Development of nanostructured bioplastic material for wound healing

Abstract

The development of new biomaterials whose characteristics are as close as possible to the properties of living human tissues is one of the most promising areas of regenerative medicine. This work aimed at creating a bioplastic material based on collagen, elastin and hyaluronic acid and studying its structure and properties to assess the prospects for further use in clinical practice. Bioplastic material was obtained by mixing collagen, hyaluronic acid and elastin in predetermined proportions with distilled water. We treated the material with photochemical crosslinking to stabilize biofilm in a liquid medium and form a nanostructured scaffold. A commercial human skin fibroblast cell culture was used to assess the biomaterial cytotoxicity and biocompatibility. The visualization and studies of the biomaterial structure were performed using light and scanning electron microscopy. It has been shown that the obtained biomaterial is characterized by high resilience; it has also a high porosity. The co-culturing of the bioplastic material and human fibroblasts did not reveal any of its cytotoxic effects on cells in culture. It was shown that the biomaterial samples could maintain physical properties in the culture medium for more than 10 days, while the destruction of the matrix was observed 3–4 weeks after the beginning of incubation. Thus, the created biomaterial can be used on damaged skin areas due to its physical properties and structure. The use of the developed biomaterial provides effective conditions for good cell proliferation, which allows us to consider it as a promising wound cover for use in clinical practice.

Ethical Publication Statement

We confirm thatwe have read the Journal’s position on issues involved in ethical publication and affirm that thisreport is consistent with thoseguidelines.

The conservative treatment of wounds of various etiologies does not always give the expected results.^1,2 In this regard, the development of new modern wound dressings that contribute to the effective treatment and regeneration of the skin is required.^3,4 Adhesiveness, plasticity, biocompatibility and maximum closeness in terms of fibroarchitectonics to body tissues and biological activity are important requirements for their creation, which allows achieving certain pharmaceutical effects from their use. Polymers (natural and synthetic), metals, inorganic materials, carbon-based materials and composites based on them are used to obtain bioplastic materials.^5,6 Polymeric materials can be divided into natural and synthetic polymers. Natural polymers can be proteins (for example, collagen and fibrin), polysaccharides (chitosan, alginate, dextran etc.), synthetic polyvinyl alcohol, polyesters, polyphosphazenes, polyanhy- drides, etc.^7,8

Currently, in the development of implantable materials for regenerative medicine, preference is given to polymers of natural origin (biopolymers) and their derivatives.^9,10 During implantation, they are split into simpler compounds that are excreted from the body or participate in metabolism at the cellular level. The advantage of biopolymers is that toxic products are not formed during their degradation.¹¹ Composite matrices that include several complementary components to obtain systems with optimal properties (porosity, swelling, elasticity, mechanical strength) have been developed. Among these products, special attention is paid to materials based on collagen and hyaluronic acid – the main components of the extracellular matrix that are involved in the restoration of damaged structures of the dermis.^12,13 This work aimed to create a new bioplastic material based on collagen, elastin and hyaluronic acid and study its structure and properties to assess the prospects for further use in clinical practice.

Materials and methods

Production of bioplastic material

Bioplastic material was produced using the following components: collagen, hyaluronic acid and elastin, mixed in a proportion equal to that in an adult human dermis: collagen 70-90%, hyaluronic acid 5-26%, elastin 1-25%. The material was subjected to photochemical crosslinking to stabilize biofilm in a liquid medium. The photochemical crosslinking was performed with an ultraviolet light source (λ < 260 nm, 30 cm distance between UV source and biomaterial). In the process of material development, we were also elaborating on the technology of hydrogel production and settling the time of hydrogel exposure to UV, in order to form the photochemical crosslinking of a future biomaterial. Optimal conditions for obtaining the biomaterial were established by testing a series of experimental samples. Substances used for biomaterial production were mixed in the following proportions: collagen - 70%, hyaluronic acid - 29% and elastin - 1%. Distilled water was added, and stirred at medium/maximum speed for 5-10 minutes using a laboratory blender. The liquid was poured over iron and hydrophobic pallets. The liquid was left in a laminar cabinet under ultraviolet radiation for 6 hours with further keeping at room temperature for 18 hours.

Assessment of physical properties of bioplastic material

The material was cut into samples of the same size (1 cm x 1 cm), placed in Petri dishes, immersed in 5 ml of culture medium and visualized under an inverted microscope. Subsequently, the material samples were transferred to a CO₂ incubator (37°C, 5% CO₂). Every 24 hours, the material specimens were weighed, and 10 μl of culture medium were selected to further study the change in optical density using a scanning spectrophotometer NanoDrop 2000C (Thermo Fisher Scientific, USA).

Assessment of cytotoxicity and biocompatibility of the bioplastic material

We studied the cytotoxicity of the bioplastic material on a commercial culture of human dermal fibroblasts (HDF) (Cell Applications, USA; Cat. # 106K-05a). Cells were cultured in СO₂ incubator (37°C, 5% CO₂) in Dulbecco's Modified Eagle’s Medium(DMEM) with 4500 mg/L D-glucose (StemCell Technology, USA; Cat.# 36250), with the addition of 10% of FBS (Biological Industries, Israel) and 2 mmol/L of L-glutamine (StemCell Technologies, USA; Cat.# 07100). The culture of cells was passaged in a d = 94 mm Petri dish, with 25×10³ cells/cm² density and cultured according to standard technique up till the formation of a monolayer. After that, we sampled a supernatant, added a new medium, put in a biomaterial sample and left it in СO₂ incubator for 72 hours. Upon expiry of this period, we sampled a supernatant and transferred it into a new Petri dish to count dead cells. We used Trypsin-EDTA, 0.25% (StemCell Technologies, USA; Catalog #07901) to get cells from the surface of the studied Petri dishes. The Countess automated counter (Invitrogen, Korea) was used to count cells and assess their viability by the manufacturer’s procedure. To study biocompatibility of the bioplastic material, we put 0.5 cm² size samples into Petri dishes and added 1 ml of cell suspension of HDF containing 12.5×10³ cells in each, forming a “drop”. After 30 minutes in СO₂ incubator, the samples were added with a growth medium and cultured for 10 days. To assess the viability of cells, we performed staining with fluorescent dyes SYTO 9 (Invitrogen, USA) and propidium iodide (Sigma-Aldrich, USA).¹⁴ The number of live and dead cells was determined according to the number of cell nuclei revealed by SYTO 9 and propidium iodide, respectively.

Fig 1.

The appearance (A) of the bioplastic material and its structure (B). A, Scale bar = 5cm; B, Scanning electron microscopy. Scale bar = 50 μm

Fig 2.

The dynamics of the bioplastic material mass change in a culture medium. Data are presented as means ± SD.

Visualization of biomaterial samples

Examination of samples of the matrix and cells attached to it was conducted using an inverted microscope Leica DM IL LED (Leica Microsystems, Germany) and a laser scanning confocal microscope LSM 510 META based on an inverted microscope Axiovert 200M (Carl Zeiss, Germany). Image analysis was perform using Zeiss 3D for LSM Version 1.4.2 software (Carl Zeiss, Germany). Scanning electron microscopy (SEM) was carried out using Camscan S2 microscope (Cambridge Instruments, UK) in SEI mode with 10-nm resolution and operating voltage of 15kV.

Statistical analysis

The data are presented as means ± SD. Statistical analysis was carried out using Student’s t-test. The level of statistical significance was set at P < 0.05.

Results & Discussion

We have developed a new bioplastic material based on collagen, hyaluronic acid and elastin and studied its properties. The resulting biomaterial is characterized by high elasticity and resilience, which allows its use on damaged skin areas with uneven relief or on mobile areas. The obtained material has also high porosity, with a pore diameter of 100–200 μm (Figure 1). Thus, the physical structure of bioplastic material allows for gas exchange of wound surface and exudates withdrawal.¹⁵ Taking into account that the obtained biomaterial can be used as a scaffold for cell culture, this structure can provide conditions for cell migration inside as well as homogeneity of cell culture conditions and mechanical properties of the structure. The study of matrix stability in culture medium revealed that this material can preserve its physical properties for more than 10 days (Figure 2). After 5 days, the biomaterial in the CO₂ incubator degraded by 15–18 %. Complete destruction of the biomaterial occurred after 3–4 weeks of incubation. Co-culturing bioplastic material and cultures of human fibroblasts did not reveal any cytotoxic effects on cell cultures. After 72 hours of incubation, no squamous cells in a culture liquid and no changes in morphological parameters of fibroblasts on the plastic surface were observed. Furthermore, we registered a higher proliferative activity of the cells in the presence of material samples than one in the control group. It was noted that all cells attached to the surface within 4-6 hours after the start of incubation when fibroblastswere cultured on the biomatrix. We havebeen culturing fibroblasts for 2 weeks to study thegrowth dynamics of cells in the matrix (Figure 3). Cellcountswere made on the images ofhorizontalprojections of a series of optical slices of thematrix ofknownthickness.The presence of dividing cellswasdetected already in 24 hours after attachment. Thetotal number of cells per mm³was 440±84 (n=10).In 5 days,the number of cells permm³ increased to 776±134(n=10, p<0.001).Themaximum number of cellswasobserved on the 10th day after attachment,whichwasupto 2155±335 cells per mm³(n=10, p<0.001). During culturing on aflatsurface, cell cultures of fibroblastsreached the monolayer on the 6th day.Due toitsproperties and structure, the developed biomaterialcould have a wide range of clinical applications. Thisbiomaterial can be applied as a mask or a patch. Thematerial can also be potentiallyused as a carrier ofdrugs,e.g.,antibacterial drugs,aswellasagentsthataffect cell differentiation, proliferation, and interactiontoproduceviableculturestoreplace lost tissue.Thisbiomaterial seems promising for 3D bioprinting thatwill create three-dimensional structures withdesirable biological, structural, andmechanical properties. Thereis an increasing demand for new biomaterials in 3Dbioprinting.¹⁶The main physical and chemicalparameters determining thesuitability of hydrogel aretheir rheological characteristics and cross-linkingmechanisms. Materialsused for 3D bioprintingmustsupport cell viability and proper functioning, by providing structural andmechanical support for themodel, biocompatibility, andeasymanipulation of thebioprinter for acquisition ofa 3D structure.^17,18Assuch, this biomaterialmeets the major requirementsforpotential usewith 3D printing. The effective solutionfor any existing problems, linked to technical andcellular aspects of this procedure,will make it possibleto obtain samples of various tissues that can in turn beused in regenerative medicine.In conclusion,the developed biomaterial can be usedon damaged skin areas due to its physical properties andstructure.The use of createdwound cover provideseffective conditions for good cell proliferation,whichallows us to consider it as a promisingbiomaterial foruse in clinical practice.

Fig 3.

Biocompatibility (A) and cytotoxicity (B) studies of the bioplastic material. 5 days of culturing. Light microscopy. Scale bar = 100 μm.

List of acronyms

DMEM

Dulbecco's Modified Eagle’s Medium

HDF

Human Dermal Fibroblasts