Immuno-SEM Characterization of Developing Bovine Cartilage

Publication Date

8-2006

Type of Culminating Activity

Thesis

Degree Title

Master of Science in Materials Science and Engineering

Department

Materials Science and Engineering

Supervisory Committee Chair

Janet M. Hampikian

Abstract

Collagen is a vital material in the tissues of living organisms. Found almost everywhere in the human body, collagen is important in connective tissues, hone growth, and cartilage. Collagen XI makes up a very small portion of the cartilaginous tissue; however, its role in cartilaginous tissue is vitally important. Collagen XI and two collagen XI isoforms, V1b and V2, are critical in the ossification process. However, the location of collagen XI, V1b, V2, and their specific function in the ossification process within developing bovine cartilage is not well characterized. In this work, the location of collagens I, II, XI and two collagen XI isoforms, V1b and V2, present in developing bovine cartilage, are investigated using the immuno-SEM technique. The viability of this technique to locate specific proteins within cartilaginous and mineralized tissue is evaluated based on locations of collagens I and II. This technique is then used to determine the locations of collagen XI, V1b, and V2 within developing bovine cartilage. Results for the locations of collagen I and II showed a high level of consistency with previous work, thus showing that the technique of immuno-SEM can be used with confidence to determine the location of various collagen types within cartilaginous and mineralized tissue. Prior work using the techniques of immuno-staining and transmission electron microscopy has shown that collagen XI is observed in the longitudinal septa, and in a restricted pericellular pattern in the resting zone. This work has shown that collagen XI is present in the lower hypertrophic region and also in a pericellular arrangement, within about two microns from cell walls, throughout the cartilaginous tissue. Prior work has shown that before primary ossification, V1b was detected only in the diaphysis, primarily adjacent to the periosteum, and not in the epiphysis. This work has shown that V1b is expressed in the articular surface, mineralized region, resting zone, and the distal edge of the diaphysis. Previous work has shown that the V2 isoform was most strongly expressed in areas of newly forming cartilage, and disappeared as chondrocyte maturation proceeded. V2 was also present in the distal edge of the epiphysis. This work showed similar results concerning the location of V2. Additionally, V2 was shown to be present in the mineralized tissue. Collagen XI and two of its isoforms, V1b and V2, are thought to play a critical role in the ossification process. However, this role is not well understood, and is still being characterized. The detection of collagen XI and two of its isoforms in the osteochondral junction as well as at a joint surface further point to collagen XI, V1b, and V2 playing a vital role in the ossification process, and warrant further research as to their specific function within the ossification process.

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