Regeneration
of large-sized acute and chronic wounds provoked by
severe burns and diabetes is a major concern worldwide. The availability
of immunocompatible matrix with a wide range of regenerative medical
applications, more specifically, for nonhealing chronic wounds is
an unmet clinical need. Extrapolating the in vitro tissue engineering knowledge for in vivo guided
wound regeneration could be a meaningful approach. This study aimed
to develop a completely human-derived and minimally immune-responsive
scaffold comprising of acellular amniotic membrane (AM), fibrin (FIB)
and hyaluronic acid (HA), termed AMFIBHA. The potential for in vivo guidance of skin regeneration was validated through in vitro dermal tissue assembly on the combination scaffold
by growing human fibroblasts, differentiated from human adipose tissue-derived
mesenchymal stem cells (hADMSCs). An effective method was standardized
for obtaining decellularized amnion (dAM) for assuring better immuno-compatibility.
The biochemical stability of dAM upon plasma sterilization (pdAM)
confirms its suitability for both in vitro and in vivo tissue engineering. The problem of poor handling
characteristics was solved by combining the dried dAM with fibrin
derived from a clinically used fibrin sealant kit. An additional constituent
HA, derived from human umbilical cord tissue, imparts the required
water absorption and retention property for better cell migration
and growth. Post sterilization, the combination scaffold AMFIBHA demonstrated
hemo-/cytocompatibility, confirming the absence of detergent residuals.
Upon long-term (20 days/40 days) culture of hADMSC-derived fibroblasts,
the suppleness of generated tissue was established by demonstrating
regulated deposition of collagen, elastin, and glycosaminoglycans
using both qualitative and quantitative measurements. Regulated expressions
of transforming growth factors-beta 1 (TGF-β1) & TGF-β3,
alpha smooth muscle actin (α-SMA), fibrillin-1, collagen subtypes,
and elastin suggest non-fibrotic fibroblast phenotype, which could
be an effect of microenvironment endowed by the AM, FIB, and HA. In
burn wound model experiments, immune response to cellular AM was prominent
as compared to untreated/sham control wounds and decellularized AM-treated
and AMFIBHA-treated wounds, ensuring biocompatibility. Wound regeneration
with complete epithelialization, angiogenesis, development of rete
pegs, and other skin appendages were clearly visualized in 28 days
after treating large-sized (4 × 4 cm2), debrided,
full-thickness third-degree burn wounds, indicating guided wound regeneration
potential of AMFIBHA dermal substitute.
