Engineering cartilage tissues with the shape of human nasal alar by using chondrocyte macroaggregate—Experiment study in rabbit model
Introduction
Cartilage repair remains an obstacle in clinical works because of poor regenerative capacity of cartilage tissues and limitation of donor sites. As an important technique for cell transplantation, tissue engineering provided a new concept for tissue regeneration and reconstruction, which has been expected to regenerate autogenic cartilage grafts by combining isolated cells and scaffold with predetermined shape (Cao et al., 1997). Over the past decade, various scaffolds including collagen, chitosan, silk protein, and synthetic degradable polymers have been developed to support the growth of chondrocytes isolated from various animal species (Hutmacher, 2001, Woodfield et al., 2002). However, these scaffolds have their respective problems including mechanical strength, cell dedifferentiation, inflammatory reaction induced by their degradation products and limited cell seeding efficiency (Sittinger et al., 2004, Britt and Park, 1998).
Strategies using natural reaggregation potential to assemble monodispersed cells in a tissue-mimicking way represent a valuable extension of current scaffold-based tissue engineering initiatives (Risbud and Sittinger, 2002). Okano et al. developed a temperature responsive culture dish, which can be used to harvest cultured cells non-invasively as intact sheets along with their deposited extracellular matrix (ECM). By transplanting monolayer or layered cell sheets, encouraging results in engineering functional myocardial patches, urothelium tissue and cornea have been achieved (Joseph et al., 2005). The principle advantage of cell sheet is that an entirely natural tissue assembled by cells, with mature ECM, can be engineered, which avoids shortcomings in scaffold based design. However, a principal drawback of cell sheet is their poor mechanical properties, which makes it hard to be fabricated into grafts with special shape, size and controllable stiffness (Ng and Hutmacher, 2006).
For cartilage tissue engineering, fabricating cartilage grafts is of great significance for reconstructive surgery in head and neck region, and three-dimensional shape of cartilage graft must be taken into consideration to achieve cosmetic contour. In addition, chondrocytes will lose their differentiated phenotype during in vitro expansion, which leads to less production of glycosaminoglycans and type II collagen (Brodkin et al., 2004, Thirion and Berenbaum, 2004). Previous studies (Benya and Shaffer, 1982; Van Osch et al., 2001, Schulze-Tanzil et al., 2002) have demonstrated that three-dimensional culture and high-density cultivation will facilitate the redifferentiation of passaged chondrocytes. Naumann et al. (2004), developed a three-dimensional in vitro macroaggregate culture system by using a cell culture insert, which allowed expanded chondrocytes to maintain good phenotype and form homogeneous cartilage graft which approximates the clinical size for cartilage defects in nose and auricle. However, due to the poor mechanical stiffness, the grafts lack stable shape to meet the need of reconstructive surgery.
Given the above, we have explored the use of a new culture system, which aimed to fabricate cell macroaggregate composed of cultured chondrocytes and self-produced ECM. Then chondrocyte macroaggregates were used to engineer cartilage tissues with special shape (nasal alar) by using “Internal support” and “External scaffold” techniques. We hypothesized that the combination of chondrocyte macroaggregates and Internal biodegradable support could provide grafts with better mechanical stability as compared with macroaggregate fabricated by External scaffold, which may facilitate the cartilage formation with special shape in immunocompetent animals. Rabbits were used to evaluate the cartilage formation in the shape of nasal alar by two different constructing approaches.
Section snippets
Animal model
Twelve New Zealand rabbits (2-month-old) were used for the experiment. The operative procedure and the care of the rabbits were performed under the regulation of Experimental Animal Centre, Fourth Military Medical University. The rabbits were acclimated for 1 week before operation and monitored for general appearance, activity, excretion and weight, then they were randomly divided into two groups: External scaffold group (n = 6) and Internal support group (n = 6).
Isolation and culture of chondrocytes
All the New Zealand rabbits were
Examination of chondrocyte macroaggregate and nasal alar grafts
At the end of the cultivation, chondrocytes-cell sheet complex was replaced by semitransparent membrane and could be easily detached from culture dish by scraping technique. The obtained chondrocyte macroaggregate was stiff enough to be handled by forceps (Fig. 2a). By confocal microscope, living cells labeled green with CFDA were observed to be embeded into macroaggregate (Fig. 2b). Safranin-O staining showed that newly formed ECM was presented as positive orange patches, and chondrocytes were
Discussion
Cartilage defect in nose, which caused by trauma, congenital microtia, or surgical operation, remains one of the most difficult problems for clinicians. Previous surgical approaches, using alloplastic prosthesis implantation or autologous cartilage grafts, may be performed to restore nasal framework (Bikhazi et al., 1997). However, these approaches were limited because of donor sites’ restriction, immunologic complications, bacterial infections, and material failure. In this study, autologous
Acknowledgements
The authors thank Mr. Jun Han for MRI examination and Miss LiJuan Shen for her assistance to the biomechanical test. This work was funded by the National Natural Science Foundation of PR China, numbers 30270373 and 30370374.
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