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Journal of Biomechanics 40 (2007) 750765 www.elsevier.com/locate/jbiomech www.JBiomech.com

Review

Osteochondral tissue engineering

Ivan Martin, Sylvie Miot, Andrea Barbero, Marcel Jakob, David Wendt
Department of Research and Institute for Surgical Research and Hospital Management, University Hospital of Basel, Hebelstrasse 20, 4031 Basel, Switzerland Accepted 13 March 2006

Abstract Osteochondral defects (i.e., defects which affect both the articular cartilage and underlying subchondral bone) are often associated with mechanical instability of the joint, and therefore with the risk of inducing osteoarthritic degenerative changes. Current surgical limits in the treatment of complex joint lesions could be overcome by grafting osteochondral composite tissues, engineered by combining the patients own cells with three-dimensional (3D) porous biomaterials of pre-dened size and shape. Various strategies have been reported for the engineering of osteochondral composites, which result from the use of one or more cell types cultured into single-component or composite scaffolds in a broad spectrum of compositions and biomechanical properties. The variety of concepts and models proposed by different groups for the generation of osteochondral grafts reects that understanding of the requirements to restore a normal joint function is still poor. In order to introduce the use of engineered osteochondral composites in the routine clinical practice, it will be necessary to comprehensively address a number of critical issues, including those related to the size and shape of the graft to be generated, the cell type(s) and properties of the scaffold(s) to be used, the potential physical conditioning to be applied, the degree of functionality required, and the strategy for a cost-effective manufacturing. The progress made in material science, cell biology, mechanobiology and bioreactor technology will be key to support advances in this challenging eld. r 2006 Elsevier Ltd. All rights reserved.
Keywords: Cartilage repair; Chondrocyte; Scaffold; Bioreactor; Mechanobiology

Contents 1. 2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Described approaches for the engineering of osteochondral grafts . . . . . . . . . . . . . . 2.1. Scaffold for bone component, scaffold-free for cartilage (Scaffold strategy A) . 2.2. Different scaffolds for bone and cartilage components (Scaffold strategy B) . . 2.3. One heterogeneous/bilayered scaffold (Scaffold strategy C) . . . . . . . . . . . . . . 2.4. One homogenous/single-layer scaffold (Scaffold strategy D) . . . . . . . . . . . . . . Towards design principles for the engineering of osteochondral composites. . . . . . . . 3.1. Size, shape of the graft and surgical approach for implantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 751 751 751 754 755 755 756 756

3.

Abbreviations: FDM, fused deposition modeling; FGF-2, broblast growth factor-2; HA, hydroxyapatite; MPC, mesenchymal progenitor cells; PCL, polycaprolactone; PEG, poly-ethylene glycol; PLA, poly-lactic acid; PGA, poly-glycolic acid; PLGA, poly-lactic-coglycolic acid; TCP, tricalciumphosphate Corresponding author. Tel.: +41 61 265 2384; fax: +41 61 265 3990. E-mail address: imartin@uhbs.ch (I. Martin). 0021-9290/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jbiomech.2006.03.008

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3.2. Cell source. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Physical conditioning of engineered osteochondral tissues . 3.4. Required in vitro maturation stage of engineered cartilage . 3.5. Manufacture of engineered osteochondral composites . . . . 3.6. Osteochondral repair by cell-free scaffolds?. . . . . . . . . . . . 4. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Osteochondral defects, typically derived by traumatic injuries or osteochondritis dissecans, are often associated with mechanical instability of the joint, and therefore with the risk of inducing osteoarthritic degenerative changes. Grafting of osteochondral units consisting of a supercial cartilaginous layer (corresponding to articular cartilage) and an underlying calcied tissue (corresponding to subchondral bone) represents a promising approach to restore the biological and mechanical functionality of the joint. Despite the encouraging results reported, the clinical use of autologous osteochondral grafts (i.e., mosaicplasty technique) suffers from several limitations, namely: (i) the amount of material available, (ii) the donor site morbidity, and (iii) the difculty to match the topology of the grafts with the injured site. Tissue engineering of osteochondral composites has the potential to overcome these limits. Threedimensional (3D) tissue grafts of pre-dened size and shape can be engineered by combining the patients own cells with 3D porous biomaterials, which provide the template for tissue development and degrade at dened rates. Tissue engineering approaches would allow the properties of the graft to be specically tailored, in order to introduce in the affected joints the structural, biological and biomechanical cues which are necessary and sufcient for a reproducible and durable repair. The progress made in material science, cell biology and bioreactor technology has been instrumental for the ourishing of a broad spectrum of approaches to the engineering of osteochondral grafts. The variety of concepts and models so far proposed and investigated by different groups for the generation of osteochondral grafts reects that understanding of the requirements to restore a normal joint function is still poor. While in principle it would be feasible to develop technical solutions to a well-dened design, the principles of the design itself still have to be dened. Based on these considerations, here we will rst review the approaches so far proposed for the fabrication of osteochondral grafts. We will then discuss some open questions that we feel should be addressed in order to establish the design criteria for engineered osteochondral composites.

2. Described approaches for the engineering of osteochondral grafts The different approaches so far proposed for the fabrication of osteochondral composite constructs can be classied based on the combination of specic strategies for the selection of the scaffold and the cell source, as schematically outlined in Fig. 1. According to this classication, osteochondral constructs have been generated using: (A) a scaffold for the bone component but a scaffold-free approach for the cartilage component; (B) different scaffolds for the bone and the cartilage components combined at the time of implantation; (C) a single but heterogeneous composite scaffold; or (D) a single homogenous scaffold for both components. These scaffolds have been (I) loaded with a single cell source having chondrogenic capacity, (II) loaded with two cell sources having either chondrogenic or osteogenic capacities, (III) loaded with a single cell source having both chondrogenic and osteogenic differentiation capacity, or (IV) used in a cell-free approach. In this section, some of the most relevant studies in osteochondral tissue engineering will be presented, following the outlined classication (see Table 1).

2.1. Scaffold for bone component, scaffold-free for cartilage (Scaffold strategy A) Composite implants can be produced by seeding and culturing a high density of chondrogenic cells directly on top of an osteoconductive biomaterial. Following this

Fig. 1. Schematic diagram of strategies used for the fabrication of osteochondral grafts. Specic approaches proposed by different research groups can be classied based on a combination of a scaffold strategy (AD) and a cell strategy (IIV). Under this classication system, for example, the approach of scaffold-free culture of chondrocytes on a cell-free osteoconductive scaffold would be designated as A/I.

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Table 1 Summary of osteochondral studies grouped according to the repair strategy described in Fig. 1 Strategy Chondral component Subchondral component Assembly of chondral and subchondral components Chondrocytes seeded & cultured on the surface of bone substitutes Chondrocytes seeded & cultured on the surface of osteoconductive scaffold Chondrocytes seeded & cultured on the surface of osteoconductive scaffold Model system for osteochondral development In vitro culture of cylindrical composite constructs In vitro pre-culture of cylindrical composites, with subsequent mechanical conditioning In vitro culture of cylindrical composite constructs & further implantation in osteochondral defects in sheeps In vitro culture of cubical composite constructs in medium supporting both chondrogenesis and osteogenesis In vitro culture of cylindrical & patella-shaped osteochondral constructs In vitro culture of cylindrical composites in a perfused bioreactor system In vitro culture of cylindrical composites in a doublechamber bioreactor Reference

A/I

Porcine articular chondrocytes (scaffold-free) Bovine articular chondrocytes (scaffold-free)

Cell-free PLA or collagen/HA

Wang et al. (2004)

A/I

Cell-free porous calcium polyphosphate

Waldman et al. (2003)

A/I

Sheep articular chondrocytes (scaffold-free)

Cell-free porous calcium polyphosphate

Kandel et al. (2006)

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A/III

Cell pellet of human MPC press-coated onto PLA scaffold and pre-cultured in chondrogenic medium Bovine articular chondrocytes in agarose gel Bovine articular chondrocytes cultured in PGA/PLA eeces Porcine chondrocytes cultured in gelatin scaffold

Osteogenic induced human MPC seeded onto opposite face of pre-cultured cartilage component Devitalized bovine trabecular bone Natural or synthetic calcium carbonate Calcium phosphate derived from bovine cancellous bone (cell-free)

Osteogenic MPC seeded onto opposite face of pre-cultured PLA construct

Tuli et al. (2004)

B/I

Cell-agarose suspension added to, & partially penetrating into, the surface of the bone Chondrocyte seeded eeces attached to ceramic with brin-cell solution Gelatin region of composite partially inltrated into pores of bone component; composite assembled prior to cell seeding. Engineered cartilage sutured to bone substitute

Hung et al. (2003)

B/I

Kreklau et al. (1999)

B/I

Chang et al. (2004)

B/I & B/II

Rabbit articular chondrocytes cultured in PGA meshes

Collagen-hydroxyapatite sponge: cell-free or with absorbed bone marrow Bovine periosteal cells cultured in PLGA/PEG sponges Osteogenic rat MPC seeded in porous calcium phosphate ceramics

Composite blocks implanted into critical-sized osteochondral defects in adult rabbits In vitro culture of cylindrical composites in osteogenic medium Composite construct blocks implanted subcutaneously in mice

Schaefer et al. (2002)

B/II

Bovine articular chondrocytes cultured in PGA meshes Chondrogenic rat MPC seeded in hyaluronic acid sponges

Engineered components sutured together following different pre-culture times Freshly seeded components sealed together with brin

Schaefer et al. (2000)

B/III

Gao et al. (2001)

C/I

Ovine articular chondrocytes cultured in PLGA/PLA region of composite scaffold Primary porcine articular chondrocytes seeded into PLA sponge PLGA based scaffold (w/ or w/o a PGA ber reinforcement), cell-free or seeded with goat rib chondrocytes Human rib chondrocytes cultured in one half of PCL scaffold (manufactured by FDM technique) Chondrogenic rat MPC embedded in photopolymerizable PEG hydrogel Polyethylene ber scaffold coated with HA, impregnated with type I collagen gel & FGF-2 (cell-free)

PLGA/TCP region of composite scaffold

A single but heterogenous scaffold with gradient of materials and porosity between compartments Components of composite scaffold combined with PLA prior to cell seeding A single homogenous or heterogenous scaffold; heterogeneous composites glued with solvent prior to cell seeding Homogeneous PCL scaffold with gap between the two compartments. Chondrocytes seeded into empty half of precultured MPC-PCL construct Sequential loading & photopolymerization of chondrogenic & osteogenic hydrogel suspensions within condyle-shaped mold A single & homogenous cellfree scaffold

In vitro culture of cylindrical composites

Sherwood et al. (2002)

C/II

Human broblasts transduced with adenovirus expressing BMP-7, suspended in brin & seeded into HA PLGA based scaffold (w/ different additives)

Freshly seeded cylindrical composites implanted subcutaneously in mice Cylindrical composites implanted into both high & low weight bearing sites in goats In vitro culture of composite construct blocks

Schek et al. (2004)

C and D I and IV

Niederauer et al. (2000)

I. Martin et al. / Journal of Biomechanics 40 (2007) 750765

D/II

Human MPC pre-cultured in one half of PCL scaffold (manufactured by FDM technique) Ostoegenic rat MPC embedded in photopolymerizable PEG hydrogel Polyethylene ber scaffold coated with HA, impregnated with type I collagen gel & FGF-2 (cell-free)

Cao et al. (2003)

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D/III

Human articular condyleshaped osteochondral composites implanted ectopically in mice Scaffold blocks implanted into patellar groove of rabbits

Alhadlaq et al. (2004)

D/IV

Fukuda et al. (2005)

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Fig. 2. In vitro engineered osteochondral composites. Histological appearance of osteochondral composites generated by seeding and culturing a high density of porcine articular chondrocytes directly on top of a porous osteoconductive scaffold made of either poly-L-lactid (A) or collagen-HA [Col-HA] (B) for 11 weeks (strategy A/I according to Fig. 1) (Wang et al., 2004). A cartilaginous matrix positively stained for Alcian blue was obtained on top of both scaffolds, but neo-cartilage integrated more extensively into the Col-HA scaffold. Images have been kindly provided by Dr. P. Mainil-Varlet, University of Bern, Switzerland (scale bar 500 mm).

approach, Wang et al. seeded primary porcine articular chondrocytes directly onto the top surface of three commonly used biomaterials: poly-L-lactide, poly-D,Llactide or collagen-hydroxyapatite (HA) (Fig. 1: A/I) (Wang et al., 2004). After 7 weeks of in vitro culture, chondrocytes cultured on each of the biomaterials produced extracellular matrix (ECM) containing a large proportion of collagen type II and glycosaminoglycans (GAG) that was partially integrated with the subchondral base (Fig. 2). Constructs produced using collagen-HA were superior in terms of cell viability, construct shape and cellular integration, although the authors pointed out that the outcome on the stability of the osteochondral composites could be different under physiological loading in an orthotopic model. In this regard, promising results were recently reported in a sheep model using cartilage tissues grown on top of porous ceramic scaffolds (Kandel et al., 2006). Using human trabecular bone-derived mesenchymal progenitor cells (MPC) in conjunction with poly-D,Llactic acid (PLA) scaffolds, Tuli et al. followed a similar approach to generate osteochondral grafts (Fig. 1: A/ III) (Tuli et al., 2004). A pellet of MPC was press-coated on a PLA scaffold and cultured in a dened medium supplemented with transforming growth factor beta-1 (TGF-b1) to support chondrogenesis. In parallel, MPC from the same patient were expanded and cultured in monolayer with factors inducing osteogenesis (b-glycerophosphate and dexamethasone) and then seeded on the opposite face of the cartilage-PLA construct. The bilayered constructs were then cultured in a cocktail medium containing insulin and osteogenic factors to support/promote chondrogenesis and osteogenesis simultaneously. The resulting composites consisted of both a hyaline cartilage-like layer and a dense bone-like component, with a well developed transition zone between the two compartments.

2.2. Different scaffolds for bone and cartilage components (Scaffold strategy B) In an alternative approach, different scaffolds can be used for the cartilage and bone components. Cartilaginous and/or bone-like tissues can then be engineered in vitro within the respective scaffold layer and combined into a single composite graft by suturing or adhering together the two layers. Schaefer et al. seeded and cultured differentiated bovine articular chondrocytes into poly-glycolic acid (PGA) meshes and periosteal-derived cells into polylactic-coglycolic acid/poly-ethylene glycol foams (PLGA/ PEG) to independently generate the cartilage and bone layers (Fig. 1: B/II) (Schaefer et al., 2000). Following 14 weeks of independent culture, the generated cartilaginous and bone-like tissues were then sutured together and cultured for an additional period in osteogenic medium. The authors showed that chondrocytes within the PGA meshes produced extracellular matrix containing high amounts of GAG and collagen type II during isolated culture and had maintained their specic phenotype during the subsequent composite culture. Furthermore, periosteal cells within the PLGA/PEG foams deposited mineralized matrix and bone specic proteins (osteocalcin and osteopontin). This study demonstrated the possibility of generating composites of cartilaginous and bone-like tissues in vitro, and pointed out that the maturation and integration of the two components can be modulated by the cultivation time. In addition, this was the rst evidence that articular chondrocytes can remain phenotypically stable when cultured with factors typically used to promote osteogenesis and adjacent to osteogenic cells producing mineralized matrix. Using a single cell source, Gao et al. generated composite osteochondral grafts from hyaluronic acidbased sponges and porous ceramics (Fig. 1: B/III) (Gao et al., 2001). In this study, rat bone marrow-derived

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MPC were rst committed in monolayers with chondrogenic (e.g., TGF-b1) or osteogenic (i.e., bglycerophosphate and dexamethasone) culture supplements and then seeded, respectively, on the sponges or porous ceramic. The two parts were sealed together using brin glue and implanted subcutaneously into nude mice. While lamellar bone was abundant in the ceramic, brocartilage, and not hyaline cartilage, lled the pores of the hyaluronic acid sponge. Osteochondral composites based on different scaffolds were rst used to repair a critical-sized osteochondral defects (7 5 5 mm) in a rabbit model (Schaefer et al., 2002). In this study, engineered cartilage, generated in vitro from autologous articular chondrocytes cultured in PGA meshes, was sutured to a collagen-HA sponge, loaded or not with autologous MPC at the time of implantation (Fig. 1: B/II and B/I, respectively). The resulting composite was then press-t into a surgically created femoropatellar groove defect. Engineered cartilage withstood physiologic loading and remodeled over 6 months into osteochondral tissue with characteristic architectural features and Youngs moduli close to native tissue (engineered cartilage: 0.680.80 MPa; explant tissue: 0.84 MPa). Constructs integrated well with host bone but not with adjacent host cartilage. The initial loading of MPC in the osteoconductive material had no advantage over the cell-free bone layer, likely due to the presence of osteoprogenitor cells in the bleeding subchondral bone during implantation. This study demonstrated for the rst time that functional engineered cartilage grafts combined with an osteoconductive support, even if the latter is not loaded with osteogenic cells, provide a template permitting the orderly repair of critical-sized osteochondral defects in adult rabbits. 2.3. One heterogeneous/bilayered scaffold (Scaffold strategy C) As opposed to generating composite osteochondral grafts by combining independent cartilaginous and bone-like components, heterogeneous scaffolds have been proposed, composed of two distinct but integrated layers for the cartilage and bone regions (Fig. 3). Following this approach, Schek et al. seeded porcine articular chondrocytes and human gingival broblasts transduced with adenovirus expressing BMP-7 into the two regions of a poly-L-lactic acidHA (PLAHA) composite scaffold (Fig. 1: C/II) (Schek et al., 2004). A thin lm of PGA was deposited at the interface between the two biomaterials to prevent cell migration between the components. The scaffold shape and pore architecture were dened using an image-based design method (which could also be used to design patient-specic implant geometries) and was manufactured using a solid-free-form fabrication technique. Following ectopic implantation in mice, the cartilage layer contained ECM

Fig. 3. Heterogeneous composite scaffold for osteochondral repair. Example of a composite scaffold, consisting of two distinct but integrated layers corresponding to the cartilage and bone components (Scaffold strategy C according to Fig. 1). The implant is comprised of: (i) a top layer of the elastomeric copolymer poly(ethylene glycol)terephthalate/poly(butylene)-terephthalate (PolyActive), having mechanical properties comparable to native cartilage (Miot et al., 2005), and (ii) a bottom layer composed of HA and tricalcium phosphate, displaying both osteoconductive and osteoinductive properties (Yuan et al., 2002). The image has been kindly provided by Dr. J. Pieper, IsoTis B.V., Bilthoven, The Netherlands.

rich in GAG, the bone layer contained regions with blood vessels, marrow stroma and adipose tissue, and a mineralized interface was often present at the junction. Using the TheriFormTM 3D printing process, Sherwood et al. developed an innovative osteochondral composite scaffold, promoting preferential cell attachment and matrix deposition within the cartilage portion (Fig. 1: C/I) (Sherwood et al., 2002). The composite consisted of a cartilage region of 90% porous D,L-PLGA/ L-PLA with macroscopic staggered channels, a bone region of 55% porous L-PLGA/tricalciumphosphate (TCP), and a transition zone between the two regions containing a gradient of materials and porosity to prevent delamination at the interface. The bone region had tensile and compressive strengths (1.6 and 2.5 MPa, respectively) similar in magnitude to fresh cancellous human bone ($8 and 1020 MPa, respectively) and was fabricated in a cloverleaf shape, with channels to increase the surface contact with bone marrow and to facilitate cell migration. 2.4. One homogenous/single-layer scaffold (Scaffold strategy D) The use of a single homogeneous scaffold and two cell types having chondrogenic and osteogenic capacity (Fig. 1: D/II) was adopted by Cao et al. to engineer

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osteochondral composites (Cao et al., 2003). Using a fused deposition modeling (FDM) technique, polycaprolactone (PCL) scaffolds were fabricated with a honeycomb-like geometry (in a 01/601/1201 lay-down pattern), with a well dened pore volume, structure and porosity, partitioned vertically into two halves. Human bone marrow derived MPC were rst seeded in one-half of the partitioned scaffold and pre-cultured. Human rib chondrocytes were subsequently seeded into the other half, and the composite constructs were co-cultured in vitro in medium containing osteogenic supplements. Using scanning electron microscopy, the authors observed different extracellular matrices in each compartment, however, no further characterization of matrix composition or cell phenotype was performed. The authors concluded that the use of slow-degrading PCL in conjunction with the FDM technique allowed for the fabrication of a scaffold with sufcient mechanical strength to endure initial in vivo loading. Alhadlaq et al. also reported the use of a single scaffold, a PEG hydrogel, but seeded with a single cell source (Fig. 1: D/III) in order to engineer a human shaped articular condyle (Alhadlaq et al., 2004). Rat bone marrow-derived MPC were expanded, induced separately to chondrogenic or osteogenic differentiation using specic culture supplements and then loaded in two stratied and integrated hydrogel layers that were photopolymerized in a human condylar mold. After 4 weeks implantation in immunodecient mice, most of the differentiated chondrogenic and osteogenic cells had synthesized corresponding cartilaginous and bone-like matrices. A cell-free approach was undertaken by Fukuda et al. who used a single 3D fabric (3DF) for the repair of a large full-thickness osteochondral defect (6 6 3 mm) in the patellar groove of rabbits (Fig. 1: D/IV) (Fukuda et al., 2005). The scaffold, consisting of an ultra-high molecular weight polyethylene ber and an unsintered HA coating, was impregnated with a type I collagen gel with or without broblast growth factor-2 (FGF-2). After 48 weeks in vivo, FGF-2 promoted biological resurfacing with hyaline-like cartilage and induced subchondral bone formation within and around the implanted graft. The approach of using a cell-free scaffold impregnated with growth factors is a promising strategy for the treatment of large osteochondral defects, even if a future clinical application will necessitate to dene optimal concentrations of released growth factor and to use more controllable delivery systems.

3. Towards design principles for the engineering of osteochondral composites 3.1. Size, shape of the graft and surgical approach for implantation Resurfacing of an irregular defect with multiple cylindrical plugs, as in the mosaicplasty technique, is

associated with the generation of cartilage-to-cartilage interfaces, known to have a limited capacity to integrate, and of incongruities in the articular surface, known to lead to signicantly increased contact pressures (Koh et al., 2004). As an alternative to cylindrical plugs, the generation of anatomically shaped constructs that reproduce the contour of the articular surfaces has the potential to reduce the overall interfacial area between opposing cartilage surfaces and to restore normal load distributions across the joint when implanted. The need for a custom-shaped graft is even more relevant when the defect is unconned, as described in a recent case report where the lesion was located on the edge of the lateral femoral condyle (Adachi et al., 2004). In this study, the authors rst restored the normal contour of the joint by implanting a cortical bone block, trimmed to adjust to the shape of the condyle bone, and then grafted a layer of engineered cartilage. The feasibility to culture anatomically shaped grafts aimed at replacing the entire articular surface of a diarthroidal joint was demonstrated by the generation of bilayered constructs consisting of chondrocyte-seeded agarose on natural trabecular bone (Hung et al., 2003). In that study, the geometry of the articular cartilage layer, previously acquired from human cadaver joints, was used in conjunction with computer-aided design and manufacturing technology to create anatomically accurate molds. As previously described in Section 2.4, a human-shaped condyle has also been generated through the photopolymerization of two stratied and integrated hydogel layers within a negative mold of a condyle (Alhadlaq et al., 2004). Based on the development of rapid prototyping techniques to fabricate not only plastic molds, but also porous scaffolds directly from computer-aided design (Woodeld et al., 2004; Yeong et al., 2004), it was proposed that 3D computed tomography, coupled with 3D computer aided design and rapid prototyping, could be used to design and engineer customized femoral and tibial cartilaginous implants made of synthetic polymers in dened pore architectures (Fig. 4). The concept of customized osteochondral composites is in principle extremely interesting, but likely difcult to be implemented in practice. Challenges to be overcome include the need of a precise t into a defect whose shape may change over time and the denition of a surgical method for graft xation. Also to be considered are the estimated high costs of the procedure and the fact that it would not allow to be performed under minimally invasive conditions. A promising approach which might allow arthroscopic implantation of anatomically shaped grafts is based on the use of shape memory materials, which would be fabricated in a condensed state, delivered via minimally invasive surgery and would then acquire the programmed shape in situ (Lendlein and Langer,

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Fig. 4. Rapid prototyping for anatomically shaped scaffolds. Using a 3D model generated by computed tomography scans of a rabbit knee joint (A), porous scaffolds made of poly(ethylene glycol)-terephthalate/poly(butylene)-terephthalate (PolyActive) were produced by 3D ber deposition (B) (Woodeld et al., 2004). The images have been kindly provided by Dr. T. Woodeld, University of Twente, The Netherlands (scale bar 5 mm).

2002). Shape memory alginate hydrogels were recently used to demonstrate the possibility of cartilage tissue formation in predened geometries following ectopic delivery through a small catheter (Thornton et al., 2004). The scaffolds, initially reduced to less than 5% of the predened volume, returned to their programmed size upon rehydration with a solution containing chondrocytes, which resulted in efcient cell seeding and in cartilage structures maintaining the predened geometry up to 6 months. Although a number of different classes of shape memory materials has already been described, the approach has not yet been tailored for the generation of osteochondral composites or the repair of joint defects. From a surgical standpoint, the ideal consistency of a graft would be that of a paste, allowing the embedding of chondrocytes and/or MPC, which might be delivered arthroscopically and shaped in situ. Several promising materials have been proposed to ll gaps in cartilage tissue (Sittinger et al., 2004), including chitosan-based gels (Chenite et al., 2000) and chondroitin sulfate photopolymerizing hydrogels (Li et al., 2004). However, none of these has yet been demonstrated to provide a substrate with the appropriate biomechanical properties and the capacity to resorb/remodel, which could promote immediate joint loading and durable regeneration of osteochondral defects. 3.2. Cell source The variety of concepts on the cell types proposed for the repair of osteochondral defects has already been

introduced earlier in this review. Considering the presence of osteoprogenitor cells in bleeding subchondral bone, paralleled by a generally high efciency of bone regeneration in the subchondral regions, it is likely that fabrication of osteochondral composites will require only one cell type, to generate their cartilaginous part. Articular chondrocytes have been the most popular source of cells to engineer cartilage grafts, but in the majority of the studies cells were used immediately after isolation from the native cartilage of different animal species. For the clinical implementation of the approaches proposed, instead, human articular chondrocytes would have to be rst expanded in culture, which is typically associated with cell de-differentiation, leading to the downregulation of cartilage-specic genes (Benya and Shaffer, 1982; Binette et al., 1998). The reduced capacity of expanded chondrocytes, particularly if of human origin, to re-differentiate and produce cartilage-specic extracellular matrix, undermines the success to engineer functional cartilage tissues. One possible strategy that could be undertaken to overcome this limitation consists in expanding chondrocytes in the presence of specic growth factors. The presence of FGF-2, TGF-b1 and platelet-derived growth factor-bb during the expansion of human articular chondrocytes was shown not only to reproducibly increase cell proliferation rate, but also to enhance the cell capacity to re-differentiate following expansion (Barbero et al., 2003; Jakob et al., 2001), particularly if the cells were from young adults below 40 years of age (Barbero et al., 2004), and to respond to differentiating factors present in the synovial uid (Jakob et al., 2004). A critical issue associated with the use of autologous articular chondrocytes is the procurement of the biopsy from the patient. A cartilage biopsy in the joint, even if harvested from a non-load bearing site, represents an additional injury to the cartilage surface, and has been reported to be detrimental to the surrounding healthy articular cartilage (Lee et al., 2000). To overcome this problem, one alternative approach would be based on the use of chondrocytes obtained from non-articular cartilage tissues. For example, biopsies of nasal or rib cartilage can be harvested under local anesthetic and by a less invasive procedure than removing tissue from specic areas of the joint. Morbidity is also reduced by the fact that the donor site is not subjected to high levels of physical forces, as in the joint. Several studies have shown that chondrocytes derived from human nasal septum or ear cartilage proliferate and generate cartilaginous tissue after monolayer expansion with similar or superior capacity to those derived from articular cartilage (Kaenah et al., 2002; Tay et al., 2004; Van Osch et al., 2004). However, to demonstrate whether the tissue generated by non-articular chondrocytes is adequate for cartilage repair at articular sites, extensive

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data from in vivo orthotopic experimental studies and/ or from in vitro loaded models will be needed. To avoid the limitations of de-differentiated chondrocytes, several groups have proposed the use of MPC to generate cartilaginous tissues. MPC have been found in a variety of human adult tissues, including bone marrow, periosteum, trabecular bone, synovial membrane, skeletal muscle, dermis, blood, and adipose tissue (Tuan et al., 2003). The most extensively studied MPC are those isolated from bone marrow, also called bone marrow stromal cells (BMSC). However, BMSC differentiated towards the chondrogenic lineage were shown to express markers specic of hypertrophic chondrocytes (e.g.: type X collagen and MMP-13) (Mackay et al., 1998; Winter et al., 2003) and to further mineralize the deposited matrix when exposed to osteogenic stimuli (Mackay et al., 1998; Muraglia et al., 1998), thus indicating a potential instability of the acquired chondrocytic phenotype. Despite a series of recent studies reporting the use of BMSC for osteochondral defect repair in different animal models (Gao et al., 2001; Oshima et al., 2004; Uematsu et al., 2005) and in a few clinical cases (Wakitani et al., 2004), the long-term efcacy of BMSC and their contribution to the regeneration of hyaline cartilage which does not remodel into bone in the long term, still has to be demonstrated. Yet another approach to bypass the difculties associated with chondrocyte expansion would be the use of a limited number of freshly isolated articular chondrocytes. Recent studies indicate that non-expanded chondrocytes can induce chondrogenic differentiation of other cell types (Hendriks et al., 2005) and that even undigested cartilage tissue, minced into small particles, can be used to repair experimental cartilage defects in goats and horses (Frisbie et al., 2005; Lu et al., 2005). These preliminary results are particularly interesting, since they would open the possibility of an intraoperative fabrication of osteochondral grafts, with obvious logistic and economic advantages. 3.3. Physical conditioning of engineered osteochondral tissues It is well known that physical stimuli can modulate the metabolism of chondrocytes and osteoblasts, and when applied with specic magnitudes and frequencies, may upregulate the production of extracellular matrix components. Consequently, to simulate specic physiological forces during joint loading, numerous tissue engineering groups have developed bioreactors to apply mechanical stimuli to cell-seeded scaffolds/hydrogels in an effort to enhance cell differentiation and/or tissue development. A number of studies have indeed reported stimulated chondrocyte metabolism and/or enhanced cartilage ECM production in response to dynamic compression, although these responses were greatly

dependent upon the specic magnitude and/or frequency applied (Buschmann et al., 1995; Davisson et al., 2002a; Lee et al., 2003; Lee and Bader, 1997; Kisiday et al., 2004). Likewise, enhanced osteogenic differentiation and mineralized matrix deposition was reported when human BMSC, pre-cultured within partially demineralized bone matrix, were subjected to physiological strain magnitudes in a four-point bending bioreactor (Mauney et al., 2004). In addition to these studies, which focused on engineering either chondral or bone tissues, mechanical conditioning has also been applied to composite osteochondral constructs. Implementing intermittent regimes of either dynamic compression or dynamic shear to composite constructs (i.e., chondrocytes pre-cultured on top of calcium polyphosphate cylinders), Waldman et al. found that with as little as 6 min of mechanical stimulation every other day during 4 weeks of culture, the accumulation of ECM and the equilibrium moduli were increased as compared to unstimulated constructs (compression stimulated 80 kPa, shear stimulated 112 kPa, unstimulated 20 kPa) (Waldman et al., 2003). Applying a different regime of intermittent dynamic compression to composite constructs, Hung et al. also reported an increase in the GAG content and Youngs modulus throughout 4 weeks of culture, although to a lower extent than in chondral constructs stimulated in the absence of the bone substrate (Hung et al., 2004). This could be explained by the fact that the underlying bone substrate signicantly altered the strains, hydrostatic pressure, and ow elds within the chondral region, as assessed by computational modeling (Lima et al., 2004). While mechanical conditioning appears to have the potential to improve the structural and functional properties of engineered tissues, little is currently known about which specic mechanical forces, or regimes of application (i.e. magnitude, frequency, continuous or intermittent, duty cycle), are most stimulatory. Indeed, since strain transfer to the cells is initially dominated by the scaffold system and is progressively modulated at increasingly important degrees by the ECM being deposited and organized (Hung et al., 2004), different regimes of conditioning might be required by tissues at different stages of development (see Section 3.4; a more in-depth discussion of mechanobiology can be found in the review of Van der Meulen and Huiskes, 2002). Mass transfer limitations of nutrients and metabolic waste products are often considered one of the greatest challenges in the engineering of cartilage and bone tissues of clinically relevant sizes. Although which particular species is/are limiting is not decisively known, insufcient oxygen transport has been associated, both experimentally and computationally, with inhomogeneous development of cartilaginous tissues (Malda et al., 2004; Lewis et al., 2005). External mass transfer limitations of oxygen and other nutrients can be reduced

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by culturing constructs in a stirred-ask, where turbulent eddies are associated with high shear stresses, or within rotating wall vessels, reported to enhance the mass transfer of nutrients and wastes while generating low levels of turbulence and shear (Martin et al., 1999; Vunjak-Novakovic et al., 1999). Recently, Chang et al. designed an innovative double-chamber stirred bioreactor to engineer osteochondral constructs (Chang et al., 2004). The ultimate goal of the bioreactor was to facilitate the co-culturing of chondrocytes and osteoblasts simultaneously within respective sections of a single-unit composite scaffold. The bioreactor was separated by a silicone membrane into two compartments, connected only through the pore structure of the composite scaffold, which traversed the membrane. Since each chamber contained independent media recirculation systems and media stirring mechanisms, different cocktails of factors could be used for the culture of each specic cell type within their respective compartment of the composite scaffold. While widely used convective systems such as stirred asks and rotating vessels may improve mass transfer to/from the surface of the construct during prolonged tissue culture, direct perfusion of culture medium through the scaffold pores can mitigate mass transfer limitations within the construct as well. Perfusion of chondrocyte-seeded scaffolds was shown to support elevated GAG synthesis and deposition (Davisson et al., 2002b; Pazzano et al., 2000), as well as a uniform distribution of viable human chondrocytes and extracellular matrix (Wendt et al., 2006). Perfusion ow has also been associated with enhanced growth, differentiation and mineralized matrix deposition by rat marrow stromal cells (Bancroft et al., 2002; Goldstein et al., 2001; Sikavitsas et al., 2005) and with the generation of highly osteoinductive grafts starting from human bone marrow nucleated cells, without prior monolayer isolation and expansion (Braccini et al., 2005). A perfusion system has also been used for the culture of a chondrocyte seeded composite scaffold (PGA/PLLA eece adhered to calcium carbonate), resulting in the formation of new ECM which adhered to the underlying ceramic biomaterial (Kreklau et al., 1999). Optimizing a convective system for the engineering of cartilaginous and osteoinductive grafts will have to address the balance between the mass transfer of nutrients and waste products to and from cells, the retention of newly synthesized extracellular matrix components within the construct, and the uid-induced shear stresses within the scaffold pores. The optimal ow conditions of a bioreactor should not be determined through a trial-and-error approach, but rather should be supported by computational uid dynamics (CFD) models to simulate the velocity and shear proles within bioreactors and at the surface of engineered constructs (Sucosky et al., 2004; Williams et al., 2002),

as well as within the scaffold pores to better characterize the local environment seen by the cells (Ciof et al., 2006; Porter et al., 2005; Raimondi et al., 2002, 2004). 3.4. Required in vitro maturation stage of engineered cartilage A key question to be addressed in tissue engineering is how developed an engineered graft should be prior to implantation to support an optimal repair. While particular biomechanical parameters have been proposed as design criteria for functional cartilage grafts (Hung et al., 2004), including local mechanical properties reecting cartilage heterogeneity (Kelly et al., in press), these have yet to be validated, and existing data do not allow to reach a consensus. In the most basic cell/scaffold-based approach to cartilage repair, cells are seeded onto the scaffold and immediately, or soon thereafter, implanted. When using this method, however, the number of cells actually retained within the scaffold can be signicantly lower than when pre-culturing constructs in vitro prior to implantation, possibly due to enhanced cell-scaffold adhesion or to the ECM stabilizing and protecting the cells (Ball et al., 2004). Yet, only minor differences in the ectopic in vivo development of engineered septal cartilage were reported when scaffolds were either immediately implanted after seeding or pre-cultured for 3 weeks (Rotter et al., 2002). On the contrary, human articular chondrocyte-based constructs precultured in a medium containing factors supporting chondrogenic differentiation were reported to contain more cartilaginous ECM and have higher equilibrium moduli following ectopic implantation than those implanted directly after seeding (Moretti et al., 2005a). Given the physiological load bearing requirements that an osteochondral graft must support once implanted, the role of biomechanical signals will likely determine the key design criteria of a cartilage graft. A well-developed cartilaginous ECM could allow for early post-operative rehabilitation and enhance the success rate of the graft survival when subjected to physiologic mechanical loads. In addition, considering the role of ECM in modulating the chondrocyte response to mechanical forces (Buschmann et al., 1995; Demarteau et al., 2003), a more developed ECM could provide a more physiological environment that supports further in vivo development of the graft. Using an in vitro model system, Demarteau et al. showed a correlation between the effect of dynamic compression and the amount of GAG contained within the engineered construct at the onset of compression (Fig. 5) (Demarteau et al., 2003). With the immature constructs, dynamic compression induced a down regulation in the GAG metabolism (i.e., GAG synthesis and accumulation), while only in the most mature constructs was the loading advantageous

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2.5 Fold difference from unstimulated 2.0 1.5 1.0 0.5 0.0 0.0 (A) 1.4 Fold difference from unstimulated 1.2 1.0 0.8 0.6 0.4 0.0 (B)

GAG Synthesized p=0.002 r=0.912

5.0

10.0

15.0

GAG content before compression

more mature constructs is consistent with the limited integrative capacity of native cartilage and could be the result of low cell proliferation, cells being entrapped within their dense ECM, or the anti-adhesive properties of GAG (Obradovic et al., 2001). Nevertheless, given the potential adverse consequences of physiological mechanical loading on an immature cartilaginous graft, methods to improve the integrative capacity of mature cartilaginous grafts should be investigated. The use of reproducible and controlled model systems to investigate specic mechanisms of integrative cartilage repair will be essential in this endeavor (Moretti et al., 2005b). 3.5. Manufacture of engineered osteochondral composites One of the major challenges to bring an autologous cell-based osteochondral product into routine clinical practice would be to translate research-scale production models into clinically applicable manufacturing designs that are reproducible, clinically effective, and economically acceptable while complying with good manufacturing practice (GMP) requirements (Ratcliffe and Niklason, 2002). Production techniques for tissue engineering products currently rely on manual cell culture procedures, which suffer from high costs, complicated logistics and little standardization: thus, innovative and low-cost bioreactor systems might play a key role in the successful exploitation of an engineered osteochondral product for wide-spread clinical use (Martin et al., 2004; Kino-Oka et al., 2005). A promising manufacturing concept is based on a de-centralized and closed bioreactor system, such as the on-site hospital based ACTESTM (Autologous Clinical Tissue Engineering System), under development by Millenium Biologix (www.millenium-biologix.com). As a fully automated bioreactor system, ACTESTM will digest a patients cartilage biopsy, expand the chondrocytes, seed and culture the cells onto the surface of an osteoconductive porous scaffold, thereby generating the CartiGraftTM osteochondral graft within a single closed bioreactor system. Bioreactor systems such as ACTESTM would eliminate logistical issues of transferring specimens between locations, eliminate the need for large and expensive GMP tissue engineering facilities, and minimize operator handling, with the likely nal result of reducing the cost of engineered osteochondral grafts. An interesting option to bypass the cultivation of cells in the manufacture of osteochondral grafts is based on the concept of an in vivo bioreactor. The approach lies in the deliberate creation and manipulation of a space between the tibia and the periosteum, a mesenchymal layer rich in MPC, in such a way that the bodys healing mechanism is leveraged in the engineering of neo-tissue (Emans et al., 2005). In a recent study, Stevens et al. demonstrated that by controlling angiogenesis it is possible to determine the formation of

GAG Accumulated

p=0.035 r=0.783

5.0

10.0

15.0

GAG content before compression

Fig. 5. Correlation plots of the effect of dynamic compression and maturation stage of engineered cartilage. Dynamic compression was applied to pre-cultivated engineered cartilage constructs containing different amounts of GAG at the onset of stimulation. In relation to unstimulated constructs under freeswelling conditions, the effect of dynamic compression on GAG synthesis (A) and GAG accumulation (B) correlated with the initial amount of GAG within the constructs. Only in the most mature constructs did compression result in an upregulation in the GAG synthesized and accumulated as compared to unstimulated constructs (i.e., points lying above the dashed line) (quantities of GAG are reported following normalization to corresponding DNA amounts (mg GAG/mg DNA)) (Demarteau et al., 2003).

to the construct development. Bioreactors such as the one described by Demarteau et al. could be used as controlled model systems to assess when a construct is biomechanically ready to be implanted, and could thus help to answer the question how good is good enough. While a more mature cartilaginous graft may impart greater biomechanical functionality, less mature grafts may have a greater capacity to integrate with the adjacent bone-substitute and the surrounding native cartilage tissue. Using an in vitro model system, Obradovic et al. showed that while cartilaginous constructs pre-cultured for 5 days had lower compressive stiffness than those pre-cultured for 5 weeks, the immature constructs integrated better with adjacent cartilage explants during in vitro culture (Obradovic et al., 2001). Likewise, integration at the cartilage/bone interface of osteochondral composites was better when less mature cartilaginous constructs were sutured to the bone-substitute and cultured together in vitro (Schaefer et al., 2000). The inferior integrative capacity seen with

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cartilage or bone tissues, thus opening the way to the ectopic engineering of osteochondral composites, which can then be transplanted into joint defects (Stevens et al., 2005). 3.6. Osteochondral repair by cell-free scaffolds? From a surgical and commercial standpoint, an ideal graft for osteochondral defect repair would be an offthe-shelf product, possibly free of the component which typically introduces the largest variability, namely cells. Is it possible for a cell-free implant to be sufciently intelligent to bring into the joint the appropriate cues to induce orderly and durable tissue regeneration? The different approaches so far investigated to fabricate cellfree osteochondral composites have been developed based on a variety of fundamental principles, but with the common goal to provide the correct regenerative signals to local cells. Mesenchymal cells from the synovial membrane have been shown to be recruited into partial-thickness cartilage defects by local treatment with chondroitinase ABC, followed by lling of the defect with a brin clot containing TGF-b1 (Hunziker and Rosenberg, 1996). In order to extend the approach to osteochondral defects, it was proven to be necessary to include an additional component to prevent rapid upgrowth of osseous tissue and vascular buds in the cartilage compartment, namely a blood vessel-excluding membrane (Hunziker et al., 2001) or an anti-angiogenic factor (Hunziker and Driesang, 2003). MPC from the subchondral bone have been considered as the target cells in an approach where microfracture was combined with the delivery of a chitosan-based gel, which stabilized clot formation and resulted in improved quantity and quality of repair cartilage in sheep (Hoemann et al., 2005). In order to selectively differentiate MPC recruited from the bone marrow, the use of specic scaffold compositions has also been explored. In this direction, a short-term study performed in rabbits indicated that type I collagen-based cell-free matrices efciently recruited osteoprogenitor cells from the subchondral bone, whereas type II collagen-based matrices were more efcient to direct cells into a chondrogenic phenotype (Buma et al., 2003). Based on these observations, the authors proposed the concept of a composite cell-free matrix consisting of a deep layer of collagen type I and a supercial layer of collagen type II. Another study reported the promising potential of multiphase implants based on polylactide-co-glycolide and various additives, used to vary the stiffness of the supercial and deep regions, for treatment of osteochondral defects (Niederauer et al., 2000). Interestingly, when implanted into a high weight-bearing region (goat condyle), scaffolds containing a cartilage layer with higher compressive stiffness (32 vs. 12 MPa) ranked higher in terms of overall repair, leading the authors to

speculate that it will be important to consider not only the composition, but also the mechanical properties of the scaffold. The latter concept is in line with theories for mechano-regulation of tissue differentiation, mostly developed to model and predict patterns of fracture healing (Carter et al., 1988; Lacroix and Prendergast, 2002), and recently applied to the repair of osteochondral defects (Duda et al., 2005). In particular, a mechano-regulation model for tissue differentiation was recently used to determine the optimal mechanical properties of a construct to properly recruit, proliferate and differentiate MPCs from the bone marrow within osteochondral defects, in order to induce orderly formation of hyaline and bone tissues rather than of brous tissue (Kelly and Prendergast, 2004). The developed criteria indicate that the bone region of the composite scaffold should be rigid enough to stabilize the defect (e.g., Youngs modulus of 50 MPa) and have a permeability sufciently low (e.g., 2E15 m4/N s) to reduce large magnitudes of uid ow that may prevent osteogenesis at between the graft and native bone. In addition, the chondral region should possess a supercial layer (i) sufciently rigid and impermeable to prevent cell death and brous tissue formation at the articular surface, and (ii) with a gradual reduction in stiffness (e.g., Youngs moduli from 60 to 10 MPa) and increase in permeability (e.g., from 1E16 to 2E15 m4/ N s) from the surface to the base of the chondral phase to avoid inducing the dedifferentiation of chondrocytes in other regions of the defect (Kelly, 2003). While the developed model includes several and sometimes arbitrary assumptions, the study is a key milestone in the eld, since it represents a serious engineering effort to develop quantitative principles of design for engineered osteochondral composites, which should direct the setting up of hypothesis-driven experimental studies.

4. Conclusions By describing the wide variety of approaches currently under investigation for the engineering of osteochondral grafts, we have here provided an evidence of the fact that an outcome-driven consensus over design principles is still far to be reached in this relatively new eld. It should also be emphasized that the variability of the type and size of the defect in clinical cases, often not considered in standardized animal models, is expected to inuence the suitability of the design. For example, it is possible that for small and conned osteochondral lesions a cell-free approach with appropriate scaffolds is sufcient, whereas for more extended injuries the delivery of growth factors is necessary for local cell recruitment, and where the wound bed is further compromised the use of a cell-

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762 I. Martin et al. / Journal of Biomechanics 40 (2007) 750765 human articular chondrocytes in vitro. Journal of Orthopaedic Research 16 (2), 207216. Braccini, A., Wendt, D., Jaquiery, C., Jakob, M., Heberer, M., Kenins, L., Wodnar-Filipowicz, A., Quarto, R., Martin, I., 2005. Threedimensional perfusion culture of human bone marrow cells and generation of osteoinductive grafts. Stem Cells 23 (8), 10661072. Buma, P., Pieper, J.S., van Tienen, T., van Susante, J.L., van der Kraan, P.M., Veerkamp, J.H., van den Berg, W.B., Veth, R.P., van Kuppevelt, T.H., 2003. Cross-linked type I and type II collagenous matrices for the repair of full-thickness articular cartilage defects a study in rabbits. Biomaterials 24 (19), 32553263. Buschmann, M.D., Gluzband, Y.A., Grodzinsky, A.J., Hunziker, E.B., 1995. Mechanical compression modulates matrix biosynthesis in chondrocyte/agarose culture. Journal of Cell Science 108 (4), 14971508. Cao, T., Ho, K.H., Teoh, S.H., 2003. Scaffold design and in vitro study of osteochondral coculture in a three-dimensional porous polycaprolactone scaffold fabricated by fused deposition modeling. Tissue Engineering 9 (Suppl. 1), S103S112. Carter, D.R., Blenman, P.R., Beaupre, G.S., 1988. Correlations between mechanical stress history and tissue differentiation in initial fracture healing. Journal of Orthopaedic Research 6 (5), 736748. Chang, C.H., Lin, F.H., Lin, C.C., Chou, C.H., Liu, H.C., 2004. Cartilage tissue engineering on the surface of a novel gelatincalcium-phosphate biphasic scaffold in a double-chamber bioreactor. Journal of Biomedical Materials Research 71B (2), 313321. Chenite, A., Chaput, C., Wang, D., Combes, C., Buschmann, M.D., Hoemann, C.D., Leroux, J.C., Atkinson, B.L., Binette, F., Selmani, A., 2000. Novel injectable neutral solutions of chitosan form biodegradable gels in situ. Biomaterials 21, 21552161. Ciof, M., Boschetti, F., Raimondi, M.T., Dubini, G., 2006. Modeling evaluation of the uid-dynamic microenvironment in tissueengineered constructs: A micro-CT based model. Biotechnology & Bioengineering 93 (3), 500510. Davisson, T., Kunig, S., Chen, A., Sah, R., Ratcliffe, A., 2002a. Static and dynamic compression modulate matrix metabolism in tissue engineered cartilage. Journal of Orthopaedic Research 20 (4), 842848. Davisson, T., Sah, R.L., Ratcliffe, A., 2002b. Perfusion increases cell content and matrix synthesis in chondrocyte three-dimensional cultures. Tissue Engineering 8 (5), 807816. Demarteau, O., Wendt, D., Braccini, A., Jakob, M., Schafer, D., Heberer, M., Martin, I., 2003. Dynamic compression of cartilage constructs engineered from expanded human articular chondrocytes. Biochemical and Biophysical Research Communications 310 (2), 580588. Duda, G.N., Maldonado, Z.M., Klein, P., Heller, M.O., Burns, J., Bail, H., 2005. On the inuence of mechanical conditions in osteochondral defect healing. Journal of Biomechanics 38 (4), 843851. Emans, P.J., Surtel, D.A.M., Frings, E.J.J., Bulstra, S.K., Kuijer, R., 2005. In vivo generation of cartilage from periosteum. Tissue Engineering 11 (3/4), 369377. Frisbie, D., Lu, Y., Colhoun, H., Kawcak, C., Binette, F., McIlwraith, C., 2005. In vivo evaluation of a one step autologous cartilage resurfacing technique in a long term equine model. Transactions of the 51st Annual Meeting of the Orthopaedic Research Society, vol. 30, p. 1355. Fukuda, A., Kato, K., Hasegawa, M., Hirata, H., Sudo, A., Okazaki, K., Tsuta, K., Shikinami, Y., Uchida, A., 2005. Enhanced repair of large osteochondral defects using a combination of articial cartilage and basic broblast growth factor. Biomaterials 26 (20), 43014308. Gao, J., Dennis, J.E., Solchaga, L.A., Awadallah, A.S., Goldberg, V.M., Caplan, A.I., 2001. Tissue-engineered fabrication of an

based approach becomes mandatory. In the selection of specic strategies for the fabrication of osteochondral grafts, it is important to consider that animal studies may be important to evaluate the safety and surgical feasibility of a procedure, or to test a general paradigm of tissue repair. However, due to the highly different biochemical and biomechanical milieu in animal and human joints, efcacy-driven guidelines could only be derived from prospective, randomized clinical trials. Finally, it is important to highlight that the patient population targeted for treatment with engineered osteochondral grafts is currently restricted to young individuals affected by traumatic injuries or by osteochondritis dissecans: future studies in the eld should address if and how the same paradigm could be extended to treatment of degenerative joint pathologies in the ageing population.

Acknowledgments This work was partially supported by the Sixth European Framework Program (Integrated Project STEPS, Grant no. NMP3-CT-2005-500465).

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