Regenerative medicine of the airway cartilage : A morphological and immunohistochemical study with focus on cricoid cartilage defects treated with BMP-2

Abstract: Background. Cartilage provides flexible support for upper airway. Induction of cartilage healing is required for the success of many surgical interventions in the head and neck that are used as treatments for several diseases, such as subglottic stenosis and tumors of the larynx and thyroid. The BMPs constitute a large family of factors that is part of the transforming growth factor beta superfamily. BMP-2 is a well-known modulator of skeletal patterning and, more specifically, of cartilage differentiation. Aims. We have investigated the effects of rhBMP-2 on the regeneration of cricoid cartilage and of respiratory epithelium in a rabbit model. We have examined the integration between host cartilage and repair tissue induced by rhBMP-2. Furthermore, we have evaluated the role of cricoid perichondrium in the repair of cricoid cartilage defects treated with rhBMP-2. We have also attempted to determine what tissue or anatomical structure is a potential source of progenitor cells for the formation of new bone and cartilage induced by rhBMP-2. We have also evaluated the possible differences between young and adult rabbits in chondrogenesis and osteogenesis. We have evaluated the ultrastructural characteristics of new bone and cartilage in order to obtain a more detailed analysis of the cellular repair pattern. Methods. The quantitative and qualitative characteristics of the healing pattern of the laryngeal wound were evaluated by histological techniques including histomorphometry, and immunohistochemistry. Tissue sections were stained with hematoxylin-eosin, alcian blue or periodic acid-Schiff for general morphological assessment (including the presence of chondrogenesis, osteogenesis, and general aspects of the vascular pattern); toluidine blue or safranine O/fast green for determining cartilage proteoglycan content; and they were immunostained with an antibody for tissue inhibitor of metalloproteinases-1 (TIMP-1), Ki-67 antibody, BMP receptor-IB antibody, and BMP receptor-II antibody. The distributions of collagen type I, collagen type II, and collagen type X were also determined. The ultrastructural characteristics of repair tissue were examined by means of TEM. Results. Regeneration of both the epithelial layer and of cartilage was significantly better in rabbits treated with rhBMP-2. The cricoid cartilage defect was completely repaired by new bone and cartilage in rabbits treated with rhBMP-2 four weeks after surgery. Furthermore, there were no discontinuities or gaps at the margins of the cartilage defects. Proteoglycans were synthesized in newly formed cartilage, and they were present four weeks after surgery. Cartilage formation was only induced from host perichondrium that adhered to cricoid cartilage in rabbits treated with rhBMP-2. A cell proliferation marker (Ki-67) was strongly expressed in granulation tissue of specimens treated with rhBMP-2 and moderately expressed in muscle that was adjacent to cricoid cartilage in both control and rhBMP-2 treated specimens. BMP receptors were strongly expressed in cartilage, and moderately expressed in adjacent muscles. The areas of newly formed cartilage were not significantly different in young rabbits from those of adult rabbits, both at one week and at four weeks after surgery. However, the segments of cricoid perichondrium that responded to the rhBMP-2 were significantly larger in young rabbits than in adult rabbits. Collagen type I was strongly expressed in the fibrous layer of the host cricoid perichondrium of the adult rabbits, and it was moderately expressed in the fibrous layer of the young rabbits. Moreover, cricoid perichondrium had a larger proliferative zone in young rabbits. Ki-67 was expressed in the proliferative layer of cricoid perichondrium of young rabbits but not in adult rabbits. TEM revealed that mineralized collage type I matrix, osteoblasts, and osteocytes were already present one week after surgery. Mineralized cartilage matrix invaded by newly formed blood vessels and osteoblasts were present two weeks after surgery. Well-structured bone trabeculae, and growth plate-like structures were present four weeks after surgery. New bone and cartilage were not induced in control rabbits. Conclusions. rhBMP-2, delivered on an absorbable collagen sponge, induces the regeneration and the repair of rabbit cricoid cartilage defects. The relining of airway epithelium is more rapid when rhBMP-2 is administered than it is in control rabbits. The repair tissue induced by rhBMP-2 consists of new bone and cartilage integrated well with the host tissue. The newly formed cartilage matures and produces proteoglycans. Perichondrium of cricoid cartilage is a potent source of progenitor cells. Newly formed cartilage appears to originate only from the progenitor cells of the host perichondrium that adheres to cricoid cartilage in rabbits treated with rhBMP-2. New bone may originate from local skeletal muscle, and new chondrocytes support bone formation at the site of the cricoid cartilage defects. rhBMP-2 induces temporal and quantitative patterns of new bone formation at the site of cricoid cartilage defects in young rabbits that are similar to those in adult rabbits, while chondrogenesis is qualitatively different. Appositional cartilage growth is more easily induced from the cricoid perichondrium of young rabbits than from the cricoid perichondrium of adult rabbits. Intramembranous and endochondral osteogenesis take place at the site of cricoid cartilage defects treated with rhBMP-2. Endochondral osteogenesis follows intramembranous osteogenesis. Progenitor cells of host perichondrium of cricoid cartilage form a growth plate-like structure similar to the epiphyseal growth plate.