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Injectable engineered cartilage that maintains a predictable shape and volume would allow recontouring of craniomaxillofacial irregularities with minimally invasive techniques. This study investigated how chondrocytes from different cartilage sources, encapsulated in fibrin polymer, affected construct mass and volume with time. Swine auricular, costal, and articular chondrocytes were isolated and mixed with fibrin polymer (cell concentration of 40 x 10 cells/ml for all groups). Eight samples (1 cm x 1 cm x 0.3 cm) per group were implanted into nude mice for each time period (4, 8, and 12 weeks). The dimensions and mass of each specimen were recorded before implantation and after explantation. Ratios comparing final measurements and original measurements were calculated. Histological, biochemical, and biomechanical analyses were performed. Histological evaluations (n = 3) indicated that new cartilaginous matrix was synthesized by the transplanted chondrocytes in all experimental groups. At 12 weeks, the ratios of dimension and mass (n = 8) for auricular chondrocyte constructs increased by 20 to 30 percent, the ratios for costal chondrocyte constructs were equal to the initial values, and the ratios for articular chondrocyte constructs decreased by 40 to 50 percent. Constructs made with auricular chondrocytes had the highest modulus (n = 3 to 5) and glycosaminoglycan content (n = 4 or 5) and the lowest permeability value (n = 3 to 5) and water content (n = 4 or 5). Constructs made with articular chondrocytes had the lowest modulus and glycosaminoglycan content and the highest permeability value and water content (p < 0.05). The amounts of hydroxyproline (n = 5) and DNA (n = 5) were not significantly different among the experimental groups (p > 0.05). It was possible to engineer injectable cartilage with chondrocytes from different sources, resulting in neocartilage with different properties. Although cartilage made with articular chondrocytes shrank and cartilage made with auricular chondrocytes overgrew, the injectable tissue-engineered cartilage made with costal chondrocytes was stable during the time periods studied. Furthermore, the biomechanical properties of the engineered cartilage made with auricular or costal chondrocytes were superior to those of cartilage made with articular chondrocytes, in this model.

A large-deflection elasticity model was used to describe the mechanical behavior of cartilaginous tissues during three-point bending tests. Force-deflection curves were measured for 20-mm long x 4-mm wide x approximately 1-mm thick strips of porcine auricular and costal cartilage. Using a least-squares method with elastic modulus in bending as the only adjustable parameter, data were fit to a model based on the von Karman theory for large deflection of plates. This model described the data well, with an average RMS error of 14.8% and an average R(2) value of 0.98. Using this method, the bending modulus of auricular cartilage (4.6 MPa) was found to be statistically lower (p < 0.05) than that of costal cartilage (7.1 MPa). Material features of the cartilage samples influenced the mechanical behavior, including the orientation of the perichondrium in auricular cartilage. These methods also were used to determine the elastic moduli of engineered cartilage samples produced by seeding chondrocytes into fibrin glue. The modulus of tissue-engineered constructs increased statistically with time (p < 0.05), but still were statistically lower than the moduli of the native tissue samples (p > 0.05), reaching only about a third of the values of native samples.

This study evaluated the biomechanical and physical properties of newly formed cartilage engineered from isolated chondrocytes in combination with matrix components. Four groups of constructs were studied. Group A consisted of lyophilized articular cartilage chips mixed with a cell-fibrinogen solution and thrombin to obtain constructs made of fibrin glue, chondrocytes, and cartilage chips. Group B constructs were prepared using fibrin glue and cartilage chips without cells. Group C contained chondrocytes in fibrin glue without chips, and group D comprised constructs of fibrin glue alone. Specimens were implanted in the subcutaneous tissue of nude mice for 9 weeks. At necropsy the specimens were examined grossly, physically, biomechanically, and histologically. The original, preimplantation mass of the constructs was retained only in experimental group A. Histological analysis of specimens in experimental groups A and C demonstrated the presence of newly formed cartilaginous matrix, whereas only fibrotic tissue was observed in control groups B and D. Biomechanical analysis demonstrated higher mean values of equilibrium modulus in the experimental samples of group A with respect to all control groups. This study demonstrated that adding lyophilized cartilage chips to a fibrin glue-engineered cartilage construct maintains the biomechanical properties and the original mass after medium-/long-term in vivo transplantation.

This study evaluated the biomechanical characteristics of newly formed cartilaginous tissue synthesized from isolated chondrocytes and seeded onto devitalized cartilage in an extended study in vivo. Cartilage from porcine articular joints was cut into regular discs and devitalized by multiple freeze-thaw cycles. Articular chondrocytes were enzymatically isolated and incubated in suspension culture in the presence of devitalized cartilage discs for 21 days. This procedure allowed the isolated chondrocytes to adhere to the devitalized matrix surfaces. Chondrocyte-matrix constructs were assembled with fibrin glue and implanted in dorsal subcutaneous pockets in nude mice for up to 8 months. Histological evaluation and biomechanical testing were performed to quantify the integration of cartilage pieces and the mechanical properties of the constructs over time. Histological analysis indicated that chondrocytes grown on devitalized cartilage discs produced new matrix that bonded and integrated individual cartilage elements with mechanically functional tissue. Biomechanical testing demonstrated a time dependent increase in tensile strength, failure strain, failure energy, and tensile modulus to values 5-30% of normal articular cartilage by 8 months in vivo. The values recorded at 4 months were not statistically different from those collected at the latest time point, indicating that the limits of the biomechanical property values were reached after four months from implantation.

Articular chondrocytes can synthesize new cartilaginous matrix in vivo that forms functional bonds with native cartilage. Other sources of chondrocytes may have a similar ability to form new cartilage with healing capacity. This study evaluates the ability of various chondrocyte sources to produce new cartilaginous matrix in vivo and to form functional bonds with native cartilage. Disks of articular cartilage and articular, auricular, and costal chondrocytes were harvested from swine. Articular, auricular, or costal chondrocytes suspended in fibrin glue (experimental), or fibrin glue alone (control), were placed between disks of articular cartilage, forming trilayer constructs, and implanted subcutaneously into nude mice for 6 and 12 weeks. Specimens were evaluated for neocartilage production and integration into native cartilage with histological and biomechanical analysis. New matrix was formed in all experimental samples, consisting mostly of neocartilage integrating with the cartilage disks. Control samples developed fibrous tissue without evidence of neocartilage. Ultimate tensile strength values for experimental samples were significantly increased (p < 0.05) from 6 to 12 weeks, and at 12 weeks they were significantly greater (p < 0.05) than those of controls. We conclude that articular, auricular, and costal chondrocytes have a similar ability to produce new cartilaginous matrix in vivo that forms mechanically functional bonds with native cartilage.

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