Smart Bio-Polymeric Matrix for Accelerated Wound Healing and Tissue Regeneration

Research Article

Austin J Biomed Eng. 2021; 6(1): 1045.

Smart Bio-Polymeric Matrix for Accelerated Wound Healing and Tissue Regeneration

Dhasmana A1,2*, Singh L3 and Malik S4

1Department of Polymer & Process Engineering, Indian Institute of Technology Roorkee, India

2Department of Biotechnology, School of Applied and Life Sciences, Uttaranchal University, India

3Department of Pharmacology, Kharvel Subharti College of Pharmacy, India

4Department of Biotechnology, Amity Institute of Biotechnology, Amity University Jharkhand, Ranchi, Jharkhand, India

*Corresponding author: Archna Dhasmana, Department of Polymer & Process Engineering, Indian Institute of Technology Roorkee, India; Department of Biotechnology, School of Applied and Life Sciences, Uttaranchal University, Dehradun, Uttarkhand-248007, India

Received: September 01, 2021; Accepted: October 08, 2021; Published: October 15, 2021


Traditionally in Chinese medicine, animal sources and their by-products widely used for surgical and healing purposes. Eggshell Membrane (ESM) has been potentially used as grafting material for wound covering and healing due to its fibrous mesh enriched with collagen and glycoproteins. However, the fragile nature of ESM limits applicability for small and superficial wounds. Therefore, acellular matrix/scaffold fabricated from the allogeneic or xenogeneic tissues widely used as grafting material for the repairing and regeneration tissue. Here, we modified an acellular scaffold in different concentrations of ESM protein (ESMP)-5, 7.5 and 10%, and studied synergistic effect for intensifying the tissue healing and regeneration process. Modified Scaffolds (ESMP-AGDS) were evaluated for tissue regeneration by subjecting it through physicochemical and biological characterization i.e., biochemical assay, FTIR, FESEM, in vitro and in vivo analysis. The study revealed proper interaction between the ESMP and acellular matrix 3D interconnected pores structure (57.69±15.65 μm) with good porosity (60.56±9.78%) for better cell and nutrient diffusion. In vitro studies revealed good biodegradability and biocompatibility of modified scaffold with 3T3 mouse fibroblast cells. At the very least concentration of 5% ESMP, acellular matrix showed excellent proliferation and attachment of fibroblast with the progression of time. Similarly, in vivo study showed a full-thickness excisional wound in the albino mice model healed within 14 days along with hair follicles regenerated neo-skin tissue, without any immunogenicity and inflammation. Thus, the study confirmed ESMP and acellular matrix synergistic effect results in a cost-effective, biodegradable, biocompatible smart material potentially applicable for tissue regeneration.

Keywords: Eggshell membrane; Cadaver goat-skin; Acellular scaffold; Skin; Wound healing; Tissue engineering


Eggshell Membrane (ESM) is a natural biopolymer similar to native ECM, which is widely used as biotemplate for wound healing applications [1,2]. ESM mainly consist of 80-85% organic matter (70- 75% are glycoproteins i.e., hyaluronic acid, sialic acid, GAGs, and 10% collagen I, V and X), which aid in would healing process3-5. ESM protein (ESMP) is biocompatible and biodegradable. Besides, it has anti-ageing, antimicrobial and anti-inflammatory property [3,6,7]. The potential value of ESMP is huge, especially in biomedical applications, viz., skin grafts, wound healing, plastic surgery, dental implants, angioplasty sleeves, cornea repair, treatment of osteoporosis as well as food casings and film emulsions [2]. Several researchers demonstrated the ESM as an ideal scaffold [8-10]. Yang and co-workers experimentally proved that ESM shows antiinflammatory response and significantly better wound healing rate as compared to commercially available skin-substitute (BiobraneTM) [8]. Thus ESMP or ESM, containing various wound healing growth factors, is a potential biomaterial for wound healing, and an ESM or ESMP based skin-graft will be a promising skin substitute [3,10- 13]. However, the ESM is very thin, difficult to handle and lacks in flexibility and durability, which limits its applicability for large area wound coverage and treating full thickness wounds. On the other hand, skin grafts can easily be developed by decellularizing cadaveric allogeneic and xenogeneic skin, which can be applied for large area wound coverage and treating full thickness wounds. Some acellular skin grafts are already available in the market, e.g., OrCel®, Apligraf®, Matriderm®, PermacolTM and Oasis® [14-17]. During decellularization of native tissue, there is a loss of biomolecules to some extent, which results in loss of bioactivity of the acellular skin graft-reducing the overall wound healing potential of the graft [16]. To overcome the above limitation and to enhance the graft’s bioactivity, many scientists have designed growth-factor incorporated grafts [18-21]. But, the high cost of growth-factors limits its applicability for tissue engineering applications. The tissue sources, used to fabricate largesized acellular skin grafts, include mainly porcine, bovine and caprine (goat) [20-25]. Among these three, goat tissue is less immunogenic and less susceptible to viruses and prions, and had not yet been reported about transmission of any cattle disease to human [26,27]. Therefore, skin graft from cadaveric goat tissue will be a better and safer alternative in comparison to the porcine or bovine-based graft. Combination of ESMP with acellular ECM rich graft (ESMP-AGDS) will synergistically accelerate the wound healing and overcome the limitations of the ESM and acellular grafts. Moreover, the graft can be used for repairing/regenerating full thickness wound of large area. The ESMP-AGDS will be cost-effective as both the ESMP and AGDS is cheap and easily available. Therefore, in this study, we want develop an ESMP-AGDS hybrid skin graft which will be a cost-effective solution for rapid repairing/regenerating full thickness wound of large area.



Raw eggshells were collected from the Campus’s Hostel mess, under aseptic condition. For the fabrication of acellular dermal graft, fresh cadaver goat-skin was brought from slaughterhouse. All the reagents/chemicals of animal grade (nutrient culture media, buffers, enzymes, antibiotics) for graft fabrication and characterization were purchased from Himedia, India. Other solvents-acetic acid, dimethyl sulphoxide, chloroform, methanol, ethanol, phenol, isoamyl alcohol, formaldehyde solution were purchased from Sigma Aldrich.

Preparation and characterization of soluble ESMP

Raw membrane-bound eggshells were collected from mess were immediately cleaned them with deionized water. Cleaned eggshell immersed in 70% aqueous Acetic Acid (CH3COOH) for 24h under continues mixing on magnetic stirrer to dissolve residual eggshell. Separated ESM were rinsed with deionized water repeatedly and dried in the oven at 50oC. ESM was further ground to prepare fine powder using cryomill. After that soluble ESMP was prepared following the protocol as explained earlier by Strohbehn and coworkers [28]. Briefly, ESM powder was dissolved in alkaline solution of 12% Sodium Hydroxide (NaOH) for 12h at 37oC. Subsequently, dissolved ESM solution was centrifuged to precipitate the remaining calcium carbonate (CaCO3) and neutralize separated the supernatant containing soluble ESMP (pH 7) with 10% CH3COOH solution at 4oC. The ESMP solution was freeze dried and molecular weight was determined by SLS by following the protocol explained earlier by other researchers [21,29]. Briefly, the molecular weight of soluble ESMP was determined by Static Light Scattering (SLS) on a Wyatt EOS (λ) at 682nm wavelength, multiangle light scattering detector, operated in batch mode with water as solvent at room temperature. ESMP solution serial diluted in different concentration and all the samples were syringe filtered (PTFE filter, 0.22μm) before analyzing and determined using high sensitivity of the detector. The refractive index increment was measured with differential refractometer DnDc- 2010 (WGE Dr. Bures) and Differential refractometer Software Ver. 3.24 (Brookhaven Instruments). SLS data were obtained by using a self-built of the laser scattering system. By using Zimm plot (plotted the intensity of the scattered light against the scattering angle) the weight-average Molecular Weight (Mw), Radius of Gyration (Rg), and second virial coefficient (A2) of the prepared sample was determined.

Fabrication of hybrid ESMP-AGDS

Acellular Goat-Dermal Scaffold (AGDS) was fabricated using physio-chemoenzymatic methods [21]. Briefly, after removal of hair, epidermis and subcutaneous tissue from the native cadaver goatskin, dermis was subjected to decellularization. The native dermal tissue was placed in the solution containing 0.25% Trypsin-EDTA in 1X PBS and 1% antibiotic (antimycotic) for 12h at 25oC. The tissue sections were further treated with 0.1% SDS in 1X PBS for 6h at 37oC, subsequently followed by agitating in enzymatic solution- RNase (20μg/ml) and DNase (0.2mg/ml) solution in 1:1 ratio for 24h at 37 oC. Finally, thoroughly washed the obtained acellular dermal scaffold with 1X PBS by gently shaking and then lyophilized it. Dried scaffold pieces were sterilized with 70% ethanol for 30min followed by UV (λ=260nm) treatment for 4h. ESMP modified acellular scaffold (ESMP-AGDS) pieces were fabricated by dip coating or soaking them in different concentration of ESMP solution- 5%, 7.5 % and 10% for 30 min and subsequently divided them as sample 1, 2 and 3 respectively (Figure 1) . After ESMP coating modified acellular scaffold were then removed from the respective solutions and lyophilized. All the lyophilized hybrid ESMP-AGDS samples were stored and characterized for wound healing and tissue regeneration.