Biomedical Engineering Reference
In-Depth Information
tion (Powell et al. 2008). In a variant of the original procedure cultured epithelial
autografts (rather than disaggregated KC) were grafted onto DRT. Although newly
synthesized epidermis and dermis showed almost normal histological appearance,
formation of BM proteins was delayed; however, at a later time of observation, the
newly synthesized skin was very durable and the two tissue layers could not be
separated (stripped) even though they still lacked certain BM proteins (Matsumura
et al. 2013).
5.4.3
In Vitro to In Vivo Synthetic Routes
In this section we describe processes that include a significant component of in
vitro processing prior to an attempt to induce regeneration in vivo, i.e., processes
in which the investigators attempted to synthesize a significant fraction of skin tis-
sues in cell culture prior to implantation at the appropriate anatomical site. The
various protocols are presented in approximate order of increasing complexity of
the implant, which often corresponds to increased extent of in vitro processing. In
the preceding sections an ECM analog (DRT) was prepared in a cell-free state and
was optionally seeded with KC; however, cell culture was rarely used and was not
critical during the preparation process in protocols described above.
A protocol based on in vitro culture of KC into DRT prior to grafting also led
to simultaneous synthesis of an epidermis and a dermis. Synthesis of an epidermis
was observed after day 11 (Boyce and Hansbrough 1988; Boyce et al. 1988; Cooper
and Hansbrough 1991; Cooper et al. 1993). The epidermis comprised about ten
stratified epidermal layers, the outermost among them being cornified (keratinized);
frequent desmosomal connections between cell layers were also observed (Boyce
and Hansbrough 1988). In a related study, DRT was modified prior to grafting by
laminating with a layer of a nonporous version of DRT (laminated DRT) in order to
localize the keratinocyte culture on one surface of the porous matrix, thereby sepa-
rating KC from fibroblasts that were also cultured (Boyce et al. 1988). Laminated
DRT was cultured with KC and fibroblasts in vitro to yield a “composite graft”
(Cooper and Hansbrough 1991; Cooper et al. 1993). The epidermis synthesized in
vitro using the composite graft protocol comprised stratified, cohesive sheets of
epithelium, approximately four to five cell layers thick (Cooper et al. 1993).
Use of the “composite graft” in a clinical study focused on covering defects in
burn patients. The defects were prepared by excision to subcutaneous fat or fascia
(dermis-free defects). Prior to placing on the defect, the composite graft consisted
of a layer of cultured autologous KC on the laminated surface of the DRT sheet; au-
tologous fibroblasts had been cultured inside the DRT pores on the opposite surface
of the graft. Several days after grafting, a fully stratified epidermis with a stratum
corneum had been synthesized. The components of the BM, including hemidesmo-
somes, a continuous lamina lucida and lamina densa, and anchoring fibrils, were
synthesized; rete ridges were also formed. Histological evidence for synthesis of
a dermis was presented; skin appendages did not form (Hansbrough et al. 1989).
