Biomedical Engineering Reference
In-Depth Information
10.3.1 Structural analyses
Collagen represents the major component of most ECMs, including that of dermis
where collagen type I is the major component. Besides providing the ECM with its
primary structural support, it plays a key role in the overall organization of other
ECM molecules including other collagens, proteoglycans, laminin, as well as
numerous others. Collagen contains specific recognition sequences for the cell-
surface integrins that regulate cellular activities such as adhesion, migration, and
differentiation. Further, it binds soluble proteins including growth factors which
are directly involved in the regulation of cell growth. In order to retain these
functions, a processed acellular RTM should possess a matrix composition as close
to that of the native ECM as possible.
Collagen accounts for greater than 75% of the dry mass of RTM as determined
by hydroxyproline content (Table 10.1). Total hydroxyproline is variably distrib-
uted throughout acid soluble, pepsin soluble and pepsin insoluble compartments.
This distribution is typical of normal dermal tissue and reflects the relative
amounts of newly synthesized, mature and mature cross-linked pools of collagen.
This data is consistent with independent analysis of RTM (Derwin
et al
., 2006) and
fresh dermal samples (Poulsen and Cramers, 1982). Soluble collagen prepared
following pepsin digestion reveals collagen type I and III to be the predominant
forms existing as single chains as well as higher order structures resulting from
intra-molecular cross-linked dimeric (
) collagen polypeptides
(
Fig. 10.3(a
)). The molecular complexity of the collagen matrix is further shown
by treating pepsin insoluble fractions with cyanogen bromide. The intricate
banding pattern observed reveals the expected
β
) and trimeric (
γ
1(III) peptide
fragments characteristic of a mature collagen ECM (Fig. 10.3(b)). Taken together,
these data demonstrate that the collagen organization in AlloDerm RTM is typical
of a mature dermal ECM complete with the levels of structural complexity
provided for by intra- and inter-molecular cross-linking.
In addition to collagen, the RTM retains the important proteoglycans decorin,
α
1(I),
α
2(I) and
α
Table 10.1
AlloDerm RTM collagen content. Collagen from AlloDerm was
extracted through sequential treatments with 0.5 M acetic acid and pepsin.
Hydroxyproline was quantified in each extract and used to estimate total
collagen content assuming that the hydroxyproline content of the collagen I and
III enriched ECM was 12.5%. Each value represents the mean ± SD (standard
deviation) of six different donor lots
Hydroxyproline
Collagen
(µg/mg dry weight)
(µg/mg dry weight)
Total collagen
97.5 ± 7.4
780.0 ± 59.5
Acid extract
10.2 ± 6.6
81.7 ± 52.5
Pepsin extract
48.2 ± 17.1
385.3 ± 137.0
Pepsin insoluble
39.1 ± 22.6
313.0 ± 181.2
