INTRODUCTION:

Wound healing is a complicated and regulated sequence of several well-coordinated biochemical and cellular phenomena to restore the integrity of the skin. Wound healing involves three main overlapping phases: inflammation, proliferation and maturation¹. In patients who have a large area of skin affected and those suffering from chronic wounds, healing process does not progress in a timely and orderly manner. In such cases, healing stall in the inflammation phase. And it is characterized by excessive levels of pro inflammatory cytokines, proteases, senescent cells as well as existence of persistent infection². Hence the therapeutic intervention is to reduce inflammation and initiate tissue regeneration in a faster and effective manner. Currently, most therapies for treating the wounds are symptomatic using analgesics, anti –inflammatory agents along with antibiotics to prevent infection and do not promote tissue regeneration. It has become a major challenge to health care systems worldwide^3,4.

Tissue engineering is an emerging interdisciplinary field that applies principles of both biology and engineering. The basic concept of tissue engineering lies in its ability to utilize and extensively exploit living cells. It can be used to facilitate the growth of damaged/diseased tissues by applying a combination of biomaterials and bioactive molecules. Among them, biodegradable polymeric scaffolds have received much attention as they offer temporal and spatial environment for tissue growth⁵.

SCAFFOLDS:

Scaffolds are 3D artificial porous structure in which extracellular matrix, drugs and growth factors combine to regenerate tissue. Scaffolds serve as a platform for cellular localisation, adhesion and differentiation as well as a guide for the development of new functional tissues. 3D scaffold material assist in mimicking natural extracellular material and accommodates the cells in their natural milieu. Extracellular matrix (ECM) consists of various growth factors, characteristic proteins and glycoproteins. It play a pivotal role in controlling cell adhesion, migration, proliferation, and differentiation. Typically scaffolds consist of polymers, bio-ceramics, and hybrid materials. Success of scaffolds depends on finding appropriate polymer materials. Scaffolds prepared with singe polymer shows poor physicochemical properties which find difficulty in handling, storage and application⁶.

Table 1: Currently commercially available scaffolds and its polymers.

Brand name	Scaffold material
Apligraf	Bovine collagen
Orcel	Bovine collagen sponge
Tissue tech autograft system	Hyaluronic acid membrane
Laserskin or Vivoderm	Hyaluronic acid membrane
Bioseed S	Fibrin sealant
Hyalograft	Hyaluronic acid membrane

Scaffolds can be manufactured using a number of approaches including lyophilisation, leaching, phase separation, electrospinning, stereolithography and 3D printing. An ideal scaffold should actively direct tissue formation, prevent scarring and finally materials should be degraded naturally during and after healing process ⁷.

ADVANTAGES:

1. Promote cell adhesion and ECM deposition

2. Promote cell – biomaterial interactions

3. Permit sufficient transport of gases, nutrients and regulatory factors.

4. Biodegradable, allow cell survival, proliferation and differentiation^8,9.

Table 2: Natural biomaterials intended for skin substitution which are currently under investigation.

Brand Name	Scaffold material
Permaderm /Cincinnati shriners skin substitute	Bovine collagen
Acudress	Fibrin substrate
Allox	Fibrin substrate
Cyzact (ICX –PRO) [ chronic wound repair]	Fibrin gel
Biodegradable polyurethane microfibers	Biodegradable polyurethane microfibres
Silk fibroin and alginate	Silk fbroin /alginate blended sponge
Bovine collagen cross linked with microbial transglutaminase	Bovine collagen cross linked with microbial transglutaminase
Collatamp	Multilayer bovine collagen matrix

Novel biodegradable scaffolds can be manufactured using natural extracts and polymers for enhanced wound healing and better tissue regeneration. Plant extracts and their phytoconstituents are promising wound healing agents since ancient times. These natural extracts can be preferred over synthetic products due to high efficacy, low- cost and limited adverse effects. But its use has fallen owing to its poor bioavailability and stability issues. Natural extracts are incorporated in biological based polymers that aid in wound healing and tissue regenerative process. scaffolds with combination of natural extracts and polymers can enhance skin regeneration while limitations of individual components can be overcome simultaneously ¹⁰ The advantageous efficacy of the medicinal plants incorporated scaffolds stimulated the authors to provide a comprehensive review of the plant based scaffolds obtained by different methods and their effective role in wound healing.

Aloe Vera derived nanofibrous scaffolds:

Aloe Vera which is rich in components like Mannose -6- phosphate and acemannan, have been used for wounds, burns, skin inflammation, insects stings since ancient times. Antioxidant, anti- inflammatory, antimicrobial and immunomodulatory properties highlight its biomedical applications. It mediate cell signalling pathway for proliferation of fibroblast. It promotes epithelization and collagen synthesis for effective wound healing^11,12,13. Silk fibroin, a natural protein, obtained from silk worm (Bombyx morri) has proteins such as sericin and fibroin. Fibroin has higher elastic strength, strong mechanical properties, biocompatibility and slow degradability and hence being used for biomedical applications. Additionally fibroin can mimic extracellular matrix and efficiently support cell attachment and proliferation of fibroblasts^14,15. Thus aloe vera and silk fibroin is an attractive material for tissue engineering. Scaffolds can be prepared by combining their unique properties using electro-spinning technique. The nanofibrous scaffolds shows finer morphology expressing amino and esteric groups with improved hydrophilicity and favourable tensile strength of 116% which is desirable for skin tissue engineering. Biological studies show that favourable fibroblast proliferation compared to control, which almost increased linearly by 34.6% on day 3 and 97.86% on day 9 with higher expression of CMFDA (5-chloromethylfluroescin diacetate), collagen, F-actin properties. Accordingly fibroblast adopt an elongated morphology stimulating large disruptions in ECM at the wound site. Human fibroblast cells adhere and proliferate well on these matrixes owing to different components present in aloe Vera (promoting cell proliferation) and silk fibroin (providing stable environment) and thus promote regenerative process of human fibroblast cell which is suitable for dermal application¹⁶. Suganya et al., 2014 synthesized nanoscale fibre scaffolds using electrospinning technique with polycaprolactone (PCL)containing lyophilized powder (5% and 10%) aloe vera (AV). It is compared with PCL /Collagen blend. PCL –AV 10% nanofibre scaffold shows finer fibre morphology with improved hydrophilic properties and tensile strength. These matrixes favour cell proliferation compared to other scaffolds. CMFDA dye expression, secretion of collagen and F-actin expressions were also high. Studies conclude that PCL –AV 10% scaffold in comparison with scaffolds of PCL /collagen will serve as a better tissue engineering scaffold because of relatively low cost, natural origin and water retention properties¹⁷. Jouybar. A et al., 2017 investigate the potential capacity of poly–L-lactic acid (PLLA) nanofibrous scaffolds coated with aloe vera gel for wound dressing applications. Electrospinning method was used for preparing PLLA nanofibers and its influence on wound healing was investigated with/without aloe vera gel. SEM and MTT assays confirm the nanometer size and biocompatibility of nanofibrous scaffolds. Macroscopic evaluation shows that gel coated scaffolds enhance the wound healing process compared with other groups. It can accelerate wound healing by shortening inflammatory phase, increasing fibroblast maturation, tissue proliferation, collagen formation and epithelium production¹⁸.

Frutalin scaffolds:

Plant lectins are carbohydrate binding proteins which can interact with cell surfaces to start anti-inflammatory pathways and immune-modulatory functions. Frutalin is tetrameric lectins isolated from Artocarpus incisa seeds popularly known as breadfruit lectin. Frutalin are proved to be most effective in fibroblast migration. Frutalin have several other biological activities. It is cytotoxic to tumour cells, chemotactic for rats and human neutrophils and is a potent mitogen of human lymphocytes. It is an inhibitor of orofacial noception in acute and chronic pain mediated by TRPA1, TRPV1, TRPM8 receptors. It is having antidepressant like actions and protect against ethanol induced gastric lesions^19,20. Polysaccharides including galactomannans, hemicellulose are found mainly in endosperm of legume seeds. Galactomannans are polymers with a β-(1,4)-D mannose backbone and D galactose side units linked α-(1,6). These polymer containing systems are now receiving wider global interest owing to its structural features, rheology and negligible biological toxicity²¹. De Sousa et al., 2019 incorporated frutalin into an immobilisation matrix of galactomannan. These lectin scaffolds have an average membrane porosity of 100µm which is sufficient to ensure water vapour permeability. It enhances angiogenesis, fibroblast and keratinocyte proliferation. Frutalin/galactomannan hybrid scaffold formulation can be an effective health care for wounds, burns in surgical patients at lower cost than current treatment plans²².

Curcumin loaded collagen scaffolds:

Curcumin, also known as diferuloylmethane, is a naturally derived active agent of perennial herb Curcuma longa. Its anti- inflammatory, antimicrobial, antioxidant properties significantly improve wound healing and protect tissues from oxidative damage. However low bioavailability and permeability hinders its application^23,24,25. Chitosan is one of the most plentiful polysaccharides existing in nature.it is a deacetylated derivative of chitin. It is biologically renewable, biodegradable, bio-compatible, antimicrobial, non-antigenic, non-toxic and bio functional. Collagen is the most abundant protein that has been used extensively for scaffold fabrication. It is degraded by the enzyme collagenase and leads to the formation of gelatinized fragments. These fragments are cleaved by several non-specific proteases. It results in cellular infiltration of fibroblast and can synthesize new extracellular matrix components for tissue regeneration. One of the disadvantage of collagen to be utilized as a scaffold is its biological instability. Chemical cross linking or hybridisation with natural polysaccharides are regarded as efficient methods to reduce easy degradation of collagen and to enhance its weak mechanical property. Curcumin chitosan nanoparticles impregnated into collagen alginate scaffolds can be prepared by emulsification method. These nanofibrous scaffolds shows good water uptake, biocompatibility and sustained release of drug. In vivo wound closure analysis reveals that nanohybrid scaffold treated wounds contracted much faster and complete epithelization with granulation tissue formation occurs. Hence syngersitic combination of curcumin, chitosan, collagen, alginate have better wound healing capability²⁶. Rezaei et al., 2018 synthesized and characterized scaffolds with curcumin nanoparticles and biocompatible polymers namely collagen (C) and chitosan (Ch). Research conclude that scaffolds with lower collagen content have more homogenous pore sizes. Curcumin nanoparticles incorporated C1Ch2 scaffolds demonstrate best physiochemical characteristics. Both lower content of collagen and higher content of chitosan in the scaffolds is an ideal parameter in the wound healing process. Invivo and invitro wound healing studies present a great closure of wounds with enhanced histological parameters in the wounds treated with CN –C1Ch2 scaffolds compared to other scaffolds. Thus the results indicate that curcumin nanoparticle incorporated collagen/chitosan scaffolds could significantly improve wound healing²⁷.

Centella asiatica impregnate dermal scaffolds:

Centella asiatica extracts are rich in tannins and phenolic derivatives and own antioxidant and wound healing property. However delivery of extract becomes a matter of concern due to its poor bioavailability and stability²⁸. Centella asiatica extract incorporated collagen scaffold ensures better wound healing by acting as a physical support for cellular proliferation. Collagen acts as a wound healing agent possessing biodegradable and biocompatible properties. Studies on male whistar rats with 1.5% centella asiatica extract incorporated collagen scaffolds reveals the high amount of hydroxylproline content as compared to marketed formulation. Histopathological examination shows more production of collagen and rise in fibroblasts resulting in epithelial gap reduction²⁹.

Silver nanoparticle loaded collagen scaffolds:

Silver nanoparticle is an effective antimicrobial agent and is a promising method for burn treatment³⁰. Silver nanoparticle loaded collagen chitosan scaffolds enhance wound healing by regulating fibroblast migration and macrophage activation. Combination of silver nanoparticle and dermal scaffold serves as a carrier for particles to preserve their insitu functions. It empower the scaffolds with ability to stimulate immune response, cell migration and achieve a rapid regeneration and high quality repair. Wound healing studies on Sprague Dawley rats showed increased levels of pro-inflammatory agents, scar related factors, α- SMA factors. On day 60 scaffold’s regenerated skin had a similar structure to normal skin³¹.

Lentil seeds loaded bioscaffolds:

Lentil seeds are edible legume seeds of lens esculenta and lens culinaris. Lentil seed have good free radical scavenging activity³². Lentil seed extracts loaded chitosan/sodium alginate bioscaffolds have significant mechanical properties. Studies on albino rats confirms significant difference in antibacterial and wound healing activity when compared to blank composite scaffolds. These are applied externally on wounds without any need of removal after healing. Moreover, as the concentration of sodium alginate increases the mechanical properties of scaffolds were remarkably improved³³.

Lithospermii radix extract containing nanofibre scaffolds:

Lithospermii radix (LR) extract were obtained from root of Lithospermum erythrorhizon and have been practicised for treating wounds for hundreds of years. The main ingredients of Lithospermii radix (LR) extract are shikonin, isobutyl shikonin, β-hydroxyl–isovaleryl–shikonin, α-methyl –n-butyl –shikonin and quinones. Shikonin, a derivative of napthaquinone have several pharmacological properties including antibacterial, wound healing and anti-inflammatory effects. The wound healing effects of shikonin is related to epithelial mesenchymal transition (EMT)which is a cell trans differentiation process important for wound healing. TGF–β signaling play an important role in EMT³⁴. Gelatin, a collagen derivative contains Arg-Gly-Asp like aminoacid sequences that promote cell adhesion and migration³⁵. Lithospermii radix extract were added to biocompatible gelatin solution. Various ratios of gelatin and collagen were added onto chitosan scaffolds to manufacture bilayer scaffolds. Porous chitosan scaffolds with a high swelling ratio show exudate absorption ability. In vivo wound healing studies in Sprague Dawley rats conclude that CGF9IL bilayer scaffolds give highest wound recovery rate. CGF91 gelatin nanofibers electrospun at a constant flow rate 0.1ml/hour and voltage of 20Kv display optimal characteristics necessary for cell attachment and skin tissue regeneration³⁶.

Dual layered 3D nanofibrous scaffold:

Fibrin is a good hemostatic agent that aids in tissue rebuilding, absorption of exudates and act as one of the key components in biomaterial development³⁷. Keratin, an insoluble protein with higher cysteine residues have a significant role in physical and biological property of biomaterial³⁸. Poly β–hydroxybutyric acid is a highly biocompatible non -toxic hydrophobic polymer³⁹. Keratin, fibrin, gelatin 3 D sponge loaded with muciprocin was prepared using freeze drying method with naturally derived materials of bovine origin. Poly β-hydroxy butyric acid, gelatin solution loaded with curcumin were electrospun to get dual drug loaded dual layered nanofibrous spongy 3D scaffold. It promotes gaseous exchange, absorption of exudates and facilitates interaction with host tissue. In-vivo assessment using silicone splint animal model shows increased collagen deposition and granulation tissue formation⁴⁰.

Calendula officinalis loaded nanocomposite scaffolds:

Calendula officinalis is commonly used for skin wounds treatment⁴¹. Among natural polymers, gum Arabic (acacia gum) is a polysaccharide that have been generally used for tissue engineering scaffolds. It has many advantages in repairing and regenerating the skin⁴². Calendula officinalis loaded PCL gum arabic nanofibrous scaffolds can be prepared by electrospinning and possess diameter distribution in the range of 85- 290 nm. The tensile strength and elongation were in the range of 2.13-4.41 MPa, 26.37-74.37% respectively which are very suitable for skin tissue engineering. Porosity of scaffold was higher than 60% and was appropriate for proliferation of fibroblast cells. Accordingly cell culture indicated that both calendula officinalis and gum arabic promote cell attachment and proliferation and thus well suited for regenerating skin⁴³. Ronald et al., 2014 modified collagen scaffold with polymeric microparticles and hydroglycolic extract of calendula officinalis flowers are subsequently loaded microparticles made of gelatin and collagen were produced by cross linking method. Invitro assessment indicate that incorporation of gelatin- collagen microparticles increase the resistance of scaffolds to enzymatic degradation A sharp decrease in cytotoxicity and prolonged release of extract was attained with these scaffolds. Results support the potential of scaffolds to develop innovative dermal substitutes with improved features⁴⁴.

Lawsonia inermis nanofibrous scaffolds:

Lawsonia inermis, which is also henna, contains several polyphenolic compounds. Recently several pharmacological benefits have been reported for Lawsonia inermis such as immunomodulatory, antioxidant, wound healing and anti-microbial properties. No gentotoxicity risk has been reported for Lawsonia inermis⁴⁵. Gelatin is one of the most commonly used polymers. But gelatin nanofibers lack desired mechanical properties. Biocompatibe polymers like polylactic acid (PLLA) have been used to fabricate tissue engineering applications. It offers desired mechanical properties⁴⁶. Lawsonia inermis was incorporated into gelatin and it was eletrospun with PLLA to form hybrid nanofibrous scaffolds. Lawsoina inermis nanofibers scaffold can kill both gram positive and negative bacteria within 2 hours and it shows good biocompatibility on fibroblast cells. PLLA/Gelatin Lawsonia inermis nanofibers can be utilised as a wound dressing to prevent infection and accelerate wound healing⁴⁷.

Electrospun scaffold of Panchvalkal powder:

Panchavalkal consists of stem barks of five trees namely Ficus bengalensis, Ficus lacor, Ficus recemosa, Ficus religeosa and Thespesia populnea in equal proportions. Electrospun scaffold was developed using biodegradable polymer and Panchvalkal powder (PV). Positively charged smaller particle size (40nm) is preferred for greater penetration through epidermal barrier. In vivo study using albino rats shows better wound healing efficiency as it shows higher wound area contraction, minimum inflammation, faster epithelization and vascularization. Complete biodegradation confirms its green nature. Elongated and spreaded morphology of PLA –PV scaffolds indicates much better cell attachment in scaffolds than other systems. It has benefits over traditional systems as it is an unstable dispersion. Histological analysis supports excellent epithelization and vascularisation in scaffolds treated wounds as opposed to solution and films. Clinical trials conducted demonstrate very good wound healing using the developed scaffold⁴⁸.

CONCLUSION:

Natural extracts have been practised for wound treatment since ancient times. Natural agents were loaded into scaffolds of biological polymers. Herbal biodegradable scaffolds show that the plant can be used as natural agent for the treatment of different skin damages with potential applications of tissue engineering. Chemical and synthetic polymers are less preferred over biological based polymers due to its harmful effects on environment and human health. Here we reviewed on scaffolds produced using completely environment friendly herbal extracts and biological polymers. Scaffolds with combinations of various natural extracts and biological based polymers augments wound healing and promote tissue regeneration. Currently commercially available tissue engineered scaffolds cannot fully replace the functional properties of native skin after wound healing. Rapid progress in tissue engineering and different approaches to design a scaffold, mainly including a natural extract is a promising one in coming years.

CONFLICT OF INTEREST:

The authors declare no potential conflicts of interest.

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Received on 28.03.2020 Modified on 15.05.2020

Research J. Pharm. and Tech 2021; 14(3):1805-1810.

DOI: 10.5958/0974-360X.2021.00320.6