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.