Tissue Engineering of Autologous Cartilage Grafts in Three-Dimensional in Vitro Macroaggregate Culture System
In the field of tissue engineering, techniques have been described to generate cartilage tissue with isolated chondrocytes and bioresorbable or nonbioresorbable biomaterials serving as three-dimensional cell carriers. In spite of successful cartilage engineering, problems of uneven degradation of bi...
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Published in | Tissue engineering Vol. 10; no. 11-12; pp. 1695 - 1706 |
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Main Authors | , , , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
Mary Ann Liebert, Inc
01.11.2004
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Subjects | |
Online Access | Get full text |
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Summary: | In the field of tissue engineering, techniques have been described to generate cartilage tissue with
isolated chondrocytes and bioresorbable or nonbioresorbable biomaterials serving as three-dimensional
cell carriers. In spite of successful cartilage engineering, problems of uneven degradation of
biomaterial, and unforeseeable cell-biomaterial interactions remain. This study represents a novel
technique to engineer cartilage by an
in vitro
macroaggregate culture system without the use of biomaterials. Human nasoseptal or auricular chondrocytes were enzymatically isolated and amplified
in conventional monolayer culture before the cells were seeded into a cell culture insert with a tracketched
membrane and cultured
in vitro
for 3 weeks. The new cartilage formed within the
in vitro
macroaggregates was analyzed by histology (toluidine blue, von Kossa-safranin O staining), and
immunohistochemistry (collagen types I, II, V, VI, and X and elastin). The total glycosaminoglycan
(GAG) content of native and engineered auricular as well as nasal cartilage was assayed colorimetrically
in a safranin O assay. The biomechanical properties of engineered cartilage were determined
by biphasic indentation assay. After 3 weeks of
in vitro
culture, nasoseptal and auricular
chondrocytes synthesized new cartilage with the typical appearance of hyaline nasal cartilage and
elastic auricular cartilage. Immunohistochemical staining of cartilage samples showed a characteristic
pattern of staining for collagen antibodies that varied in location and intensity. In all samples,
intense staining for cartilage-specific collagen types I, II, and X was observed. By the use of von
Kossa-safranin O staining a few positive patches-a possible sign of beginning mineralization within
the engineered cartilages-were detected. The unique pattern for nasoseptal cartilage is intense
staining for type V collagen, whereas auricular cartilage is only weakly positive for collagen types
V and VI. Engineered nasal and auricular macroaggregates were negative for anti-elastin antibody
(interterritorially). The measurement of total GAG content demonstrated higher GAG content for
reformed nasoseptal cartilage compared with elastic auricular cartilage. However, the total GAG
content of engineered macroaggregates was lower than that of native cartilage. In spite of the mechanical
stability of the auricular macroaggregates, there was no equilibrium of indentation. The
histomorphological and immunohistochemical results demonstrate successful cartilage engineering
without the use of biomaterials, and identify characteristics unique to hyaline as well as elastic cartilage. The GAG content of engineered cartilage was lower than in native cartilage and the biomechanical
properties were not determinable by indentation assay. This study illustrates a novel in
vitro macroaggregate culture system as a promising technique for tissue engineering of cartilage
grafts. Further long-term
in vitro
and
in vivo
studies must be done before this method can be applied to reconstructive surgery of the nose or auricle. |
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Bibliography: | ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2 |
ISSN: | 1076-3279 1557-8690 |
DOI: | 10.1089/ten.2004.10.1695 |