# data[].attributes.body
Group Members :
Wan Nooranis bt Wan Zaid (A18SC0491)
Nur Isyafatira bt Japri (A18SC0446)
Nur Amilia bt Ahmad Sukri (A18SC0439)
Nurul Shazleen Natasha bt Mohd Miskam (A18SC0465)

Article 1: Current Status and Potential Applications of Underexplored Prokaryotes

- Uncommon prokaryotes and hard-to-cultivate microorganisms were still under discovery which led to inadequate fundamental knowledge and has limited application.
- Researchers neglected the uncommon prokaryotes and hard-to-cultivate microorganisms.
- There are some methods to cultivate the underexplored prokaryotes such as focusing on isolation and growth preparation, reconsider sampling sites and modifying cultivation methods
- The exploration of underexplored prokaryotes should be done more to enhance their application in industry or human health.
-Some rare prokaryotes might offer great advantages to the industrial application but the obstacle to cultivate or to exploring the rare prokaryote might give the limitation to the researcher to work on it.

Article 2: Epidermal and Fibroblast Growth Factors Incorporated Polyvinyl Alcohol Electrospun Nanofibers as Biological Dressing Scaffold
- Several conventional wound dressings do not allow the typical physiological environment to facilitate the wound healing process due to lack of biological activities and responses.
- Electrospinning techniques which are absorption or immobilization and coaxial or multiaxial techniques are complicated and require particular attention to material selection, solvent combination and specific volatility.
-The wrong technique chosen to fabricate scaffold will affect the efficacy scaffold properties where the fiber will lack of smoothness and there will be present of bead in the electrospun fibers.

# data[].attributes.body
Group member :
Hannah Nadiah binti Zulkefli
(A18SC0393)
Nurasyura Najla binti Basri (A18SC0456)
Fatin Nabilah binti Rakib (A18SC0391)
A'dlina binti husny @ Badrul Hisham
(A18SC0373)

Article 1 : Current Status and Potential Application of Underexplored Prokaryotes
- Researchers have been paying less attention to uncommon prokaryotes and hard-to-cultivate microorganisms
- By exploring these prokaryotes; their  novelties, function and application in industry can be discovered
- Suggestions on how to isolate or cultivate underexplored prokaryotes :  modify method of isolation and growth preparation , reconsider the sampling sites (location with minimum human activities) and modify cultivation strategy (in situ and ex situ cultivation)
-DNA sequencing strategies like MAG, SAG and WGS enable researchers to discover the genomic contents of underexplored prokaryotes - MAG is the most effective way
- The type and preparation of samples have a significant impact on the quality of MAGs and it needs computational ability to process large amounts of sequencing read data and perform downstream analysis.
- Limitation  functional-based metagenomics is expensive and is not affordable on a larger scale

Article 2 : Epidermal and Fibroblast Growth Factors Incorporated Polyvinyl Alcohol Electrospun Nanofibers as Biological Dressing Scaffold
-Skin regeneration and reconstruction are currently a demand healthcare area due to the global population growth. The skin’s external and internal inhibitory factors can delay wound healing and may trigger a chronic wound construction that will increase medical treatment cost.
-There are various techniques have been utilized to fabricate scaffolds such as solvent casting, freeze-drying, phase separation, gas foaming, electrospinning, fibre bonding, melt moulding, prototyping, powder compaction, supercritical fluid and fibre mesh.
-However, Electrospinning is an inexpensive and appropriate way to fabricate nanofiber meshes.
-There are several method to obtain the result, for example, characterization analyses, wettability analysis, tensile analysis and Cell culture of human dermal fibroblasts (HDFs)

# data[].attributes.body
Groupmate’s name:

1) AZRIE FAUZY B ABDULLAH

2) MUHAMMAD SUHAIL B MOHD ARIFFIN

3) IMRAN QASHFI B ISMAIL

4) MOHAMMAD AL-FAYYADH B MD NOOR

5) NOR TAIB ARASH B YUSOFF

Article 1: Current Status and Potential Applications of Underexplored Prokaryotes

Massive quantities of experimental findings and knowledge generated from the well-known prokaryotic strains. Therefore, this study provides an update for uncommon and unculturable prokaryotes, compiling various research efforts.

Researchers are required to comply with most, if not all requirements of Minimum Information about a Single Amplified Genome (MISAG) before submitting SAG sequences to databases.

It is less understood that certain underexplored prokaryotes also have biological roles in the environment, in particular in geochemical cycles of carbon, nitrogen, and sulfur.

Article 2: Epidermal and Fibroblast Growth Factors Incorporated Polyvinyl Alcohol Electrospun Nanofibers as Biological Dressing Scaffold

Several conventional wound dressings do not allow the typical physiological environment to facilitate the wound healing process due to lack of biological activities and responses

Both absorption/immobilization and coaxial/multiaxial techniques are complicated and require particular attention to material selection, solvent combination and specific volatility

the nanofiber the crosslinked PVA nanofiber still exposing the tissue to additional toxicity risks

The variations found to reflect the capability of the utilized PVA in this study to carry the proteins to the burned tissue within an acceptable period can not confirm the design influence as the difference exists but can not differentiate the most influencing design.

# data[].attributes.body
MASHITOH BINTI AHMAD (A18SC0409)

There are many unpopular prokaryotes and microorganisms still remain under-explored and usually the researchers neglected it .Apart from that, there are many challenging / obstacles due to the lack of information related to the microorganisms or prokarryotes.

# data[].attributes.headline
Group Discussion on Research Problem                                                

# data[].attributes.subject
Group Discussion on Research Problem                                                

# data[].attributes.body
Group Members:

1. YEOH SHIR LING A18SC0495

2. LOIS LO TZU HUI A18SC0408

3. TAN ZEN DONG A18SC0487

4. PUA LEI WEN A18SC0467

5. TAN JIA YONG A18SC0485

Journal_Epidermal and fibroblast growth factors incorporated polyvinyl alcohol electrospun nanofibers as biological dressing scaffold

Free association approach

1. Problem with skin and reconstruction

2. High medical treatment cost problem

3. Problem with not unique scaffold properties

4. Fabrication of scaffold technique problem

5. Epithelialization and proliferation of dermal fibroblast

Small conclusion:
The study of epidermal and fibroblast growth factors to improve Electrospinning nanofibers technique in solving demanding high cost medical treatment issues towards skin regeneration and reconstruction.

Journal_Current Status and Potential Applications of Underexplored Prokaryotes

Free association approach

1. Time-consuming in exploring rare prokaryotes

2. High research cost

3. Requiring new advance biotechnology to look for potential microbe

Small conclusion:
The Discovery of rare prokaryotes and its potential functions and applications for future use are less. Therefore, problems and suggestions in culturing the underexplored prokaryotes need to be taken more actions.

# data[].attributes.body
Group Members :
Norarina Suraya (A18SC0431), Norazrin Anis (A18SC0432),
Nur Aisyah (A18SC0437),
Nor Azlia Aziera (A18SC0428),
Nur Anis Nadia (A18SC0440)

Article 1: Current Status and Potential Applications of Underexplored Prokaryotes

Discovery of underexplored culturable and unculturable prokaryotes which may clarify their unspoken novelties and function has always been abandoned.

These rare prokaryotes can be unculturable microorganisms since researchers found that it is hard to cultivate the prokaryotes under laboratory condition.

The rare prokaryotes are still under studied and explored because of the limitations in their discovery and application.

This is because there is insufficient information about underexplored prokaryotes and hard-to-cultivate microorganisms that are actually important to link in terms of biodiversity and their biological roles in the environment.

Several recommendations for cultivating unexplored prokaryotes include paying attention to isolation and growth preparation, reconsidering sampling sites, and changing cultivation methods.

Article 2: Epidermal and Fibroblast Growth Factors Incorporated Polyvinyl Alcohol Electrospun Nanofibers as Biological Dressing Scaffold

There are many limitations in conventional wound dressing and it opens new study and research of skin regeneration and reconstruction by using biological scaffold as a tool to improve in every aspect such as healing time, process of treatment and effectiveness.

Some conventional wound dressing do not allow the typical physiological environment to facilitate the wound healing process due to their insufficiency in biological activities and response which can be addressed by scaffold utilization.

Absorption/immobilization, coaxial/multiaxial and emulsion techniques are complicated because of their material selection, solvent combination and specific volatility while the emulsion technique requires specific properties of molecules, polymers and solvent combinations.

Since PVA’s solubility limited its application in the pharmaceutical field, especially for the partially-hydrolysed PVA, it is crosslinked before utilization to reduce its hydrophilicity. However, crosslinked PVA nanofiber can still expose the tissue to toxicity risks.

# data[].attributes.headline
Current Status and Potential Applications of Underexplored Prokaryotes

# data[].attributes.subject
Current Status and Potential Applications of Underexplored Prokaryotes

# data[].attributes.body
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7970974/pdf/41598_2021_Article_85149.pdf

# data[].attributes.attachment_link.readable_text
You seem to have javascript disabled. Please note that many of the page functionalities won't work as expected without javascript enabled.

Received: 13 August 2019 / Revised: 5 October 2019 / Accepted: 8 October 2019 / Published: 18 October 2019

Thousands of prokaryotic genera have been published, but methodological bias in the study of prokaryotes is noted. Prokaryotes that are relatively easy to isolate have been well-studied from multiple aspects. Massive quantities of experimental findings and knowledge generated from the well-known prokaryotic strains are inundating scientific publications. However, researchers may neglect or pay little attention to the uncommon prokaryotes and hard-to-cultivate microorganisms. In this review, we provide a systematic update on the discovery of underexplored culturable and unculturable prokaryotes and discuss the insights accumulated from various research efforts. Examining these neglected prokaryotes may elucidate their novelties and functions and pave the way for their industrial applications. In addition, we hope that this review will prompt the scientific community to reconsider these untapped pragmatic resources. View Full-Text

▼ Show Figures

Figure 1

Goh, K.M.; Shahar, S.; Chan, K.-G.; Chong, C.S.; Amran, S.I.; Sani, M.H.; Zakaria, I.I.; Kahar, U.M. Current Status and Potential Applications of Underexplored Prokaryotes. Microorganisms 2019, 7, 468. https://doi.org/10.3390/microorganisms7100468

AMA Style

Goh KM, Shahar S, Chan K-G, Chong CS, Amran SI, Sani MH, Zakaria II, Kahar UM. Current Status and Potential Applications of Underexplored Prokaryotes. Microorganisms. 2019; 7(10):468. https://doi.org/10.3390/microorganisms7100468

Chicago/Turabian Style

Goh, Kian M.; Shahar, Saleha; Chan, Kok-Gan; Chong, Chun S.; Amran, Syazwani I.; Sani, Mohd H.; Zakaria, Iffah I.; Kahar, Ummirul M. 2019. "Current Status and Potential Applications of Underexplored Prokaryotes" Microorganisms 7, no. 10: 468. https://doi.org/10.3390/microorganisms7100468

Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Article Access Map by Country/Region

# data[].attributes.attachment_link.title
Current Status and Potential Applications of Underexplored Prokaryotes

# data[].attributes.attachment_link.description
Thousands of prokaryotic genera have been published, but methodological bias in the study of prokaryotes is noted. Prokaryotes that are relatively easy to isolate have been well-studied from multiple aspects. Massive quantities of experimental findings and knowledge generated from the well-known prokaryotic strains are inundating scientific publications.

# data[].attributes.headline
Epidermal and fibroblast growth factors incorporated polyvinyl alcohol electrospun nanofibers as biological dressing scaffold

# data[].attributes.subject
Epidermal and fibroblast growth factors incorporated polyvinyl alcohol electrospun nanofibers as biological dressing scaffold

# data[].attributes.body
https://www.nature.com/articles/s41598-021-85149-x.pdf

# data[].attributes.attachment_link.readable_text
Introduction

Skin regeneration and reconstruction are currently a demand healthcare area due to the global population growth1. The skin's external and internal inhibitory factors can delay wound healing and may trigger a chronic wound construction that will increase medical treatment cost. Wound healing is a challenging clinical issue that focuses on wound care management and emphasizes scavenging new treatment approaches3. Several conventional wound dressings do not allow the typical physiological environment to facilitate the wound healing process4 due to lack of biological activities and responses5. Scaffolds are polymeric three dimensional (3D) structures that have the prospect to address the limitation of traditional wound dressing materials. The unique scaffold properties allow cell attachment, cell migration, modify the diffusion of vital cell nutrients and control the behavior of cell phases which exert certain mechanical and biological influences6. Various techniques have been utilized to fabricate scaffolds such as solvent casting, freeze-drying, phase separation, gas foaming, electrospinning, fiber bonding, melt moulding, prototyping, powder compaction, supercritical fluid and fiber mesh7,8.

Electrospinning is an inexpensive and appropriate way to fabricate nanofiber meshes9,10. Numerous factors that affect the electrospinning process, including electrospinning parameters, electrospinning solution and environmental parameters11,12. Simultaneously, solution parameters of polymer concentration, solvent, viscosity and solution conductivity, and environmental parameters of temperature and humidity play roles in determining the electrospun fibers' properties. In general, all parameters are identical to be controlled to form smooth and bead-free electrospun fibers11.

There are several techniques of electrospinning that have been used by researchers, such as co-electrospinning, adsorption/immobilization, coaxial/multiaxial and emulsion electrospinning13,14. These techniques help to control drug release from the high surface area of nanofibers into the applied environment, especially for sensitive molecules or small water-soluble drugs14. Co-electrospinning is a versatile and simple technique to obtain nanofibers of different polymers with the ability to load several types of molecules in these structures15. Simultaneously, absorption or immobilization is a technique to immobilize molecules on fiber surfaces through covalent linkages that act as a specific molecular cue to control drug release14,15. In the coaxial/multiaxial technique, two and more separate solutions are electrospun using core-shell nozzles to maintain the initial bioactivity of molecules14,16. However, both absorption/immobilization and coaxial/multiaxial techniques are complicated and require particular attention to material selection, solvent combination and specific volatility14,16. The emulsion technique does not require concise apparatus15. However, it requests specific properties of molecules, polymers and solvent combination14. Therefore, in this study, the co-electrospinning technique was employed to form a polymer -based loaded growth factor (GF) as it is simple and does not require specific chemical properties.

Polyvinyl alcohol (PVA) is a synthetic polymer increasingly utilised in biotechnology, pharmaceutical, food chemistry and medicine, as a film, hydrogel or nanofibers. The significant drivers to using PVA in the medical and pharmaceutical fields are the biocompatibility, processability and hydrophilicity features17,18,19 and the Food and Drug Administration (FDA) approval17, 20. PVA is categorized into fully hydrolyzed and partially hydrolyzed21. Hence, depending on its type and level of polymerisation, the PVA's solubility limited its application in the pharmaceutical field, especially for the partially-hydrolysed PVA. Therefore, it has been crosslinked before being used to reduce the hydrophilicity of the PVAin many studies22,23,24. However, the nanofiber the crosslinked PVA nanofiber still exposing the tissue to additional toxicity risks25,26. This study used fully hydrogels PVA without crosslinked due to the hydrophobicity that makes it suitable to sustain the extracellular moist nature of the wounds and to degrade slowly compared to the hydrophilic PVA27,28. Therefore, PVA was chosen to be used in this study as a polymer matrix to form electrospun nanofibers and to carry two different types of growth factors (GFs).

Tissue development or repair regulated by growth factors proteins along with several cellular processes within the extracellular matrix (ECM)29. Among the several families of the cytokines and growth factors30 the epidermal growth factor (EGF) considered the first and foremost factor to promote wound healing31. It is mitogenic, form various epithelial cells, hepatocytes and fibroblasts32,32. It possesses high-affinity receptors that are expressed by both fibroblasts and keratinocytes. EGF utilization is mainly to improve epidermal and mesenchymal regenerations, cell mobility, proliferation and ECM synthesis, which consequently facilitate wound healing31,33. At the same time, fibroblast growth factor (FGF)32 Acidic and basic are the most known FGF classification15,32,34,35. Many functions are attributed to FGF, are due to interaction with cell surface receptors that has intrinsic tyrosine kinase activity, the development of new blood vessels, and the wound repair and hematopoiesis function34,36.

As EGF and FGF are playing roles in improving the healing capacity of skin cells and, at the same time, will provide natural remediation, both EGF and FGF in this study were incorporated into PVA to be co-electrospun for the fabrication of wound dressing. The electrospun nanofibers' wettability and elasticity were assessed through a water contact angle (WCA) and tensile test analyses. At a final stage, the nanofibers' biological activity was evaluated through in vitro cell studies with human dermal fibroblast cells and in vivo studies on a mammalian-wound rat model (burn).

Materials and methods

Sample preparation

This study's materials include a polymer of full-hydrolyzed polyvinyl alcohol (PVA), MW 145,000 (Merck, Japan). Two growth factors of EGF and FGF, phosphate-buffered saline (PBS) and Bovine serum albumin (BSA) (Sigma-Aldrich, Germany, Sigma-Aldrich, USA). The PVA has been dissolved deionized water (DI) at a concentration of 8% to prepare the polymer solution, then stirred at 80 °C for 2 h to obtain a homogeneous polymer matrix solution37,38. Amounts of 100 µg EGF and 100 µg FGF were dissolved in BSA/PBS in separate beakers. The growth factor solution mixed with the polymer solution at a temperature of 30 °C by applying a water bath concept for 30 min13,31. For all nanofibers were set at PVA:EGF:FGF, 9.5:0.5:0.5 v/v, respectively. In this study, five membranes were electrospun as stated in Fig. 1, which consisted of pure PVA (control), single layer PVA/EGF, single layer PVA/FGF, mix layer PVA/EGF-FGF and multiple layers PVA/EGF/FGF, which has been fabricated in sequence by creating the layer 1: single layer PVA/FGF, followed by layer 2: single layer PVA/EGF.

This study has utilized an electrospinning setup, as explained in Fig. 1 the used electrospinning machine from NaBond Technologies Co., Limited, Shenzhen, China. The syringe needle was connected to a 35 kV DC supply with a ground collector. For the electrospinning, the prepared solutions were introduced into the syringe and mounted on the syringe pump. The syring to collector distance was set at 15 cm, and a positive voltage of 20 kV was applied at a 0.5 ml/h volumetric flow rate in a room temperature environment38.

Characterization analyses

The nanofibers chemical functionalities were analyzed by ATR-FTIR (L1600301, PerkinElmer, UK) between wavenumbers of 500 and 4000 cm −1 at 4 cm −1 resolutions. The acquired ATR-FTIR spectra were then annotated and analyzed using Originlab software (Origin Pro 8, OriginLab Corporation, USA)10.

Then the nanofibers' morphology was visualized by SEM (JEOL, iT300LV, Japan) at a magnification of 10,000× at an acceleration voltage of 10 kV to view the formation of felt-like nanofibers, possible beads and electrospray fibers. Prior to the SEM observation, the electrospun nanofibers membranes were dried under vacuum (0.1 mbar), and a thin film of gold was coated by using a gold sputter coater (Q150R, Quorum Technologies Ltd, England). The diameter of fibers was measured with mage analysis software (JEOL, iT300LV, Japan). A total of 100 random fibers per image were examined to calculate the mean and standard deviation of fiber diameter9,10.

The nanofibers' topography was further identified with AFM (AFM5300E, Hitachi High-Technologies Corporation, Tokyo, Japan) in an intermittent tapping mode of 10 μm × 10 μm area with a back-side aluminium-coated rectangular cantilever. The acquired topography AFM images were then subjected to post-analyses of surface roughness and fiber diameter.

Wettability analysis

A contact angle instrument have been used to test the hydrophilicity and hydrophobicity (wettability) of the nanofibers(VCA, AST Product Inc, USA). The contact angle measurements were then performed at five different points by dropping 1.5 µL of deionized water on the membranes and measuring the contact angle with a drop shape analyzer.

Tensile analysis

The elasticity measurement was performed using a texture machine (CT3 Texture analyzer AMETEK Brookfield, United Kingdom). The nanofiber membranes were cut into a dumbbell shape with a nominal 22 mm, 5 mm of length and depth. The membrane thickness was in a range between 0.05 and 0.10 mm. The tensile test was conducted by applying 0.05 N load at 0.2 mm/s test speed according to ASTM standard D5034-9539. The measured yield stress (Young's modulus), and the ultimate strength of nanofibers were obtained by stress-strain curve analysis. The results were recorded as an average and standard deviation of three consecutive measurements.

Cell culture of human dermal fibroblasts (HDFs)

The materials of the cell culture include the human dermal fibroblast cells (HDFs) (ATC-PCS-201-012), 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, Dulbecco's Modified Eagle Medium (DMEM). Medium refreshed every 72 h under 95% air, 37 °C and 5% CO2 humid atmosphere. When HDFs reached 80% confluence, cells washed with 0.25% trypsin-EDTA to be collected then re-suspended in phosphate-buffered saline (PBS). The cell passage between 3 and 5 was used. All nanofibers sterilized by ultraviolet (UV) on both sides for 15 min to be used in the in vitro study. Triplicate data for the treated cells were collected, and non-treated cells were used as a control. The cells (HDFs) then seeded and incubated at a concentration of 1 × 105 cells/cm ² on each nanofibers membrane, 5% CO ₂ and 37 °C consequently, up to 7 days. The media were changed every 72 h regularly. After 1 h of culture media, the cells attachment on the nanofiber surfaces was observed.

MTT assay

The produced fibers effect on the cell viability and proliferation was determined by MTT assay 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide based on color observationafter the incubation at 1, 3 and 7 days. After 1, 3, and 7 days, the media were removed, and 1 ml fresh media were added into each well with 100 μL of MTT solution (0.5 mg/ml) following another incubation for four hours. After that, the media consisting of MTT solution were removed, and DMSO was added to dissolve the formazan products followed by 30 min incubation and with using a microplate reader (Multiskan FC, Thermo Scientific, USA) reading taken at the absorbance of 540 nm. The collected data then processed using the following equation:

Cell attachment

The physicohemical properties and roughness of nanofibers significantly influence cell behavior in term of proliferation and adhesion. Using the field-emission scanning electron microscope (FESEM) cell adhesion, the scaffolds' proliferation and morphology images have been analysed40. After the HDFs were cultured on the nanofibers and incubated on day 1 and 7, the nanofibers were washed, fixed with PBS (pH 7.4) and 2.5% glutaraldehyde for 2 h consequently. The nanofibers were undergone gradual dehydration in 30%, 50%, 75%, 90% and 100% ethanol for 10 min consecutively. Next, the nanofibers were freeze-dried overnight, coated with gold thin films. The cell morphology and adhesion were visualized under FESEM (SUPRA 35VP, Zeiss, Germany) at 5000× magnifications.

In vivo wound healing study

The in-vivo study performed according to approved animal care ethical protocols with reference number: UPM/IACUC/AUP-R011/2020 under Institutional Animal Care & Use Committee (IACUC) of Universiti Putra Malaysia (UPM). During the in vivo experiments, we ensured maintaining the animals (rats), i.e. feeding, pain management, locating, and sacrificing rats. The in-vivo mammalian model was the male Sprague-Dawley (SD) rats, weighting 240-260 g41,42, in which a burn created using controlled temperatures and contact times. After staining with hematoxylin and eosin stain (H&E), the obtained histology has been used to characterize changes and to determine the burn wound healing after exposure to the synthesized PVA-Gfs loaded scaffolds.

Wounding and preparation of tissue specimens for histological study

Pre-operatively, the rats were kept to adapt to the laboratory conditions for 7 days under a controlled diet and watered adlibitum. Prior to creating the wound, ketamine- HCl and xylazine anaesthesia (100 and 10 mg /kg body weight) were given to the rats under the observation of the veterinary (vet) physician, shaving the dorsal area of the rats. After that, one full-thickness circular burn wound created using a hot circular steel billet (ф = 25 mm; 100 °C, 30 s)43. A specialized vet physician has performed the burning procedure to assess the burn thickness and diameter preceding each experimental intervention to reduce the thickness evaluation bias created by the lack of uniform protocol for assessing the extent of tissue injury44. After the third-degree burn wound area created, we left the rats wound left to cool down with no medical treatment. Subsequently, the experiment consisted of six groups of rats, and we left the first group without treatment as a negative control (burned). The second group burn was dressed with one layer of the dry sterile scaffold of protein-free PVA as a positive control (burned) while the group three to six were treated with PVA/EGF, PVA/FGF, PVA/EGF-FGF, and PVA/EGF/FGF sequentially and fixed with a surgical suture to prevent the preventing the rats from removing it by scratching or chewing. we used individual cages for rats under a controlled ambient temperature at 23-24 °C with 12/12 h light/dark cycles during the experiments45. The control rat killed immediately on day 0; after that, the treated rats were killed on days 7, 14, 21 after the burn and photographs were taken of each wound using a digital camera and measured by a ruler. The data was calculated by adopting the equation mentioned in46:

where A ₀ was the area of initial wound and A ₁ was the area of wound at time.

Three rats of each group were euthanized, and the skin tissue was excised to evaluate skin repair at each time point for histological analysis. The specimens were prepared and washed in saline, fixed in 10% neutral buffered formalin and embedded in paraffin blocks and sequentially sectioned at 5 μm using a microtome. The hematoxylin and eosin (H&E) stain used to estimate the healing phases47. Images were taken with a camera on the microscope (T380C-10M, AmScope, USA).

Statistical analyses

The wound closures measurement has been used to identify the statistical differences among the synthesized PVA loaded GFs. The data were triplicated for statistical analysis purpose. The collected data of cell viability and the wound closure in the in-vivo study analyzed using GraphPad Prism 6.01 software (GraphPad Prism, La Jolla, CA, USA) to calculate one-way ANOVA of variance and Tukey's test for multiple comparisons at P < 0.05.

Results and discussion

Chemical composition analysis

Figure 2 shows the ATR-FTIR spectra of PVA (control), PVA/EGF, PVA/FGF, PVA/EGF-FGF and PVA/EGF/FGF nanofibers which have been electrospun at similar electrospinning parameters. Functional groups of PVA has been identified in all scaffolds. The presence of hydroxyl (OH −) peaks at 3200-3500 cm −1 were dominated by O-H stretch and O-H bend, and that existed in the chemical structure of PVA48.

The C-H stretch peaks were observed at 2850-3000 cm −1, while C-H bend identified at 1450-1470 cm −1 and C-H rock peaks were identified at 720-725 cm −1. The long hydrocarbon chain in the chemical structure of PVA is dominated to be responsible for the hydrophilic properties of PVA49. The presence of C=O stretch and the C-O stretch has indicated the hydrophobic groups of the PVA was then observed in between 1665 and 1710 cm −1 and 1000 and 1320 cm −1 , consequently50.

The PVA nanofibers incorporated with GF demonstrated the presence of amine groups, N-H stretch at 3300-3500 cm −1 38 overlapping with the OH − groups. Another identical GF peaks of N-H bend are clearly shown only on the GF incorporated membranes at 1580-1650 cm −1 51,52 which clarify the existence of GF in the nanofiber membranes.

Morphological structure assessment

The SEM visualized the surface morphology, structure and diameter of the electrospun Nanofibers. We found that all nanofibers were constructed in continuous long protruded nanofibers, imitating porous meshes. Figure 3 shows the uniform nanofibers without the bead and electrospray feature for all membranes. The nanofibers' average diameters were counted and illustrated in Fig. 3, where the PVA membranes were constructed of 230 ± 59 nm diameter nanofibers.

The mean diameters for the PVA/EGF and the PVA/FGF electrospun nanofibers were counted to be 286 ± 60 and 198 ± 37 nm, respectively. The molecular weight of GFs was capable of increasing or decreasing fiber diameter through the adjustment in solution viscosity53,54. In this study, the low molecular weight of FGF (16-34 kDa) compared to EGF (4.6-74 kDa)55,56 has led to the reduction in fiber diameter.

The incorporation of both EGF and FGF produced the nanofiber diameters of 205 ± 50 nm for the PVA/EGF-FGF and 218 ± 52 nm for the PVA/EGF/FGF. These two membranes, mix and multiple GFs, were deposited with the range of nanofiber diameter between of the PVA/EGF and the PVA/FGF. Despite the molecular weight, other parameters are also affecting the diameter of fibers including voltage, the distance between needle tip and collector, nozzle diameter, solution conductivity, environmental humidity and environmental temperature11. Decreasing the nozzle diameter and increasing the applied voltage57, the distance between the tip and the collector58, solution conductivity59, environment temperature, as well as environment humidity, will reduce the fiber diameter11. However, in this study, all electrospinning parameters were kept constant to maintain electrospun nanofibers' ejection.

Surface roughness analysis

The surface roughness analysis was performed on the topography AFM data, as shown in Fig. 4, where the PVA nanofibers were recorded with 418 ± 30 nm surface roughness. Decrement of surface roughness was observed on the PVA/EGF and the PVA/FGF with the values of 365 ± 11 nm and 305 ± 15 nm, respectively. The surface roughness was further reduced on the PVA/EGF-FGF with 295 ± 28 nm roughness and the PVA/EGF/FGF with 316 ± 22 nm roughness. There was no significant difference in the surface roughness data between PVA/EGF-FGF and PVA/EGF/FGF membranes, signifying that the incorporation of FGF and EGF, either through the electrospun of mix solution or multiple layers, did not impact the nanofiber roughness. Ahlawat et al. 60 mentioned the capability of low surface roughness to assist cell adhesion and proliferation mechanically. In this study, the low surface roughness of GFs incorporated membranes could provide a medium for cell interaction to induce cell integration and proliferation.

The fiber diameter was further verified through the post-analysis of AFM topography data. The fiber diameter value for all nanofiber membranes (Fig. 4) are identical to the increment and decrement trends of fiber diameter observed through the SEM analysis. However, the values were higher due to the back-and-forth cantilever retraction during the scanning process, cause the data projection to be wider. From the post-analysis results, the incorporation of both FGF and EGF into the PVA nanofibers led to the reduction of fiber diameter with the acquired data of 298 ± 55 nm and 277 ± 57 nm for the PVA/EGF-FGF and PVA/EGF/FGF, respectively.

Wettability analysis

The perspective of wettability properties is preferable for cell attachment, migration and proliferation due to the hydrophilic interaction between cells and material surfaces61. The PVA nanofibers produced contact angle data of 83 ± 0.88°, approaching the hydrophobic properties, as the PVA is constructed of C=O and -CH ₃ hydrophobic groups62,63. The incorporation of GFs cause the nanofiber membranes to be more hydrophilic with the records of 66° ± 2.98° and 66.08° ± 0.88° for the PVA/EGF and the PVA/FGF, respectively. The decrement of contact angle values was mainly caused by the hydroxyl and amine groups in the GFs, which tailored the properties of the nanofibers towards greater wettability64. The combination of EGF and FGF have further reduced the contact angle data to be 53° ± 2.05° for the PVA/EGF-FGF and 57° ± 2.19° for the PVA/EGF/FGF. The combination of both GFs has tuned the wettability of nanofiber surfaces to be higher compared to the singular GF incorporated membranes. These results are identical to the measurements in previous studies65,66 which testified high wettability records on the GFs incorporated polymer nanofibers (Fig. 5).

The tensile analysis

The stress-strain curves of the PVA, PVA/EGF, PVA/FGF, PVA/EGF-FGF and PVA/EGF/FGF nanofibers under tensile loading data are arranged in Table 1 and plotted in Fig. 6. The Young's modulus of PVA nanofibers was reduced to 17 MPa, when the FGF was incorporated within the PVA matrix compared to Young's modulus of pure PVA nanofibers 33.89 MPa.

Table 1 Mechanical properties of electrospun PVA, PVA/EGF, PVA/FGF, PVA/EGF-FGF and PVA/EGF/FGF nanofiber membranes.

In contrast,Young's modulus of PVA nanofibers was increased to 36 MPa when the EGF was incorporated within the pure polymer membrane. The properties of FGF, which derived from the classification of natural fibers and elastin, have increased the elasticity of the electrospun membrane, which affected the material strain, thus decreasing Young's modulus value67. The combination of FGF and EGF in constructing the PVA/EGF-FGF and the PVA/EGF/FGF cause further reduction of Young's modulus, between 18 and 20 MPa. The nanofibers (Young's modulus) in the range of preferable properties of the human skin, 15-150 MPa68, which projects a view on its utilization for skin tissue engineering application.

MTT assay

The in vitro study of HDFs goal s to evaluate the synthesized scaffolds compatibility to be implanted in the biological environment. Cell attachment and proliferation are important factors in designing scaffolds that can enhance and present regeneration of tissue69. Figure 7 shows the proliferation of HDFs and cell viability and the nanofibers of PVA, PVA/EGF, PVA/FGF, PVA/EGF-FGF and PVA/EGF/FGF at day 1, 3, and 7. A similar density of culture was seeded on all nanofibers to evaluate the cellular proliferation by MTT assay. All nanofibers were biologically active with HDFs and enhanced cell proliferation. All nanofibers did not show cytotoxic effect due to the cells viability remains more than 70%, according to Catanzano et al. and Cerchiara et al. 70,71. The cell proliferation trend has increased, starting with slow growth at day 3 to fast growth at day 7 ( P < 0.05). On day 1, the observed cells showed a significantly higher proliferation on the GFs incorporated nanofibers than the PVA with significantly greater proliferation on the PVA/EGF-FGF and PVA/EGF/FGF compared to the PVA/EGF and PVA/FGF. On day 3, a similar trend was observed, while on day 7, the more visible trend was projected with the highest HDFs proliferation on the PVA/EGF-FGF and PVA/EGF/FGF. That indicated the EGF and FGF effect in stimulating the fibroblast cell growth, which was observed much more at 7 days of the analysis with that may indicate the activity of both GFs64,72.

Cell attachment

Figure 8 presents the images of the HDFs surface and cells features (morphology), viewed under FESEM to identify the early adhesion of HDFs on the nanofibers on day 1 and 7. The images show that the HDFs were attached and disseminated more on the GFs incorporated nanofibers at respective days with different attachment trend than the PVA nanofibers. On day 1, the HDFs were slightly adhered and spread in a flat and focal form. The filopodia were elongated and stretched, clutching the porous structure of nanofibers. The HDFs were found in a small cluster on the PVA/EGF, while a more significant cell cluster was found on the PVA/FGF. On the one hand, the GFs mixture in the PVA/EGF-FGF cause the cells to attach in a small cluster following the trend on the PVA/EGF due to the direct contact between EGF and cells. On the other hand, a big cluster was viewed on the PVA/EGF/FGF due to FGF exposure to the cells, as the EGF layer was at the bottom. On day 7, the cells were significantly interacted and spread on the GFs incorporated nanofibers due to continuous EGF and FGF releases, according to Bahadoran et al. and Lin et al.69,73. It is clear that HDFs were attached to the PVA nanofibers at day 7 without spreading on the nanofiber surface, which explicated as a passive adhesion due to complex physicohemical interaction between the cell membrane and the polymer surface69.

In vivo wound healing study

The clinical evaluation of the wound characteristics and closure is shown in Fig. 8 following the visual assessment of burn depth74. Post-treatment observation and the clinical evaluation of the treated rats continue until the 21st day as it is the survival rate average of the burn-wound models due to the application of this study purpose75. The process of burn-wound healing of the in-vivo experiment in PVA/EGF, PVA/FGF, PVA/EGF-FGF, and PVA/EGF/FGF nanofibers groups at 7th, 14th, and 21st days have been thoroughly assessed. We have identified the features of the full-thickness burn tissue characterized by the dry, leathery dark brown, and we followed the wound closure within the same period of the healing compared to all the GFs loaded Nanofibers. The burn-wound healing effects of PVA/EGF, PVA/FGF, PVA/EGF-FGF and the PVA/EGF/FGF nanofiber has been visually the confirmed, compared to the positive control (PVA) treated rats and negative control (burned only rats). It was visually found that there were marginal changes in the burn-wound size in all experimental groups at 7, 14, and 21 days' (Fig. 9). It was found that although there were marginal differences in the burn-wound closure rate of the negative control and positive control, the best closure was shown in the experimental groups at 14 days and 21 days with the mixture and layered PVA/EGF-FGF, and PVA/EGF/FGF sequentially of the healing period, and the best burn-wound closure rate was shown in PVA/EGF/FGF (Fig. 9). These results are most probably related to growth factors' activity76,77.

The percentage of the wound healing (closure) measurements for the experiment groups are presented in Fig. 10. All the PVA/EGF, PVA/FGF, PVA/EGF-FGF, and PVA/EGF/FGF nanofibers could enhance burn wound healing compared to control and PVA groups, while the control group show sign of hemorrhage and inflammation in the wounds. Consistently, the quantitative data represented in Fig. 9, showing the same trend of growth with some variations in the results between the mixtures and layers PVA/EGF-FGFand PVA/EGF/FGF. PVA/EGF-FGF and PVA/EGF/FGF nanofibers displayed high rapid recovery of the wounds contrasted to that of only EGF or FGF nanofibers, which imputed the combined effects of EGF and FGF. Figure 9 detected that on the 21st post-burn wound day, the closure rate of the PVA/EGF-FGF and PVA/EGF/FGF groups (70%, 69.2%) were significantly higher compared with the PVA/EGF (41.6%), PVA/FGF (44.7%), PVA (18%), and control (15.9%). Moreover, it was found that PVA-GFs loaded nanofiber was a good scaffold for burn-wound healing as compared to PVA only nanofiber. The observed healing in the PVA only fibers might be due to the characteristics of the nanofiber that assist in working as an extracellular mesh76,77. This finding indicates the EGF and FGF inside scaffold that promoted the epithelialization, collagen synthesis, and tissue regeneration. However, the variations found to reflect the capability of the utilized PVA in this study to carry the proteins to the burned tissue within an acceptable period can not confirm the design influence as the difference exists but can not differentiate the most influencing design. Despite this observed inconsistency, we can elude that the type PVA utilized in this study has no limiting effect on its capability to be used as a protein carrier, as shown in Figs. 9 and 10.

Histological analysis of burn-wounds

Histological analyses of the burn healing process were performed by H&E stain on day 7, day 14, and day 21in Fig. 11. On day 7, the epithelial tongue was grown, which show the new regenerated epidermis in PVA/EGF, PVA/FGF, PVA/EGF-FGF and PVA/EGF/FGF whilst the necrosis was seen in control and PVA group. At Day 14, stratified epidermal layers grew more in PVA/EGF, PVA/FGF, PVA/EGF-FGF and PVA/EGF/FGF. The low level of necrosis was shown in the control and PVA group. On day 21, the papilla basement membrane was obviously seen in PVA/EGF while PVA/FGF few sebaceous glands were observed. The neovascular structure was seen in PVA/EGF, PVA/FGF, PVA/EGF-FGF and PVA/EGF/FGF. PVA/EGF-FGF and PVA/EGF/FGF were observed sebaceous gland and hair follicles and show healthier skin on day 21. However, PVA and control groups show a newly regenerated epidermis with necrosis in the control group. The control of infection in the healing wound is very important for a good progressive wound closure, specifically in the burn-wound healing process78. From the finding in this study, it is likely that the wound closure not interrupted with infections and has lead to a proliferation in the dermal cells and accelerated the healing effect. The finding was investigated that PVA/EGF-FGF and PVA/EGF/FGF can enhance collagen synthesis, re-epithelialization and vascularization with a short time of healing due to EGF stimulate epithelialization in human wound repair, and FGF promoted the proliferation of dermal fibroblasts79.

Conclusion

In this study, we fabricated and characterized Growth factors incorporated PVA electrospun mats with single, mix and multiple layers as burn-wound healing scaffold. The incorporation of EGF and FGF in PVA nanofibers, either through single, mix or multiple layers, led to the detection of O-H and N-H from the chemical groups of GFs. All nanofibers were electrospun without the bead and spray feature, where the combination of EGF and FGF has reduced the fiber diameter. The wettability, surface roughness and mechanical properties of the GFs incorporated PVA nanofiber membranes were also improved, which fit its utilization as a biological dressing scaffold.

The synthesized fibers have been examined for feasibility to be used as a wound-healing dressing. The burn-wound healing effect was examined by digital camera observation and histological analyses. The GFs incorporated PVA nanofibers display no cytotoxic effect and cell proliferation within the nanofibers' structure regarding in vitro studies. The in vivo study evaluatin shows the GFs incorporated PVA nanofibers accelerated the burn wound healing process that enhances epithelialization and proliferation of dermal fibroblasts.

References

1. 23.
Google Scholar

2. 28.
Saputra. A., H. & Apriliana N., H. Polyvinyl alcohol (PVA) partially hydrolyzed addition in synthesis of natural hydrogel carboxymethyl cellulose (CMC) based from water hyacinth. In: MATEC Web of Conferences. EDP Sciences. 01007(2018).‏

3. 45.
R. R. P. 20: Guidelines for the Housing of Rats in Scientific Institutions. Animal Research Review Panel, Sydney. (2007).‏

4. 51.
Google Scholar

5. 59.
Google Scholar

6. 63.
Zhang, W., Zhang, Z. & Wang, X. Investigation on surface molecular conformations and pervaporation performance of the poly(vinyl alcohol) (PVA) membrane. J. Colloid Interface Sci. 333, 346-353 (2009).

ADS CAS PubMed Article PubMed Central Google Scholar

7. 71.
Cerchiara, T. et al. Spanish Broom ( Spartium junceum L.) fibers impregnated with vancomycin-loaded chitosan nanoparticles as new antibacterial wound dressing: Preparation, characterization and antibacterial activity. Eur. J. Pharm. Sci. 20, 20 (2017).

Google Scholar

Download references

Acknowledgements

The authors would like to acknowledge the Ministry of Higher Education in the Kingdom of Saudi Arabia for financial support. The authors also appreciate the research team, Advanced Diagnostics and Progressive Human Care Research Group and laboratory staff support at University Technology Malaysia and Ministry of Higher Education Malaysia for the financial support of Grant with reference no: GUP-UTMER PY/2020/04225.

Author information

Affiliations

1. Department of Biosciences, Faculty of Science, Universiti Teknologi Malaysia, 81310, Johor Bahru, Johor, Malaysia

Amnah Asiri & Mohd Helmi Sani

2. IJN-UTM Cardiovascular Engineering Centre, Institute of Human Centered Engineering, Universiti Teknologi Malaysia, 81310, Johor Bahru, Johor, Malaysia

Syafiqah Saidin

3. Department of Clinical Sciences, School of Biomedical Engineering and Health Science, Universiti Teknologi Malaysia, 81310, Johor Bahru, Johor, Malaysia

Rania Hussien Al-Ashwal

4. Advanced Diagnostics and Progressive Human Care (Diagnostic) Research Group, Health and Wellness Research Alliance, Universiti Teknologi Malaysia, 81310, Johor Bahru, Johor, Malaysia

Rania Hussien Al-Ashwal

Contributions

A.A. performed the experiments, acquisition, analysis, interpretation of the data and wrote the main manuscript. S.S. co-supervised the work, reviewed and edited part of the manuscript. M.H.S. co-supervised the work, reviewed and part of the manuscript manuscripts. A. R. supervisor of the work designed the research, analyzed the data reviewed, edited the manuscripts and prepared the manuscript for dissemination.

Corresponding author

Correspondence to Rania Hussien Al-Ashwal.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
Reprints and Permissions

About this article

Download citation

# data[].attributes.attachment_link.title
Epidermal and fibroblast growth factors incorporated polyvinyl alcohol electrospun nanofibers as biological dressing scaffold

# data[].attributes.attachment_link.description
In this study, single, mix, multilayer Polyvinyl alcohol (PVA) electrospun nanofibers with epidermal growth factor (EGF) and fibroblast growth factor (FGF) were fabricated and characterized as a biological wound dressing scaffolds. The biological activities of the synthesized scaffolds have been verified by in vitro and in vivo studies.