Cell-Engineered Human Elastic Chondrocytes Regenerate Natural Scaffold In Vitro and Neocartilage with Neoperichondrium in the Human Body Post-Transplantation

Abstract

We have developed a unique method that allows us to culture large volumes of chondrocyte expansion from a small piece of human elastic cartilage. The characteristic features of our culturing method are that fibroblast growth factor-2 (FGF2), which promotes proliferation of elastic chondrocytes, is added to a culture medium, and that cell-engineering techniques are adopted in the multilayered culture system that we have developed. We have subsequently discovered that once multilayered chondrocytes are transplanted into a human body, differentiation induction that makes use of surrounding tissue occurs in situ, and a large cartilage block is obtained through cartinogenesis and matrix formation. We have named this method two-stage transplantation. We have clinically applied this transplantation method to the congenital ear defect, microtia, and reported successful ear reconstruction. In our present study, we demonstrated that when FGF2 was added to elastic chondrocytes, the cell count increased and the level of hyaluronic acid, which is a major extracellular matrix (ECM) component, increased. We also demonstrated that these biochemical changes are reflected in the morphology, with the elastic chondrocytes themselves producing a matrix and fibers in vitro to form a natural scaffold. We then demonstrated that inside the natural scaffold thus formed, the cells overlap, connect intercellularly to each other, and reconstruct a cartilage-like three-dimensional structure in vitro. We further demonstrated by immunohistochemical analysis and electron microscopic analysis that when the multilayered chondrocytes are subsequently transplanted into a living body (abdominal subcutaneous region) in the two-stage transplantation process, neocartilage and neoperichondrium of elastic cartilage origin are regenerated 6 months after transplantation. Further, evaluation by dynamic mechanical analysis showed the regenerated neocartilage to have the same viscoelasticity as normal auricular cartilage. Using our multilayered culture system supplemented with FGF2, elastic chondrocytes produce an ECM and also exhibit an intercellular network; therefore, they are able to maintain tissue integrity post-transplantation. These findings realized a clinical application for generative cartilage surgery.

Biocompatibility of Human Auricular Chondrocytes Cultured onto a Chitosan/Polyvynil Alcohol/Epichlorohydrin-Based Hydrogel for Tissue Engineering Application

“…We now analyzed the biocompatibility of human auricular chondrocytes isolated from cartilage remnants and cultured onto the CS-PVA-ECH hydrogel. As expected, we were able to expand chondrocytes from microtic cartilage, these cells displayed similar morphology and regular growth when compared with normal chondrocytes previously reported (Yamaoka et al, 2006;Yanaga et al, 2012).…”

Development of Scaffold-Free Elastic Cartilaginous Constructs with Structural Similarities to Auricular Cartilage

“…Interestingly, while the formation of a cartilaginous region in the developed constructs were observed among all the investigated conditions (seeding density and NaHCO 3 supplementation), the cultures supplemented with NaHCO 3 developed more distinct perichondrium-like regions. Although the development of perichondrium-like regions in neocartilage constructs have been observed in relatively few previous studies-most notably by Yanaga and coworkers who also showed a spontaneous recapitulation of a perichondrium-like region after implantation, 46 the majority of elastic cartilage tissue engineering approaches tend to only utilize cells isolated from the cartilaginous region. 10,11,[47][48][49][50][51] The creation of engineered constructs possessing both cartilaginous and perichondrium regions, similar to that observed in native auricular cartilage, is of benefit toward the development of functional engineered constructs suitable for implantation.…”

“…[1][2][3][4] However, there are a few approaches currently available for external ear reconstruction, such as alloplastic implants and sculpted autologous cartilage grafts-both of which have their advantages and disadvantages. While the availability of alloplastic implants in the shape of the ear is typically not an issue, these implants are susceptible to both infection and immunogenic issues and, as such, their long-term durability is uncertain.…”

The burned ear; possibilities and challenges in framework reconstruction and coverage

“…Because the ear has a unique shape the scaffold will need to have a defined, preferably personalized model, and should maintain this during the development of the new cartilage tissue. Yanaga et al demonstrated that cultured autologous auricular chondrocytes needed 6 months to develop into sufficient new cartilage in a patient after re-implantation [46]. By combining cells with biomaterials cartilage, development time could perhaps be reduced but it would still be a question of months rather than weeks to create enough cartilage of sufficient quality for reconstruction.…”

“…Other researchers have also demonstrated this specific trait [57,58]. A current interesting clinical development is the work of Yanaga et al who injected in vitro expanded autologous chondrocytes in the abdominal wall of microtia patients resulting in sufficient cartilage for ear reconstruction purposes [46]. The same principle could possibly also be applied to viable cartilage remnants after burn injuries; this remains to be investigated however.…”