP-7 sequentially increased ALP activity while suppressing proliferation of MSCs,^[Reference Yilgor, Tuzlakoglu, Reis, Hasirci and Hasirci^118] highlighting the potential for benefits from temporal application of biochemical factors to cells. Growth factor delivery using a scaffold can increase efficiency of stem cell differentiation into a desired cell phenotype. Localized delivery of IGF in a PLGA scaffold increased MSC chondrogenesis in vitro,^[Reference Sundararaj, Cieply, Gupta, Milbrandt and Puleo^150] while FGF-2 and TGF-β1 increased chondrogenesis of periosteum derived cells.^[Reference Marini, Martin, Stevens, Langer and Shastri^122] GDF-5 supplemented medium as well as GDF-5 induction by adenovirus increased expression of type I collagen and scleraxis, associated with tenogensis, in stem cells seeded on a 3D scaffold.^[Reference Farng, Bs, Bs, Bs and Mcallister^128, Reference Abraham and Haut Donahue¹Reference Hardingham and Fosang^29] TGF-β3 conjugated with chondroitin sulfate increased MSC chondrogenic differentiation by increasing GAG production and expression of Sox9, COMP, aggrecan, and type II collagen genes.^[Reference Park, Yang, Woo, Yang, Na and Park^71] Controlled release of TGF-β3 also promoted chondrogenesis of human infrapatellar fat pad-derived stem cells, measured by increased production of sulfated GAGs and collagen.^[Reference Kim, Erickson, Choudhury, Pleshko and Mauck^127, Reference Almeida, Liu, Cunniffe, Mulhall, Matsiko, Buckley, O'Brien and Kelly^154]

Growth factors are useful chemical tools for generating complex materials gradients. Since growth factors can be incorporated into a biomaterial, they can be strategically placed or applied to influence cellular behavior in controlled chemical gradients for soft tissue-to-bone tissue engineering. BMP-2, delivered using microspheres in a poly(propylene fumarate) scaffold, increased bone regeneration and ACL graft fixation.^[Reference Parry, Olthof, Shogren, Dadsetan, Van Wijnen, Yaszemski and Kakar^116] An integrated gradient of BMP-2 and TGF-β1 has been shown to create a continuous material and phenotypic transition between cartilage and bone.^[Reference Tevlek, Hosseinian, Ogutcu, Turk and Aydin^50, Reference Dormer, Singh, Zhao, Mohan, Berkland and Detamore^54] Application of growth factors is especially favorable when using a single progenitor cell type in the scaffold. Rather than seeding multiple cell types, growth factors can be incorporated to initiate the differentiation of stem cells. ASCs, seeded in a porous PCL/Pluronic F127 membrane with gradients of PDGF, specifically PDGF-BB (tendon) and BMP-2, created a continuous interface between tendon and bone, with PDGF promoting tenogenesis and BMP-2 promoting osteogenesis [Fig. 6(c)].^[Reference Min, Oh, Lee, Im and Lee^69] In combination with materials processing methods and cells, growth factor gradients contribute to the development of integrated and graded regions for engineered soft tissue-to-bone interfaces.

In addition to facilitating cell–biochemical interactions, growth factors can be sequestered by ECM proteins. Proteoglycans function as physiologic regulators by sequestering growth factors and controlling release. SLRPs, such as biglycan, decorin, and fibromodulin, are capable of binding to TGF-β and are likely to regulate the availability of TGF-β to cells.^[Reference Hildebrand, Romarís, Rasmussen, Heinegård, Twardzik, Border and Ruoslahti^155] While the exact mechanism is not well understood, increased levels of type II collagen in a scaffold enhance the effect of TGF-β on chondrocytes.^[Reference Qi and Scully^156] FGFs bind to heparan proteoglycans in the ECM. For example, perlecan, a heparan sulfate proteoglycan, co-localizes with FGF thus regulating FGF availability to cell receptors.^[Reference Mongiat, Otto, Oldershaw, Ferrer, Sato and Iozzo^157] Availability of BMP-2 is regulated through sequestering with heparin and type IIA procollagen.^[Reference Ruppert, Hoffmann and Sebald^158, Reference Zhu, Oganesian, Keene and Sandell^159] Biomaterials, such as alginate, have been modified with binding peptides that specifically bind a growth factor in order to sustain growth factor availability over longer culture periods.^[Reference Aguilar, Trippel, Shi and Bonassar^130] The ability of ECM proteins to regulate growth factor availability contributes to the highly complex interplay of materials, cells, and biochemical signaling in a tissue engineered construct but also allows for the engineered regulation of cellular behavior and scaffold maturation.

Biomaterials combined with growth factors can act as powerful chemoattractants. Growth factors, connective tissue growth factor (CTGF) and TGF-β3, seeded onto a tissue engineered scaffold encouraged cellular recruitment and region-specific cell morphology in the meniscus.^[Reference Lee, Rodeo, Fortier, Lu, Erisken and Mao^81] Decellularization techniques preserve the material structure and resident biochemical components that can be used as a fully functional scaffold.^[Reference Kawecki, Łabuś, Klama-Baryla, Kitala, Kraut, Glik, Misiuga, Nowak, Bielecki and Kasperczyk^160] Decellularized and demineralized bone matrix applied to the tendon-to-bone surgical suture site improved tendon-to-bone healing, demonstrated through increased amounts of fibrocartilage and mineralized fibrocartilage in the repair site enthesis and reduced rates of tendon failure.^[Reference Sundar, Pendegrass and Blunn^64] Recent work has successfully decellularized the entire tendon-to-bone insertion,^[Reference Xu, Kuntz, Foehr, Kuempel, Wagner, Tuebel, Deimling and Burgkart^63] with significant improvements in pullout strength using the decellularized tendon-to-bone insertion over the direct suture technique.^[Reference Farnebo, Woon, Kim, Pham and Chang^161] Cells were able to repopulate the graft and exhibited a transition from a cartilage-like to tenocyte-like morphology across the interface.^[Reference Farnebo, Woon, Kim, Pham and Chang^161] Biomaterials and growth factors together serve as promising tissue engineered scaffolds that rely on cell repopulation after in vivo implantation.

Chemically driven maturation

Chemical means of driving cell maturation typically occur through media supplementation. In the case of the enthesis, bone media, cartilage media, ligamentous/tendinous media, and meniscal media are typically used individually or in combination. The majority of osteogenic media contains β-glycerophosphate and dexamethasone,^[Reference Criscenti, Longoni, Di Luca, De Maria, Van Blitterswijk, Vozzi and Moroni^52, Reference Liu, Thomopoulos, Chen, Birman, Buehler and Genin^55, Reference Ma, Smietana, Kostrominova, Wojtys, Larkin and Arruda^68, Reference Lin, Lozito, Alexander, Gottardi and Tuan^163–Reference Goldman and Barabino^165] with some including BMP-2^[Reference Lin, Lozito, Alexander, Gottardi and Tuan^163, Reference Goldman and Barabino^165] and TGF-β.^[Reference Ma, Smietana, Kostrominova, Wojtys, Larkin and Arruda^68] Dexamethasone is also included in some chondrogenic media,^[Reference Mueller, Fischer, Zellner, Berner, Dienstknecht, Prantl, Kujat, Nerlich, Tuan and Angele^126, Reference Grayson, Bhumiratana, Grace Chao, Hung and Vunjak-Novakovic^164, Reference Goldman and Barabino^165] while TGF-β is often used in media for the soft tissue portions of the enthesis.^[Reference Criscenti, Longoni, Di Luca, De Maria, Van Blitterswijk, Vozzi and Moroni^52, Reference Ma, Smietana, Kostrominova, Wojtys, Larkin and Arruda^68, Reference Mueller, Fischer, Zellner, Berner, Dienstknecht, Prantl, Kujat, Nerlich, Tuan and Angele^126, Reference Lin, Lozito, Alexander, Gottardi and Tuan^163–Reference Goldman and Barabino^165] Dexamethasone is a glucocorticoid that has been shown to increase ALP activity in MSCs.^[Reference Jaiswal, Haynesworth, Caplan and Bruder^166] β-Glycerophosphate is an organic phosphate donor classically used to induce MSC differentiation toward bone phenotypes.^[Reference Criscenti, Longoni, Di Luca, De Maria, Van Blitterswijk, Vozzi and Moroni^52, Reference Lin, Lozito, Alexander, Gottardi and Tuan^163, Reference Grayson, Fröhlich, Yeager, Bhumiratana, Chan, Cannizzaro, Wan, Liu, Guo and Vunjak-Novakovic^167, Reference Boskey and Roy^168] Application of β-glycerophosphate at the interface of a calcium polyphosphate substrate cultured with chondrocytes formed two zones, a calcified region between a calcium phosphate bony substrate and a hyaline cartilage-like zone.^[Reference Allan, Pilliar, Wang, Grynpas and Kandel^45] Furthermore, combining β-glycerophosphate and BMP-2 in the bone region of an osteochondral scaffold directed stem cell osteogenesis.^[Reference Augst, Marolt, Freed, Vepari, Meinel, Farley, Fajardo, Patel, Gray, Kaplan and Vunjak-Novakovic^124] Ascorbic acid and L-proline are added to media to promote collagen production. Ascorbic acid or ascorbate-2-phosphate (a format used to stabilize ascorbic acid in solution)^[Reference Hata and Senoo^169] has been shown to increase the hydroxylation rate of proline,^[Reference Schwarz, Kleinman and Owens^170] aiding in collagen production. These chemical stimulants can be incorporated into the scaffold design to enhance a desired cell behavior either through direct interaction with cells or complementary mechanisms to other growth factors or ECM materials present in the scaffold.

Many of the relevant published systems have been produced for the osteochondral interface, given its similarity to other soft tissue-to-bone interfaces. The majority of systems designed to promote interface formation in culture rely on diffusion-based bioreactors; the general principle being that bone phenotype-promoting media and soft tissue phenotype-promoting media will diffuse through the construct, meet in the middle, and facilitate the formation of an interface.^[Reference Chang, Lin, Lin, Chou and Liu^61, Reference Grayson, Bhumiratana, Grace Chao, Hung and Vunjak-Novakovic^164, Reference Goldman and Barabino^165, Reference Li, Qu, Han, Mauck, Han and Ph^171] Using this concept, a microfluidic bioreactor directed MSC differentiation along osteogenic and chondrogenic pathways in parallel [Fig. 7(a)]. The bioreactor consisted of an MSC-seeded agarose gel sandwiched between two other MSC-seeded agarose gels that contained channels. The channel-containing gels are perfused with osteogenic and chondrogenic media, respectively, creating a tri-layered scaffold with an interfacial region. This system produced gradients from the osteogenic to chondrogenic regions: decreasing type I collagen content, increasing type II collagen content, and decreasing Alizarin Red staining, reflecting decreasing matrix-immobilized calcium content. These gradients indicate that interfacial regions can be formed by supplying one cell type with different medias and flow conditions simultaneously [Fig. 7(a)].^[Reference Goldman and Barabino^165, Reference Abraham and Haut Donahue¹Reference Han, Heo, Driscoll, Delucca, McLeod, Smith, Duncan, Mauck and Elliott^72] Flow of chondrogenic and osteogenic media through the top and bottom of a singular osteochondral scaffold, while maintaining media separation around the scaffold using an O-ring, produced distinctive corresponding regions [Fig. 7(b)]. Osteochondral scaffolds were produced by pipetting a chondrogenic, photo-crosslinked, cell-seeded gel onto an osteogenic, photo-crosslinked, cell-seeded gel. Chondrogenic gels consisted of MSC-seeded methacrylated gelatin with hyaluronic acid and a photo-crosslinker (lithium phenyl-2,4,6-trimethylbenzoylphosphinate). Osteogenic gels had a similar composition, except with hydroxyapatite rather than hyaluronic acid. After 4 weeks of culture, the corresponding sections of the osteochondral construct showed heightened expression of chondrogenic and osteogenic markers. The construct also contained a junction between the sections of the scaffold, with visible GAG staining in the chondrogenic portion and calcium staining in the osteogenic portion [Fig. 7(b)].^[Reference Lin, Lozito, Alexander, Gottardi and Tuan^163]

Figure 7. Examples of bioreactor designs for maturation of tissue engineering interfacial tissues. (a) Osteochondral microfluidic bioreactor.^[Reference Goldman and Barabino^172] This bioreactor creates two microchannel arrays in a gel, separated by a non-channel gel slab in the center. Osteogenic and chondrogenic media are flowed through the microchannels allowing for diffusion into surrounding walls and through central slab, creating an interfacial construct. Reprinted with permission from Stephen M. Goldman, Gilda A. Barabino, Cultivation of agarose-based microfluidic hydrogel promotes the development of large, full-thickness, tissue-engineered articular cartilage constructs, John Wiley and Sons. Copyright © 2014 John Wiley & Sons Ltd.^[Reference Goldman and Barabino^172] (b) Osteochondral bioreactor.^[Reference Lin, Lozito, Alexander, Gottardi and Tuan^163] Chondrogenic and osteogenic media are continuously flowed through a scaffold, while maintaining separation of media baths through use of an O-ring. Histology shows interfacial region for construct: chondral component (CC) and osseous component (OC). Alizarin red stains for calcium, Safranin-O stains for negatively charged molecules (GAGs), and fast green stains for proteins. This schematic was reprinted from <http://pubs.acs.org/doi/abs/10.1021/mp500136b>. Further permissions related to the material excerpted should be directed to the ACS.^[Reference Lin, Lozito, Alexander, Gottardi and Tuan^163] (c) Combination mechanical, chemical, and co-culture bioreactor for culturing meniscal enthesis constructs. These constructs consist of two bone plugs seeded with mesenchymal stem cells (MSCs) injected and connected with a high density collagen gel embedded with meniscal fibrochondrocytes (FCCs). The bone plugs are anchored down in the bioreactor using the walls, and then osteogenic media and meniscal media can be applied to different portions of the scaffold. Image shows distribution of co-cultured cells on constructs, and histology shows the morphology of the interfacial region of the construct (images courtesy of Leanne Iannucci). Reprinted from Acta Biomaterialia, Volume 56, Mary Clare McCorry, Melissa M. Mansfield, Xiaozhou Sha, Daniel J. Coppola, Jonathan W. Lee, Lawrence J. Bonassar, A model system for developing a tissue engineered meniscal enthesis, 110–117, Copyright © 2016, with permission from Elsevier^[Reference McCorry, Mansfield, Sha, Coppola, Lee and Bonassar^60]

Mechanically driven maturation

The native enthesis sustains dynamic tensile, compressive, and shear loading that contributes to the development of the integrated regions. The structural transition from soft tissue-to-bone promotes gradual load transfer across the interface, indicating the importance of the structure–function relationship in the enthesis.^[Reference Abraham and Haut Donahue^1, Reference Moffat, Sun, Pena, Chahine, Doty, Ateshian, Hung and Lu^26, Reference Thomopoulos, Marquez, Weinberger, Birman and Genin^173] The native enthesis contains a dense network of collagen fibers interdigitated into bone that aid in tensile and compressive load transmission.^[Reference Villegas, Hansen, Liu and Haut Donahue^174–Reference Hu, Birman, Demyier-Black, Schwartz, Thomopoulos and Genin^177] The loading environment of the enthesis provides mechanical cues to cells that instruct matrix production and remodeling. Lack of loading during native development results in impaired mineral deposition and disorganized fiber distribution.^[Reference Kim, Galatz, Patel, Das and Thomopoulos^178] In the case of healing after tendon injury, immobilization actually increased structural, compositional, and viscoelastic properties compared with an exercised group.^[Reference Thomopoulos, Williams and Soslowsky^179] Considering the mechanical influences on tissue development is important when designing methods for tissue engineered enthesis maturation.

During development, anchoring of the meniscus to the tibia produces a static mechanical boundary condition that triggers longitudinal fiber organization in the soft tissue.^[Reference Gardner and O'Rahilly^90–Reference Mérida-Velasco, Sánchez-Montesinos, Espín-Ferra, Rodríguez-Vázquez, Mérida-Velasco and Jiménez-Collado^92, Reference Clark and Ogden^180] Mimicking this static mechanical boundary condition has shown organized fiber remodeling from several cell types including fibroblasts,^[Reference Huang, Chang, Aggarwal, Lee and Ehrlich^181–Reference Costa, Lee and Holmes^184] MSCs,^[Reference Nirmalanandhan, Levy, Huth and Butler^185–Reference Awad, Butler, Harris, Ibrahim, Wu, Young, Kadiyala and Boivin^187] cardiomyocytes,^[Reference Costa, Lee and Holmes^184] annulus fibrosis chondrocytes,^[Reference Bowles, Williams, Zipfel and Bonassar^188] and meniscal fibrochondrocytes.^[Reference McCorry, Mansfield, Sha, Coppola, Lee and Bonassar^60, Reference Puetzer, Koo and Bonassar^74, Reference McCorry and Bonassar^86] Mechanical anchoring at the bony ends of a soft tissue-to-bone model system directed longitudinal fiber formation as well as formed interdigitated fibers at the collagen–bone interface [Fig. 7(c)].^[Reference McCorry, Mansfield, Sha, Coppola, Lee and Bonassar^60] Cell monolayers mechanically fixed at the end points contracted the cell sheet into a 3D construct. Using this method, the cells organized a highly aligned and integrated enthesis for ACL repair [Fig. 6(b)].^[Reference Ma, Smietana, Kostrominova, Wojtys, Larkin and Arruda^68] While mechanically directed fiber remodeling has been shown in several systems, different cell types have also displayed varying levels of remodeling capability. When tendon and meniscal fibrochondrocytes were embedded in a collagen matrix and clamped, fibrochondrocytes formed significantly larger fibers than tendon cells.^[Reference Puetzer, Sallent, Gelmi and Stevens^189] MSCs in a tissue engineered meniscus also showed decreased fiber alignment and diameter compared with fibrochondrocytes.^[Reference McCorry and Bonassar^86] These studies indicate that the response to mechanical stimuli is highly dependent on cell type.

Active mechanical loading is applied to enthesis tissues in vivo, and these active mechanical signals guide differential tissue formation. While active mechanical loading has not been applied to in vitro enthesis tissue engineering yet, other systems have utilized active loading. Bioreactors have been designed to apply uniaxial tensile loads in bulk ligament and tendon tissue engineering (constructs excluding an enthesis).^[Reference Lee, Shin, Cho, Kang, Kim, Park and Shin^103, Reference Subramony, Dargis, Castillo, Azeloglu, Tracey, Su and Lu^104] After tensile loading of an aligned scaffold seeded with MSCs, type I and III collagen expression increased as did expression of fibroblastic markers, including tenascin-C, fibronectin, and integrins α ₂, α ₅, and β ₁.^[Reference Subramony, Dargis, Castillo, Azeloglu, Tracey, Su and Lu^104] Simultaneous tensile and compressive stimulation by compressively loading a self-assembled meniscus ring, resulted in significant increases in mechanical and biochemical properties.^[Reference Huey and Athanasiou^190] This effect was further demonstrated by loading mechanically anchored tissue engineered menisci. Dynamic compressive loading of the meniscus enhanced organized collagen fiber formation, mechanical properties, and GAG accumulation. Mimicking native mechanical loads guided heterogeneous tissue development, where tensile loads in the outer meniscus produced a fiber-containing, collagen-rich tissue, and compressive loads on the inner meniscus increased GAG development.^[Reference Puetzer and Bonassar^191] The native environment can also serve as a natural load inducer to guide tissue maturation, assuming the construct is robust enough for implantation. A cell self-assembled bone–ligament–bone construct underwent marked increases in collagen content and alignment as well as increases in stiffness after implantation, driving biochemical content and mechanical properties toward native ACL.^[Reference Ma, Smietana, Kostrominova, Wojtys, Larkin and Arruda^68] These studies collectively support active mechanical stimulation as a useful tool to the drive structural development of tissue engineered constructs.