+*van Osch, G JVM; *Bos, P K; **Duynstee, M LG; **Verwoerd-Verhoef, H L; *Verhaar, J AN
+*Dept. Orthopaedics, Erasmus university Medical Center Rotterdam, Rotterdam, the Netherlands. +31 10 4087661, Fax: +31 10 4089441, vanosch@kno.fgg.eur.nl
Introduction. The regeneration capacity of cartilage in general is very limited. Cartilage does not contain blood vessels and is thought to consist of only one cell type (chondrocytes) lacking progenitor cells. The young articular cartilage contains progenitor cells thereby expressing
“perichondrium-like properties” in culture. chondrocytes, laying isolated in a dense extracellular matrix, have very limited proliferation potential. Despite the absence of repair or regeneration of damaged articular cartilage, cartilage structures in the head and neck region do show some healing capacity. These cartilage structures are covered with a layer of perichondrium. The neocartilage formed originates from proliferation of progenitor cells in the inner layer (cambium layer) of perichondrium 1 . Articular cartilage is not lined by perichondrium and thus lacks this internal progenitor cell source. We here present two sets of different, unrelated experiments that lead us to formulate the hypothesis that the superficial layer of articular cartilage might possess perichondriumlike properties.
Materials/Methods. In the first set of experiments explants of rabbit ear cartilage were cultured for 3 weeks with the entire perichondrium intact, the entire perichondrium removed or only the outer layer of the perichondrium removed.
In the second set of experiments full-thickness articular cartilage explants of calves (6 months old) were cultured for 4 weeks under standard culture conditions. In an additional group, explants were treated with 30 U/ml collagenase the first 48 h of the culture period.
Culture medium consisted of DMEM/Ham’s F12 medium with 10% FCS and 25 µg/ml l-ascorbic acid. The explants were stained with Hematoxylin-
Eosin, Alcian Blue and Thionin. Immunohistochemical staining for collagen type II was performed using antibody II-II6B3.
Results. Explants of cartilage with intact perichondrium as well as explants with the entire perichondrium removed, never showed formation of new cartilage. On the other hand, explants where the outer (fibrous) perichondrium-layer was selectively removed thereby exposing the inner layer of the perichondrium that contains progenitor cells, demonstrated outgrowth of new cartilage. The new cartilage tissue clearly originated from the inner perichondrial layer (Fig 1).
The experiments using untreated articular cartilage explants revealed comparable images. In our culture of full thickness articular cartilage, progenitor cells from the bone marrow were not present. Another cell source suggested by Hunziker 2 to be able to repair articular cartilage defects is the synovial lining. This type of cells was also not present in our culture set-up.
We therefore were surprised that the cultured cartilage explants expressed formation of new cartilage tissue attached to the explant in half of the cultures (Fig 1).
Figure 2. Explant of joint cartilage treated with collagenase and thereafter cultured for 4 weeks.
Discussion. The superficial zone of articular cartilage is recognised to be different from the rest of the cartilage. The cells are more elongated, the collagen fibres are oriented parallel to the surface. Different protein expression patterns are found in this layer of the cartilage perichondrium-like tissue covering the cartilage 10
3-6 and the cells demonstrated different proliferation and matrix synthesis rates 7-9 . More evidence for the possible presence of a perichondrium-like layer at the articular surface was found from embryonic development. Histologic examination of articular surface or human foetuses have shown a fibrous
and it has been proposed that cells from the superficial zone of articular cartilage are derived from the fibrous perichondrial layer present during articular cavity formation, based on the types of collagen expressed in articular cartilage during development 11 . The expression of other proteins (i.e. COMP, CMP, PTHrP) in the perichondrium-like layer on top of the cartilage also showed a pattern different from the rest of the cartilage
Finally, Archer et al 14
12,13 .
demonstrated in developing cartilage of 7marsupials that the majority of growth appears appositional. This suggests the existence of progenitor cells during development in the cartilage surface.
Combining our results with knowledge of embryonic development we suggest that the superficial cartilage layer of young individuals still contains progenitor cells. These cells have the capacity to proliferate and to differentiate into neocartilage. The fact that spontaneous repair of superficial defects in mature articular cartilage is never seen can have several causes. First the perichondrium-like properties of the superficial layer can be lost due to further maturation or due to the early loss of the superficial layer in degenerative diseases. On the other hand, it may very well be that the superficial cells lack a certain stimulus to induce proliferation and differentiation. The nutritional and biomechanical circumstances may not allow in-vivo neocartilage formation, whereas invitro the circumstances are more optimal to induce proliferation and differentiation. Further research has to reveal the answers to these questions and to the question how this knowledge can be implemented in current
Figure 1.
Left: New cartilage formation around ear cartilage from which the inner perichondrium layer is exposed
Right: Cartilage formation around joint cartilage explants
Moreover, enzymatic treatment of the explants with collagenase, making it easier for cells to leave the matrix, appeared to increase the incidence and the amount of neocartilage formation (Fig 2). The newly formed cartilage appears to originate from proliferation of cells from the superficial cartilage zone. These data lead us to the hypothesis that the superficial zone of stategies to stimulate articular cartilage repair.
References.
1. Engkwist et al. (1979) Scand J Plast Reconstr Surg 13: 275; 2. Hunziker et al (1996) J Bone Joint Surg 78A:721; 3. Fang et al (2000). J Orthop Res
18:593; 4. Schumacher et al (1999) J Orthop Res 17:11-; 5 Clark (1990) J
Anat 171:117; 6. Khan et al (2001) Trans ORS 386; 7. Aydelotte et al
(1988) Connect Tissue Res 18:223; 8. Aydelotte et al (1988) Connect
Tissue Res 18:205; 9. Wong et al (1996) J Orthop Res 14:424; 10. Mahasen et al (2000) Cells Tissues Organs 166:359; 11. Treilleux et al (1992) Matrix
12: 221; 12. Vortkamp et al (1996) Science 273:613; 13. Murphy et al
(1999) Matrix Biol 18:487; 14. Archer et al (1994) AAOS publishers:491.
**Dept. Otorhinolaryngology, Erasmus university Medical Center
Rotterdam, Rotterdam, the Netherlands.