Biomaterials 23 (2002) 3183–3192
Crosslinked type II collagen matrices: preparation, characterization,
and potential for cartilage engineering
, P.M. van der Kraan
, T. Hafmans
, J. Kamp
, P. Buma
, J.L.C. van Susante
W.B. van den Berg
, J.H. Veerkamp
, T.H. van Kuppevelt
Department of Biochemistry 194, University Medical Centre Nijmegen, NCMLS, Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands
Department of Rheumatology, University Medical Centre Nijmegen, NCMLS, Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands
Department of Orthopedics, University Medical Centre Nijmegen, NCMLS, Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands
Received 13 June 2001; accepted 14 February 2002
The limited intrinsic repair capacity of articular cartilage has stimulated continuing efforts to develop tissue engineered analogues.
Matrices composed of type II collagen and chondroitin sulfate (CS), the major constituents of hyaline cartilage, may create an
appropriate environment for the generation of cartilage-like tissue. In this study, we prepared, characterized, and evaluated type II
collagen matrices with and without CS. Type II collagen matrices were prepared using puriﬁed, pepsin-treated, type II collagen.
Techniques applied to prepare type I collagen matrices were found unsuitable for type II collagen. Crosslinking of collagen and
covalent attachment of CS was performed using 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide. Porous matrices were prepared
by freezing and lyophilization, and their physico-chemical characteristics (degree of crosslinking, denaturing temperature,
collagenase-resistance, amount of CS incorporated) established. Matrices were evaluated for their capacity to sustain chondrocyte
proliferation and differentiation in vitro. After 7 d of culture, chondrocytes were mainly located at the periphery of the matrices. In
contrast to type I collagen, type II collagen supported the distribution of cells throughout the matrix. After 14 d of culture, matrices
were surfaced with a cartilagenous-like layer, and occasionally clusters of chondrocytes were present inside the matrix.
Chondrocytes proliferated and differentiated as indicated by biochemical analyses, ultrastructural observations, and reverse
transcriptase PCR for collagen types I, II and X. No major differences were observed with respect to the presence or absence of CS
in the matrices. r 2002 Published by Elsevier Science Ltd.
Keywords: Type II collagen; Cartilage; Matrix; Tissue engineering
Articular cartilage has a limited potential to restore
damage to the joint surfaces. This is related to the low
mitotic activity of chondrocytes and the avascular
nature of the tissue. Strategies like abrasion arthro-
plasty, excission, or the use of perichondreal or
periosteal autografts, generally result in the formation
of mechanically inferior ﬁbro-cartilagenous tissue which
may induce osteoarthritis [1,2]. Tissue engineering of
cartilage, in which biocompatible matrices are cultured
with chondrocytes to prepare transplantable hyaline-like
tissue, may provide a more suitable alternative [3–5].
Various insoluble biodegradable materials have been
exploited as a carrier for chondrocytes. These include
demineralized bone , hydroxyapatite composites ,
polylactic acid and polyglycolic acid [8,9], collagen
[1,4,10], and hyaluronate . The physico-chemical
characteristics of the materials considerably inﬂuence
the stability of the chondrocyte phenotype. Preservation
of the chondrocytic phenotype is essential for the
generation and maintenance of hyaline-like cartilage.
Matrices which mimic articular cartilage biochemically,
may add in preserving the differentiated state of the cells
The function of native articular cartilage as a load-
bearing tissue is based on the unique structure of an
organized extracellular network mainly composed of
*Corresponding author. Tel.: +31-24-361-6759; fax: +31-24-354-
E-mail address: firstname.lastname@example.org
(T.H. van Kuppevelt).
0142-9612/02/$ - see front matter r 2002 Published by Elsevier Science Ltd.
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