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AN ATTEMPT TO FABRICATE A NOVEL BIOLOGICAL SCAFFOLD WITH

GREEN NANOPARTICLES TO ARTIFICIAL SKIN

Balasundari Ramesh, Sanjay Cherian, Senthil kumar, Alsa , maha lekshmi, Usha,
madhu, and k.M.Cherian:

Abstract
Fabrication of 3D novel scaffolds is very challenging and critical to achieve the appropriate
function for tissue regeneration. This study aims to develop a hybrid biological nano scaffold
using lyophilized amniotic membrane, placenta derived collagen nano- fibers, and green
nanoparticles derived from sea weed Sargassum wightii, with adhesive and healing
properties. Its application as wound dressing material was explored. Moreover, an attempt
was made to develop in vitro artificial skin by seeding the scaffold with cord blood derived
MSCs and transdifferentiating them to keratinocytes and skin fibroblast like cells using
keloid foreskin conditioned media. This in vitro developed culture system can be used for
validating various cosmetic products as the scaffold mimic skin. This novel scaffold can be a
promising dressing material for non healing ulcers and burns.

Introduction:
The use of various biological dressings to cover burn wounds goes back to centuries (Ref). A
variety of techniques have been attempted in order to reduce wound sepsis, and variable
results have been reported. Initially heterografts from different animal sources ranging from
lizard to porcine skins were used (Ref). Amniotic membrane has been used with variable
success as a material for burn injury coverage. Amniotic membrane can be used for
superficial bums, deep burns, after necrectomy, on extensive granulating wound surfaces, on

autografts, in donor regions, and after dermoabrasion. Amniotic membrane is readily


available and does not present immunological problems. It does not cause allergic responses
and reduces water loss. The risk of the transmission of some viral infections is there.
Bacterial examinations performed with burn wounds covered with amniotic membrane
showed low or no bacterial colonization of the burn surface. It is concluded that amniotic
membrane should be more widely used in this particular aspect of burn treatment.
With the healthcare taking priority amongst public, recently there have been great
developments in wound care, with allografts, amniotic membranes, artificial dermis, and
synthetic and semi-synthetic dressing materials playing an important role. In superficial
burns, amniotic membrane adheres after application and remains so until epithelialization is
complete. In deep and deep dermal burns, the burned tissue needs to be removed by total burn
excision or tangential excision, and the wound is covered with an autograft. At the same time,
in burn patients who are not suitable for early excision, the raw surface is covered with an
allograft, amniotic membrane, or other dressing material in order to prevent external
contamination, with loss of fluid and electrolytes. At a later stage the allograft and the
amniotic membrane need to be replaced by an autograft. The tissue engineering laboratory of
our institution have invented a way of processing amniotic membrane so that it makes the
membrane adaptable for adhering to the wound area and thereafter enhancing the growth of
the neighbouring tissue, which further allows the foreign membrane, placed on the wound
area to merge with the neighbouring skin.
The invention in general relates to the use of amniotic membrane as dressing, as graft and for
transplantation. The uniqueness of the processing lies in the decellularisation and cross
linking of the membrane prior to the use.

Green Silver nanoparticles:


The green seaweed S. wightii was collected from Mandapam costal region (7880 E, 9170 N)
in Gulf of manner, the southeast coast of India. The seaweed was washed thoroughly thrice
with distilled water and was shade dried for 10 days. Fine powder of the seaweed was
obtained by using kitchen blender. The seaweed powder was sterilized at 120 C for 15 min,
20 g of powder was taken and mixed with 100 ml of milli Q water and kept in boiling water
bath at 60 C for 20 min. The extracts were filtered with Whatman No. 1 filter paper and
stored in refrigerator at 4 C for further studies. For the biosynthesis of silver nanoparticles, 5
ml of seaweed extract was mixed with 50 ml of AgNO3 solution and incubated at 28 C for 24
h. The bio-reduction of AgNO3 into AgNps can be confirmed visually by the change in color
from colorless to reddish brown.

Discussion:
Human amniotic membrane is one of the most effective biological dressings that is used in
burn treatment. Using human amniotic membrane incorporating silver nitrate gives a better
therapeutic effect than plain amniotic membranes. Silver nitrate incorporated into the

membranes increases their manageability, provides easier application to the burned area and
creates a bactericidal effect, therefore reducing the risk of contamination and infection. One
of the main advantages of wound coverage with amniotic membrane is that it does not appear
to discourage re-epithelization, reduces fluid, protein, heat and energy loss, increases mobility
and most important this may be the ideal wound cover next to the patient's own skin.
Therefore, it is highly recommended for the use of silver nitrate-incorporated amniotic
membrane, since it is readily available and freely obtainable, has low preparation and storage
costs that make it an ideal dressing to use, especially in countries where economic factors
prevent the purchase of other types of dressings
Amniotic membrane was chosen as the best biomaterial for a 3D scaffold. In this
pilot study, different types of scaffold designs were used to transdifferentiate
cord blood derived mesenchymal stem cells to Keratinocytes. The SEM images of
all the scaffold designs showed a good number of healthy proliferating cells
(figure 4.6.1). These cells which were growing in conditioned media for 3 weeks
(figure 4.7.3) were trypsined , their RNA were isolated for reverse transcriptase
PCR and amplified for keratinocyte primers. The agarose gel images with the
positive

bands

for

Keratinocyte

primers(figure

4.7.4)

indicate

that

transdifferentiation of cord blood MSCs must have taken place thus proving that
the biological scaffold designs could be used in future for wound healing and
burn dressing. However a comparative study in the growth and differentiation of
Mesenchymal stem cells on these typical scaffold designs cultured under
conditioned medium could give a better understanding of the properties of each
scaffolds and their specific applications in wound management. In addition
Mesenchymal Stem cells of alternative sources can also be experimented, in a
search for faster cell homing and proliferation at the wound site.

Although wound healing takes place naturally on its own, some of its
complications, such as sepsis, disruption of tissue and skin layer, maggot
formation, and extension of infection to adjacent and interior organs, occur in
major cases. To prevent extensive loss and damage to the tissue, Amniotic
membrane as biological dressings (Fatima et al., 2008) are being used. TGF-b1 is
one of the principal isoforms expressed during wound healing, promote
epithelialisation and Accelerate keratocyte proliferation and myofibroblast
transformation (Carrington et al., 2006). AM has been reported to exert
antimicrobial activity through direct contact with bacteria (Talmi et al., 1991),
suggested to be due to soluble tissue factors, such as defensins (Kjaergaard et
al., 2001) which which ensures septic conditions .

Drug-loaded dressings are prepared by incorporating drugs such as antibacterials


and antibiotics in the dressings. When applied to a wound, drug-loaded dressings
act as a barrier to microorganisms and thus prevent secondary infections, while
stimulating

the

wound-healing

environment

(Gunasekaran

et

al.,

2012).

Therefore, drug-loaded dressings are useful in preventing secondary infections


on the wound and promoting fast wound healing. Amniotic membrane coated
with Mycobacterial drugs could be used as wound dressing for leprosy patients

Lee et al. investigated the effect of silver nanoparticles in dermal contraction


and epidermal reepithelialization during wound healing and suggested that silver
nanoparticles could increase the rate of wound closure. This was achieved, on
one hand, through the promotion of proliferation and migration of keratinocytes.
Hybrid polymer scaffolds coated with silver nano particles could be the future
wound dressing that could be used to treat minor injuries to extensive unhealing
diabetic wounds

The hybrid scaffolds can be tested on an animal models and the efficacy of
differentiated cells in the process of wound healing can be estimated.
Dependingly, easy and sustainable large scale biodegradable hybrid scaffolds
can be fabricated that are cost effective. Similarly, many new ideas can be
imparted to develop better dressing materials that not only help in quick healing
but also prevent scar formation.