Tissue Engineering of Autologous Cartilage Grafts in Three-Dimensionalin VitroMacroaggregate Culture System

Abstract

In the field of tissue engineering, techniques have been described to generate cartilage tissue with isolated chondrocytes and bioresorbable or nonbioresorbable biomaterials serving as three-dimensional cell carriers. In spite of successful cartilage engineering, problems of uneven degradation of biomaterial, and unforeseeable cell-biomaterial interactions remain. This study represents a novel technique to engineer cartilage by an in vitro macroaggregate culture system without the use of biomaterials. Human nasoseptal or auricular chondrocytes were enzymatically isolated and amplified in conventional monolayer culture before the cells were seeded into a cell culture insert with a track-etched membrane and cultured in vitro for 3 weeks. The new cartilage formed within the in vitro macroaggregates was analyzed by histology (toluidine blue, von Kossa-safranin O staining), and immunohistochemistry (collagen types I, II, V, VI, and X and elastin). The total glycosaminoglycan (GAG) content of native and engineered auricular as well as nasal cartilage was assayed colorimetrically in a safranin O assay. The biomechanical properties of engineered cartilage were determined by biphasic indentation assay. After 3 weeks of in vitro culture, nasoseptal and auricular chondrocytes synthesized new cartilage with the typical appearance of hyaline nasal cartilage and elastic auricular cartilage. Immunohistochemical staining of cartilage samples showed a characteristic pattern of staining for collagen antibodies that varied in location and intensity. In all samples, intense staining for cartilage-specific collagen types I, II, and X was observed. By the use of von Kossa-safranin O staining a few positive patches-a possible sign of beginning mineralization within the engineered cartilages-were detected. The unique pattern for nasoseptal cartilage is intense staining for type V collagen, whereas auricular cartilage is only weakly positive for collagen types V and VI. Engineered nasal and auricular macroaggregates were negative for anti-elastin antibody (interterritorially). The measurement of total GAG content demonstrated higher GAG content for reformed nasoseptal cartilage compared with elastic auricular cartilage. However, the total GAG content of engineered macroaggregates was lower than that of native cartilage. In spite of the mechanical stability of the auricular macroaggregates, there was no equilibrium of indentation. The histomorphological and immunohistochemical results demonstrate successful cartilage engineering without the use of biomaterials, and identify characteristics unique to hyaline as well as elastic cartilage. The GAG content of engineered cartilage was lower than in native cartilage and the biomechanical properties were not determinable by indentation assay. This study illustrates a novel in vitro macroaggregate culture system as a promising technique for tissue engineering of cartilage grafts. Further long-term in vitro and in vivo studies must be done before this method can be applied to reconstructive surger...

The in vitro and in vivo capacity of culture-expanded human cells from several sources encapsulated in alginate to form cartilage

“…2002; Karlsson et al, 2007;Kusuhara et al, 2009;Lohan et al, 2011;Malicev et al, 2009;Naumann et al, 2004;Panossian et al, 2001;Sakaguchi et al, 2005;Seda Tigli et al, 2009;Tay et al, 2004;van Osch et al, 2004;Vinardell et al, 2012;Xu et al, 2004;Yoshimura et al, 2007;Zhang and Spector, 2009). However, precise comparison of the performance of culture-expanded human cells is lacking.…”

A Comparison of Self-Assembly and Hydrogel Encapsulation as a Means to Engineer Functional Cartilaginous Grafts Using Culture Expanded Chondrocytes

“…18 After 12 weeks of culture, this SA approach has been shown to support the generation of a hyaline-like cartilaginous tissue with biochemical and mechanical properties approaching those of native articular cartilage. Numerous other studies have investigated the SA of chondrocytes, [25][26][27][28][29][30][31][32][33] with determination of the initial cell seeding number identified as a key parameter to successfully engineer a cartilaginous graft using the SA approach. Researchers have also investigated the potential of generating cartilage grafts through SA of mesenchymal stem cells (MSCs), 19,34 with some success reported in repairing chondral defects in vivo using this approach.…”

Confocal raman microspectroscopy : applications in cartilage tissue engineering

“…In our laboratory and a number of others have studied the use of a family of block copolymers poly(ethylene oxide terephthalate) poly(butylene terephthalate) (PEOT/PBT) as a biomaterial for scaffolds and they proved to be highly biocompatible both Chapter 1 in vitro and in vivo [44][45][46][47] and some compositions have reached clinical application (PolyActive™, IsoTis Orthopaedics S.A.) as dermal substitutes [48] and bone fillers [49][50]. Currently, among scaffold fabrication techniques, rapid prototyping -and within rapid prototyping systems, 3D fiber deposition -appears to be the most promising and widely used fabrication method.…”

“…Currently, among scaffold fabrication techniques, rapid prototyping -and within rapid prototyping systems, 3D fiber deposition -appears to be the most promising and widely used fabrication method. This scaffold fabrication technique allows one to tailor the porosity, pore size and shape of scaffolds and the process results in implants which have a completely interconnected pore network and predefined shape [47,[51][52]. It has been demonstrated that scaffolds can be fabricated with such composition and architecture that they can have instructive properties for expanded human chondrocytes to enhance the formation of cartilaginous tissues [2,53].…”

“…Several studies with chondrocytes showed a correlation between aggregation and enhanced cartilaginous tissue formation [46][47][48][49].…”