在臨床上應用於耳朵軟骨膜移植實驗時，之前的文獻指出有些移植後可促進軟骨的新生，而有些卻無法有軟骨新生的現象，且年紀對於軟骨新生有一定之影響力，對於這些研究到目前並不是十分深入。目前已知在軟骨組織大致可分三層細胞，包括最外側的纖維層(fibrous layer)、軟骨組織(cartilage layer)及在軟骨組織表層的形成層(cambium layer)。
本論文之實驗目的為了解年輕與成年兔耳軟骨膜及軟骨組織細胞的特性，及其分化能力之比較。首先利用酵素萃取及組織學鑑定方法判定取得分離纖維層、形成層及軟骨組織中之細胞的一致性，並進一步分析細胞表面標誌、群落形成能力(colony forming ability)及其分化能力。
由實驗結果可知，在細胞型態上，年輕與成年兔纖維層細胞呈現類纖維母細胞型態，軟骨組織的細胞型態為多角型，而形成層的細胞型態則兩者均有。在細胞表面標誌上，年輕與成年兔形成層及軟骨組織表現較多具軟骨分化潛力之細胞標誌。在群落形成能力的比較上，此三層細胞年輕兔均高於成年兔。群落形成效率(colony forming efficiency)均為軟骨組織表現量較高一些；群落形成面積(colony forming area)則是形成層表現量較高一些。此外，在分化實驗中，纖維層分化比例均最差。在軟骨分化上，此三層細胞雖在年輕兔和成年兔均為百分之百，然而年輕兔軟骨組織分化之群落數目高於形成層2倍之多，又年輕兔形成層分化之群落數目高於成年兔形成層2倍，而年輕兔軟骨組織為成年兔軟骨組織為10倍之多。
總結而言，形成層和軟骨組織不論是年輕兔或成年兔其潛能性均較纖維層高。以年輕兔而言，軟骨組織有較高之軟骨分化潛能性；而以成年兔而言則是形成層保留較高之軟骨新生潛力。

Perichondrium transplantation has been applied clinically before and shown chondrogenic potential. However, it is not consistent due to variable factors. Age may play an important role. Thus, the researchers pay much attention to this field, but the information is still very limited. Nowadays, it was known that auricular cartilage can roughly be separated into three layers: fibrous, cambium and cartilage layer. The fibrous layer is at the outer part of perichondrium, and the cambium layer is the inner part.
The aim of this study is to investigate the characteristics of cells from perichondrium and cartilage of young and adult rabbit ears and their potentials of differentiation. In the preliminary, we tried to set up a consistent approach for isolating cambium, fibrous layer cells, and chondrocytes. Afterwards, the characteristics of these three layers, including chondroprogenitor marker profiles, colony forming efficiency, and multi-potent differentiation abilities, were analyzed.
For the cell morphology in vitro, fibrous layer cells presented as fibroblast-like shape; cambium layer showed fibroblast-like and polygonal appearance; cartilage layer only demonstrated polygonal ones. For the mesenchymal stem cell marker profiles, cambium layer and cartilage layer all expressed higher chondroprogenitor related marker profiles. Regarding colony forming potential, young rabbit is higher than that of adult. Furthermore, cartilage cell layer expressed higher colony forming efficiency than that of cambium layer; cambium cell layer expressed much colony area than that of cartilage cell layer. In the differentiation assay, fibrous layer has the least differentiation potential. In adipogenesis, young rabbit is higher than old one, and the cartilage layer is slightly higher than cambium layer. In osteogenesis, adult rabbit is higher than that of young one. The adult cambium layer is better than cartilage layer for the osteogenesis, however, there is no significant difference between cambium and cartilage layers in young rabbit. In chondrogenesis, young and adult rabbits both showed strong chondrogenic potential.
In summary, the cambium and cartilage layers both expressed much mesenchymal stem cell markers than that of fibrous layers, and the young cambium cells provided much multi-potent differentiation potential than that of the adult rabbit ears. The perichondrial progenitor cells (cells from cambium layer) did have chondrogenesis potential according to our results. Further investigation of in vitro and in vivo chondrogenesis is valuable. The experimental results obtained from this project will provide important information regarding the mechanism of cell proliferation and differentiation of perichondrial progenitors, and potentially applied in the cartilage tissue engineering in the future.

Alsalameh, S., R. Amin, et al. (2004). "Identification of mesenchymal progenitor cells
in normal and osteoarthritic human articular cartilage." Arthritis Rheum 50(5):
1522-32.
Alvarez, J., J. Horton, et al. (2001). "The perichondrium plays an important role in
mediating the effects of TGF-beta1 on endochondral bone formation." Dev Dyn
221(3): 311-21.
Arai, F., O. Ohneda, et al. (2002). "Mesenchymal stem cells in perichondrium express
activated leukocyte cell adhesion molecule and participate in bone marrow
formation." J Exp Med 195(12): 1549-63.
Baddoo, M., K. Hill, et al. (2003). "Characterization of mesenchymal stem cells
isolated from murine bone marrow by negative selection." J Cell Biochem 89(6):
1235-49.
Brittberg, M., A. Lindahl, et al. (1994). "Treatment of deep cartilage defects in the
knee with autologous chondrocyte transplantation." N Engl J Med 331(14):
889-95.
Deans, R. J. and A. B. Moseley (2000). "Mesenchymal stem cells: biology and potential
clinical uses." Exp Hematol 28(8): 875-84.
DeLise, A. M., L. Fischer, et al. (2000). "Cellular interactions and signaling in cartilage
development." Osteoarthritis Cartilage 8(5): 309-34.
Delise, A. M. and R. S. Tuan (2002). "Analysis of N-cadherin function in limb
mesenchymal chondrogenesis in vitro." Dev Dyn 225(2): 195-204.
Di Nino, D. L., M. L. Crochiere, et al. (2002). "Multiple mechanisms of perichondrial
regulation of cartilage growth." Dev Dyn 225(3): 250-9.
Dowthwaite, G. P., J. C. Bishop, et al. (2004). "The surface of articular cartilage
contains a progenitor cell population." J Cell Sci 117(Pt 6): 889-97.
Duynstee, M. L., H. L. Verwoerd-Verhoef, et al. (2002). "The dual role of
perichondrium in cartilage wound healing." Plast Reconstr Surg 110(4): 1073-9.
Edoff, K. and C. Hildebrand (2003). "Neuropeptide effects on rat chondrocytes and
perichondrial cells in vitro." Neuropeptides 37(5): 316-8.
Fickert, S., J. Fiedler, et al. (2003). "Identification, quantification and isolation of
mesenchymal progenitor cells from osteoarthritic synovium by fluorescence
automated cell sorting." Osteoarthritis Cartilage 11(11): 790-800.
Giurea, A., T. J. Klein, et al. (2003). "Adhesion of perichondrial cells to a polylactic
acid scaffold." J Orthop Res 21(4): 584-9.
Goldring, M. B., K. Tsuchimochi, et al. (2006). "The control of chondrogenesis." J Cell
Biochem 97(1): 33-44.
Grogan, S. P., A. Barbero, et al. (2007). "Identification of markers to characterize and
sort human articular chondrocytes with enhanced in vitro chondrogenic
capacity." Arthritis Rheum 56(2): 586-95.
Han, Y. S., O. S. Bang, et al. (2004). "High dose of glucose promotes chondrogenesis via
PKCalpha and MAPK signaling pathways in chick mesenchymal cells." Cell
Tissue Res 318(3): 571-8.
Hatakeyama, Y., J. Nguyen, et al. (2003). "Smad signaling in mesenchymal and
chondroprogenitor cells." J Bone Joint Surg Am 85-A Suppl 3: 13-8.
Heng, B. C., T. Cao, et al. (2004). "Directing stem cell differentiation into the
chondrogenic lineage in vitro." Stem Cells 22(7): 1152-67.
105
Hering, T. M. (1999). "Regulation of chondrocyte gene expression." Front Biosci 4:
D743-61.
Ito, Y., J. S. Fitzsimmons, et al. (2001). "Localization of chondrocyte precursors in
periosteum." Osteoarthritis Cartilage 9(3): 215-23.
Jian-Wei, X., M. A. Randolph, et al. (2005). "Producing a flexible tissue-engineered
cartilage framework using expanded polytetrafluoroethylene membrane as a
pseudoperichondrium." Plast Reconstr Surg 116(2): 577-89.
Katz, A. J., A. Tholpady, et al. (2005). "Cell surface and transcriptional
characterization of human adipose-derived adherent stromal (hADAS) cells."
Stem Cells 23(3): 412-23.
Lim, S. M., Y. S. Choi, et al. (2005). "Isolation of human periosteum-derived
progenitor cells using immunophenotypes for chondrogenesis." Biotechnol Lett
27(9): 607-11.
Majumdar, M. K., V. Banks, et al. (2000). "Isolation, characterization, and
chondrogenic potential of human bone marrow-derived multipotential stromal
cells." J Cell Physiol 185(1): 98-106.
Miralles, G., R. Baudoin, et al. (2001). "Sodium alginate sponges with or without
sodium hyaluronate: in vitro engineering of cartilage." J Biomed Mater Res
57(2): 268-78.
O'Driscoll, S. W. and J. S. Fitzsimmons (2001). "The role of periosteum in cartilage
repair." Clin Orthop Relat Res(391 Suppl): S190-207.
Ogawa, T., H. Shimokawa, et al. (2003). "Localization and inhibitory effect of basic
fibroblast growth factor on chondrogenesis in cultured mouse mandibular
condyle." J Bone Miner Metab 21(3): 145-53.
Park, J., K. Gelse, et al. (2006). "Transgene-activated mesenchymal cells for articular
cartilage repair: a comparison of primary bone marrow-,
perichondrium/periosteum- and fat-derived cells." J Gene Med 8(1): 112-25.
Pittenger, M. F., A. M. Mackay, et al. (1999). "Multilineage potential of adult human
mesenchymal stem cells." Science 284(5411): 143-7.
Ramalho-Santos, M., S. Yoon, et al. (2002). ""Stemness": transcriptional profiling of
embryonic and adult stem cells." Science 298(5593): 597-600.
Togo, T., A. Utani, et al. (2006). "Identification of cartilage progenitor cells in the adult
ear perichondrium: utilization for cartilage reconstruction." Lab Invest 86(5):
445-57.
van der Kraan, P. M., P. Buma, et al. (2002). "Interaction of chondrocytes,
extracellular matrix and growth factors: relevance for articular cartilage tissue
engineering." Osteoarthritis Cartilage 10(8): 631-7.
Verwoerd-Verhoef, H. L., J. K. Bean, et al. (1998). "Induction in vivo of cartilage
grafts for craniofacial reconstruction." Am J Rhinol 12(1): 27-31.
Watanabe, N., Y. Tezuka, et al. (2003). "Suppression of differentiation and
proliferation of early chondrogenic cells by Notch." J Bone Miner Metab 21(6):
344-52.
White, D. G., H. P. Hershey, et al. (2003). "Functional analysis of fibronectin isoforms
in chondrogenesis: Full-length recombinant mesenchymal fibronectin reduces
spreading and promotes condensation and chondrogenesis of limb
mesenchymal cells." Differentiation 71(4-5): 251-61.
Yang, I. H., S. H. Kim, et al. (2004). "Comparison of phenotypic characterization
between "alginate bead" and "pellet" culture systems as chondrogenic
differentiation models for human mesenchymal stem cells." Yonsei Med J 45(5):
891-900.
106
Youn, I., J. K. Suh, et al. (2005). "Differential phenotypic characteristics of
heterogeneous cell population in the rabbit periosteum." Acta Orthop 76(3):
442-50.