One treatment goal for large articular cartilage defects is the restoration of the anatomical contour of the joint with tissue having a structure similar to native cartilage. Since surface incongruities may limit graft success, shaped and stratified cartilaginous tissue may be a suitable graft to achieve such restoration. The objective of this study was to establish and validate a molding technique for fabrication of cartilaginous constructs that are anatomically shaped, targeting the spherically shaped hip, and biomimetically stratified with superficial and middle/deep zone chondrocyte subpopulations. We present a molding technique for fabrication of shaped cartilaginous constructs using ARCs in saucer and cup shapes with one and two surfaces molded, respectively (Fig. ). Qualitatively, the shaped constructs had surface contours different from those of the control disk constructs. Quantitatively, the saucer and cup constructs were distinct in their radii of curvature (Table ). These results demonstrate molding fabrication can generate constructs that are contoured and fabricated from only chondrocytes and their biosynthetic products. The matrix products accumulated fairly similarly regardless of the shape of the construct, demonstrating the shaping does not adversely affect chondrocyte functions (Figs. –). Additionally, this molding technique was adapted to create shaped cartilaginous constructs with biomimetic stratification (Fig. ). Thus, in this study tissue-engineered cartilaginous constructs were designed to have, and analyzed for, anatomic three-dimensional contours and biomimetic stratification.
We note several limitations. Cell source is an important consideration since these constructs were formed solely from the chondrocytes and the pericellular matrix they produced. While this study was performed with immature calf chondrocytes, which are metabolically active, this shaping technology would likely be applicable to chondrocytes from mature articular cartilage since such chondrocytes can be used to form scaffold-free disk constructs [29
]. Additionally, the cell source limitation may be circumvented by expansion of the cell source followed by three-dimensional alginate bead culture [9
Previous studies on shaped tissue engineering of cartilage have utilized molding or machining of the scaffold material to define the contour of the construct. These methods have been used for engineering of elastic and nonarticular cartilage such as auricle [7
], nose [8
], and tympanic membrane [17
]. Several shaped osteochondral constructs containing articular cartilaginous sections, for phalanx [20
] and mandibular condyle [1
], for example, have been fabricated. Bone grafts also have been constructed in various shapes, including femoral head and mandible [23
]. Many of these previous works rely on scaffolds to provide mechanical stability to the constructs and aid in the maintenance of their shapes. Like some of these previous studies, molding was used to shape the constructs in this study. However, here we sought to develop three dimensionally-shaped cartilaginous constructs supported only by the cells and its biosynthetic products, using a molding technique.
In previous studies, the shape of constructs changed qualitatively from the initial scaffold or mold shape due to growth and/or remodeling of the scaffold during culture [7
]. Additionally, many of the previous reports on shaped cartilaginous grafts did not quantify the construct shapes or contours. In the present study, we developed an analysis of the surface contour of the shaped constructs, from determining a method of accurate data acquisition and the development of the appropriate image processing protocol using MATLAB. Here, the measured radii of curvature of the shaped constructs differed from those of the hemispherical molds that were used (Table ). However, contraction of scaffold-free constructs noted in a previous study with expanded chondrocytes cultured on agarose [18
] was not observed here, possibly due to phenotype stabilization during alginate preculture. A longer shaping period with a permeable mold may allow for better retention of the initial shape and also adequate nutrient transfer. Also, reshaping by mechanical loading after the initial construct formation period may allow for finer control of construct shape [39
]. This molding technique for shaping of tissue-engineered construct may also be applied to joints with geometries and contours that are more complex than those of the hip.
A biomimetic approach to cartilage tissue engineering in terms of construct shape and structural organization may be advantageous for clinical applications. The use of tissue-engineered grafts with flat surfaces may be sufficient to approximate the normal cartilage surface for smaller defects but not larger defects. Surface incongruity between osteochondral grafts and the surrounding native cartilage results in local mechanical stresses that may be unfavorable for the graft survival and treatment efficacy [27
The applicability of this shaping technology to constructs with a biomimetic cellular organization suggests the possible application to tissue with multiple layers. Previously, stratified cartilaginous constructs with S chondrocytes atop M chondrocytes maintain the zonal characteristics typical of their source, such as proteoglycan 4 and differential matrix production [26
]. Such differential spatial characteristics result in inhomogeneous biochemical and biomechanical properties of native cartilage with respect to depth and may be important in the maturation and, ultimately, function of tissue-engineered cartilaginous constructs as well [24
]. The stratification can be customized with different types and arrangements of cells and materials for various target application and location.
The biochemical content of the shaped constructs in this study is consistent with previously described constructs [26
]. Like many tissue-engineered cartilaginous constructs, the constructs here had lower extracellular matrix content, especially collagen, compared to that of native cartilage. The slightly elevated deposition of collagen in cup-shaped constructs may be due to enhanced nutrient transport provided by the thinner cup constructs as compared to thicker disk and saucer-shaped constructs. It is also possible the thicker agarose support on the bottom of cup constructs helped to retain more of the matrix products.
The shaping technique presented here has the potential to facilitate treatment of larger articular cartilage defects where recreation of surface contour is important. Such shaping methods may be coupled with noninvasive three-dimensional imaging to determine the surface contour of subchondral bone and/or of contralateral cartilage to appropriately tailor the shape of the construct [37
]. Improved control over shaping and stratification would be useful in clinical translation of these shaped, stratified, scaffold-free grafts. Larger constructs that are needed for repair of large articular cartilage defects may become feasible with expansion and redifferentiation methods for the cells in conjunction with a bioreactor for improved construct growth and maturation. To attach such constructs to the surrounding native tissue, such shaped grafts may either be fixed in the defect area with fibrin glue, as with current tissue-engineered grafts like MACI [11
], or be fabricated in vitro atop a boney substance, which can integrate into the surrounding native bone after implantation as with current allo- or autograft techniques. In vivo implantation of these grafts is needed to better assess the functionality and durability of such biomimetic, tissue-engineered grafts with appropriate shape and stratification.