Performance evaluation of femtosecond laser processed natural bioremediation membranes

doi:10.21203/rs.3.rs-3327868/v1

Background

Natural bioremediation membranes are excellent materials for skin repair. Traditional mechanical processing methods have limitations, including low processing speed and heat accumulation. Therefore, scholars have proposed the femtosecond laser as a novel processing technique to improve processing speed, accuracy, and quality at the same time reducing the heat effect.

Methods

In this study, a smoother and flatter surface without carbonization was obtained by optimizing the laser parameters, then compared with mechanical processing in terms of physicochemical properties and biocompatibility.

Results

The results revealed consistent physicochemical properties of the femtosecond laser interface with wound dressing requirements; moreover, the collagen biocompatibility remained preserved, which was beneficial to the adhesion and proliferation of human dermal fibroblasts.

Conclusions

Therefore, this work validates the feasibility of femtosecond laser in processing natural bioremediation membranes and addresses the challenges of slow processing speed, low precision, and heat accumulation predominant in conventional processing methods. The findings not only provide insights and methods for processing natural bioremediation membranes but also other medical regenerative materials.

Skin injury

Natural bioremediation membrane

Femtosecond laser

Physical and chemical properties

Biocompatibility

Skin injury is a common clinical condition, and the use of skin grafts has increased patient healing rates. The natural decellularized dermal matrix, which retains both collagen components and the natural tissue structure of the dermis, promotes cell proliferation and growth and has attracted research attention[1, 2]. This study focuses on the natural bioremediation membrane, which is a bovine-derived decellularized dermal matrix. Although perforation and cutting of bioremediation are necessary before leaving the factory, mechanical cutting and perforation inevitably result in problems including tool wear and heat accumulation [3]. This may lead to unstable processing quality, and collagen denaturation. Femtosecond laser processing is a newly proposed processing method with extremely short pulse width, therefore, extremely high peak power can be obtained with correspondingly low energy in a single pulse [4]. The adverse effects of mechanical cutting, drilling tools, continuous-wave, and long-pulse laser cutting can be overcome using ultra-short pulse laser due to its non-contact nature during processing. During thermal diffusion of the material, the ultra-short pulse laser can transfer energy to the target material, ensuring "cold processing", thus generating clean and neat edges [5–9]. The ultra-short pulse laser can be used for processing biological tissue, inflammable and explosive materials, etc. Moreover, the femtosecond laser can process a wide range of materials, essentially any material, and brittle material processing does not generate cracks [10–14].

The preparation of microns or nanostructures on biomaterials has significant potential in the biomedical field. Due to the incompatibility of biomaterials with conventional micro and nanofabrication processing techniques, femtosecond lasers are considered for processing, fabricating, and customizing materials for organic photonics, electronics, medicine, and bioengineering [15–19]. Yu et al [20] discovered that femtosecond lasers could achieve defect-free and crack-free micropatterning without damaging protein crystals. Ashforth et al [21] comprehensively studied femtosecond laser microsurgery on bovine and sheep cortical bone using a femtosecond laser, which was completely free of heat shock-induced damage compared to mechanical tools that increase the temperature when cutting.

Since the early days of research, femtosecond laser processing has rapidly developed in various fields, changing the conventional concept of laser processing, and gradually developing into a powerful and accurate processing method. Therefore, this work uses femtosecond lasers to cut biological membranes, where the structure of natural collagen is extremely unstable and easily denatured by temperature and UV light. A series of experiments were conducted before measuring the real-time processing temperature, which varied between 30 and 40°C (not instantaneous) during the femtosecond laser processing. Consequently, the collagen denaturation temperature in the solution state was approximately 38°C. As the collagen water content decreases, the denaturation temperature increases, hence the dry collagen denaturation temperature should be higher than 38°C. Further, we evaluated the physicochemical properties and biocompatibility of the processed bioremediation membranes to assess the feasibility of studying natural bioremediation membrane processing mediated by femtosecond laser. As a consequence, we confirmed that the physicochemical properties of the femtosecond laser-processed bioremediation membranes met the requirements of medical wound dressings and retained the three-stranded helical structure of collagen; this helps in the adhesion and proliferation of human dermal fibroblasts. Therefore, we believe that the femtosecond laser processing of bioremediation membranes is feasible, although it should be confirmed in upcoming trials.

2.1 Experimental reagents and equipment

KBr, Acetic acid, Deionized water, Collagenase (BR Ⅰ,> 125CDU/mg), Phosphate buffer salt solution, Natural bioremediation membrane, Femtosecond laser (FemtoYL-50), Scanning electron microscope (SEM) (Regulus8220, Fourier infrared spectrometer (FTIR) (Nicolet10), Circular dichromatic spectrometer (CD) (Chirascan), Electronic universal testing machine (AGS-X5KN), Freezing grinder (RETSCHCryomill), Human dermal fibroblasts, Special culture medium for fibroblasts, 0.25% trypsin, CCK-8 reagents, Cakcein-AM/EthD-1(calcein/ethingot homopolymer) mixed solution, 48/96-well cell culture plate, Cell counter, Cell incubator, microplate reader, Inverted fluorescence microscope inverted phase-contrast microscope.

2.2 Experimental design

Femtosecond laser processing samples

Figure 1a shows the experimental setup. The experiment used a FemtoYL-50 femtosecond laser system, and a bovine collagen film sample (Yantai Zhenghai Biotechnology Co., Ltd.) with 0.4–0.8 mm thickness. The parameters were chosen to maximally prevent collagen denaturation and eliminate visible carbonization from the processed interface, primarily based on visual and scanning electron microscopy and ultra-deep field 3D microscopy to observe the extent of carbonization.

The quality of the process is influenced by several factors. The amount of defocusing affects the size of the laser spot, which in turn affects the width and taper of the slit. The cutting speed and laser pulse frequency affect the width of the slit and the surface flatness. Processing power affects the width and depth of the cut. The power was initially set at 18.45W based on the material and thickness of the bioremediation membrane, the scanning speed at 300mm/s, frequency at 200KHz, the number of scans at 30, and the focal length at 44.94mm. Figure 1(b) shows the first processing, the out-of-focus amount was set at 2mm, and the surface of the processed sample exhibited visible carbonization, and there was melting. Other parameters remained constant and the defocusing amount was set at 0. The sample was processed again, and the surface of the processed sample had no obvious carbonization to the naked eye in Fig. 1(d). Figure 1(e) shows no melting of the processed edges and a relatively well-maintained fiber structure.

Based on the above processing parameters, we increased the processing speed by increasing the processing power, laser frequency; and scanning speed.

(1) Increasing the processing power

First, the processing power was increased to 25.12W and the results did not improve the processing speed at the same time maintaining the processing quality. In contrast, Fig. 1(f) shows that the carbonization of edges was more serious and the edges became black when observed under a microscope.

(2) Increasing the laser frequency

By adjusting the laser frequency to 300 kHz and keeping the rest of the parameters unchanged, the processing at a suitable interval did not trigger carbonization at the edges, and remained flat (Fig. 1(h)); however, more burrs were in Fig. 1(i) than the laser frequency of 200 kHz.

(3) Increasing the scanning speed

The scanning speed positively correlated with the instantaneous temperature generated during processing, i.e., the faster the scanning speed, the higher the processing temperature. During processing, the borders were easily carbonized to varying degrees after increasing the scanning speed (Fig. 1(j)); thicker areas requiring more than 30 repetitive scans were more severely carbonized (Fig. 1(k)), which was analyzed due to too fast scanning speed. This was due to too fast scanning speed and increased processing temperature.

Through the previous testing phase, balancing all factors, we eventually selected the power of 18.45W, scanning speed of 300mm/s, frequency of 200kHz, out-of-focus amount of 0, and 30 or 60 scans (30 scans in the thinner areas and 60 scans in the thicker areas due to uneven thickness of the sample film) as the optimized parameters to process subsequent samples. The cut surface of the bioremediation membrane cut by this set of parameters was smooth and burr-free, with no carbonization, resulting in a good-quality machined surface.

The mechanical method utilizes a mechanical tool for contact scribing and is currently the most established and commonly used method for processing bioremediation membranes. All test subjects in subsequent experiments were set up as laser and mechanical groups.

The elements of the processing area

The composition of the laser-processed sample was tested before subsequent tests. First, scanning electron microscopy was used for energy spectrum analysis. Sample films of the mechanical and laser group were obtained, respectively. The rough surfaces of the two groups of samples were fixed on the loading platform, and the samples were sprayed with gold at low temperatures. The loading platform was subsequently fixed on the SEM sample table, and the elements in the film and two different processing interfaces were analyzed qualitatively.

FTIR analysis: The samples from the mechanical and laser groups were cut 1cm from the processing interface, and ground to a powder, before mixing with KBr at a ratio of 1:150. The well-mixed sample mixture was transferred to a mold and pressed into ingots under high-light transmission. The spindle was placed on a measuring table before switching on the Fourier transform infrared spectrometer; the scanning interval was set to 4000 − 500 cm^− 1 and the resolution was 4 cm^− 1. A total of 16 scans were taken for each sample and the two sets of bioremediation membranes were replaced in turn.

UV-Vis absorption spectroscopy

A sample solution with 0.5 mg/ml concentration was prepared, and 0.05 mol/L acetic acid solution was used as the reference; acetic acid and sample solutions of the mechanical and laser groups were injected into the quartz cuvette respectively; the wavelength range was set to 190–300 nm; the scanning interval was 1 nm, and the scanning speed was 100 nm/min.

CD analysis

A 30mg mechanical sample and a 30mg laser sample were dissolved in 0.05mol/L acetic acid solution and 0.5mg/mL sample solution, respectively, before injecting into a 1mm diameter quartz cell at 1000L/h flow rate. The flow rate of the nitrogen flow meter was adjusted to 400L/h. The scanning rate was 100 nm/min, and 3 scans were performed for the reference and sample in sequence.

Molecular particle size

A sample solution with a concentration of 0.5mg/ml was prepared and the trend of the particle size with temperature was measured using a Zeta potential and a nanoparticle size tester, respectively. The mean particle size was the average of the three parallel tests.

Tensile strength

The samples were cut into 60mm×10mm rectangular specimens by mechanical and laser methods, and two sets of membrane samples were subjected to tensile testing using an electronic universal testing machine, with the loading speed set at 2mm/min and the sample membrane fractured from the middle third. Twenty samples were measured in each group and the arithmetic mean was taken.

The experimental results were computed based on the following formula:

Tensile strength: σ = F_b / S₀

Elongation at break: σ_h = (L_h − L₀) / L₀ × 100%

In vitro degradation capacity

The samples were then weighed and the mass m₁ was recorded. The samples were placed in test tubes, sealed, and placed in a constant temperature water bath set at 37°C. The samples were removed at 6h, 10h, 24h, 30h, 34h, and 48h of degradation, washed several times with deionized water, blotted with filter paper, and dried in a vacuum oven at a temperature of 25°C for 24h. The sample film was removed and weighed (recorded as m₂).

The degradation rate of the sample was calculated using the formula.

D = (m₁ − m₂) / m₁ × 100%

Hygroscopicity test

Six groups of sample films (2.5cm×2.5cm) were dried in an oven for 4h, then the dry weight m₁ was weighed and recorded. Six 50×30 weighing bottles were labeled as the mechanical group and laser group in half, and the sample films were placed into the corresponding numbered weighing bottles, respectively. Exactly 10ml deionized water was added to each weighing bottle, and soaked for 24h. One corner of the membrane was lifted using tweezers until there were no drops. An electronic balance was used to accurately weigh and record the mass m₂.

The bioremediation membrane water absorption was computed by the following water absorption formula:

W= (m₂ − m₁) / m₁ × 100%

Determination of pH

Exactly 1g of each sample film of the mechanical group and laser group was soaked in 10ml of deionized water at 37℃ for 12h.The degradation solution of biofilm degraded in PBS solution for two months was also taken. The pH values of the different samples were measured using an electronic precision pH meter on five parallel samples from each group. The electrode was cleaned with deionized water; the electrode surface was dried; the pH meter was preheated for more than 5 min before calibration; the electrode was cleaned with deionized water and the solution to be measured, inserted into the sample solution, before reading the pH value after digital stabilization. The measurement was repeated three times for each sample solution.

Co-culture of fibroblasts and natural bioremediation membrane

The bioremediation membrane was processed in the ultra-clean table by drilling into the circular bioremediation membrane with a diameter of 6mm as the mechanical group, whereas the bioremediation membrane with a diameter of 6mm was cut by laser as the laser group. The two groups of membrane materials were immersed in 75% alcohol for 2h for disinfection; the sample materials were then rinsed 3 times with PBS, and irradiated with a UV lamp for 30min. Each group of triple holes was placed at the middle bottom of the 96-well plate. The suspension of the third generation of fibroblasts was inoculated at the center of the sample surface at a concentration of 2×104 cells/mL, 500µL per well, and placed in a cell culture box at 37℃ for compound culture. The medium was changed every 2–3 days. At 12h, 24h, 48h, and 72h of culture, the surface of the material was gently washed with PBS, and the adhesion and growth of the membrane surface were observed under a phase-contrast microscope.

CCK-8 cell proliferation detection

In the ultra-clean table, the bioremediation membrane was processed into a circular bioremediation membrane with a diameter of 6mm through-hole drilling as the mechanical group. Bioremediation membranes with a diameter of 6mm were cut by laser as the laser group. In the ultra-clean bench, 0.5 ml of PBS solution was added to each well plate of the 96-well plate, placing two groups of membrane samples at the bottom center of the well plate. A blank group without membrane samples was set up, and the pre-added PBS buffer solution was discarded in the well plate before adding the fibroblast special medium. The medium in the well plate was absorbed and discarded after incubating for 12h in a CO₂ cell incubator (37℃, CO₂ 5.0%).

The third-generation fibroblasts, which had grown into a dense monolayer, were removed from the cell culture chamber, and the original culture medium was vacuumed with a pipette. Exactly 1ml trypsin digestion solution was added to the T-bottle, and the T-bottle was placed in the cell culture box at 37℃. After the cells became round and fell off from the T-bottle, a fibroblast special culture medium was added to the T-bottle to terminate digestion. The cells were then blown away and the cell concentration was adjusted to 2×104 cells/mL with a fibroblast special medium. The suspension of the third generation of fibroblasts was inoculated on the sample surface at a concentration of 2×104 cells /ml, 500µL per well, and combined culture was conducted in the cell incubator. The medium was changed every 3 days, and on the 1st, 3rd, 5th, and 7th days of culture, respectively. Exactly 10µL 10% CCK-8 reagent was added into 3 wells from each group. The well plate was placed into the saturated humidity cell culture box for further cultivation for 4h. Afterward, the absorbance value at 450nm was measured with a microplate reader; the blank well was adjusted to zero. CCK-8 reagent was also added to the blank control group without membrane samples. Experiments were conducted under similar conditions. Eventually, the time and absorbance values were used as coordinates to draw the cell proliferation curve.

Double fluorescence staining with calcein/ethidium homodimer

In the ultra-clean table, the bioremediation membrane was processed into a round bioremediation membrane with a diameter of 10mm through-hole drilling as the mechanical group. The bioremediation membrane with a diameter of 10mm was cut by laser as the laser group. They were placed at the middle bottom of 48-well plates, and a blank control group without membrane samples was set. The third-generation fibroblast suspension was inoculated on the surface of the sample at a concentration of 2×104 cells /ml, 500µ L per well, and the medium was changed every 2 days. On the 7th day of culture, 6 wells were taken respectively, cleaned twice with PBS, and a suitable amount of calcein/ethidium homodimer mixed dye was added into the Wells for staining. Subsequently, the cell surface was washed with PBS, and the cell growth as well as adhesion on the material surface were observed under blue excitation light (490nm) in an inverted fluorescence microscope.

3.1 Structural characterization

3.1.1 Analysis of element variation in processing regions

Table 1

Element contents of the two groups
Sample number	N(%)	C(%)	H(%)	O(%)
Machinery	15.63 ± 0.22	42.19 ± 0.31	6.65 ± 0.12	32.54 ± 0.26
Laser	15.58 ± 0.19	42.22 ± 0.25	7.00 ± 0.10	33.43 ± 0.29

Energy dispersive spectroscopy was used to characterize the material surface elements and qualitatively analyze the elemental species of bioremediation membranes at a specific range for both processing methods. Figure 2 shows the analysis of cutting-edge element types in the bioremediation membrane sample. The results showed that besides C, N, and O, the bioremediation membrane materials had element H (Table 1) No difference in the contents of each element was found between the two groups. The content percentage of each element displayed by the elemental analyzer was consistent with that of the energy spectrum. The content of C was the highest, followed by O, N, and H. Therefore, the laser processing process will not alter the elements of the material.

3.1.2 SEM observation of edge structure

The bioremediation membrane requires burr-free when detecting the edge. SEM was adopted to characterize the boundary of the bioremediation membrane processed in two approaches. As shown in Fig. 3 from the microscopic perspective, both groups had a small amount of burr on the cut edges, with no fiber melting occurring, and the laser group had smoother edges. Thus, we believe the boundary of the laser-processed bioremediation membrane has a higher quality.

3.1.3 FTIR analysis

As shown in Fig. 4 by FTIR, the two groups have apparent infrared absorption near 2929cm^− 1, a characteristic absorption frequency of the vas (CH₂) group, and can be the difference between collagen molecules and other protein molecules. The absorption band near 1240cm^− 1 is due to the amide III band and amide II bands. The absorption band of collagen protein is highly responsive to alterations in its secondary and tertiary structures. This sensitivity is closely linked to the collagen's triple helical arrangement, where the ratio at 1450 cm-1 can elucidate the integrity of this structure. When the ratio hovers around 1, it indicates the preservation of the intact triple helical structure of collagen [22]. The ratio of denatured collagen was approximately 0.5. As shown in Table 2, the ratio was 1.04 for the laser group and 1.02 for the mechanical group. The characteristic absorption frequencies in the infrared spectra of the two groups of samples were similar, and consistent with collagen characteristics. Therefore, laser processing does not cause collagen degeneration and meets bioremediation membrane processing requirements.

3.1.4 UV-Vis absorption spectroscopy

Collagen contains almost no tryptophan, which absorbs strongly at 280 nm, whereas the C = O, -COOH, CO-NH₂ groups in the collagen molecule, which produce large light absorption near 220nm, can be used as a method to identify collagen and other proteins. This can be used as a method to identify collagen and other proteins.

As shown in Fig. 5, the UV absorption profiles of collagen in the mechanical and laser groups were not different, with maximum absorption peak in the laser group at 221 nm. The maximum absorption peak in the laser group was 221 nm. The maximum absorption peak in the mechanical group was 222 nm. Therefore, we tentatively concluded that both groups matched the characteristic values of natural collagen.

3.1.5 CD analysis

The disruption of the three-stranded helical structure of collagen can influence wound repair. Figure 6 shows the CD of the two membrane groups. As shown, both groups showed positive and negative peaks in the scan range, with positive peaks at 222 nm in both groups, both of which did not disappear, and negative peaks at 197 nm, which did not undergo a red shift.

The ratio of the positive and negative peak intensities of the collagen CD (Rpn) can be used to confirm the triple-stranded helical structure of the collagen solution. The Rpn values computed from the data in Table 3 were 0.1186 for the laser group and 0.1256 for the mechanical group. This indicates that the femtosecond laser exerts an effect on the hydrogen bonds within the collagen molecules and some of the three-stranded helical structure was partially loosened. Both mechanical and laser processing could retain the three-stranded helical structure of collagen well, and the optimized laser processing had nearly no effect on the three-stranded helical structure of collagen molecules.

Table 3

Location of characteristic absorption peaks of the two groups
	Positive peak wavelength (nm)	negative peak wavelength (nm)	Rpn
Machinery Group	222	197	0.1256
Laser Group	222	197	0.1186

3.2 Performance analysis

3.2.1 Variation of molecular particle size with temperature

Laser particle size and zeta potentiometry can precisely measure the particle size of materials in the 0.6nm-6000nm range. Within a certain interval, as the temperature increases, collagen molecules with a stable three-stranded helical structure can be interconnected by intermolecular forces of hydrogen bonding to form collagen microfibrils, further forming a higher spatial structure of collagen fibril bundles and increasing collagen particle size.

Figure 7 shows the trend of the average particle size of the mechanical and laser group solutions with temperature. The results show that the average particle size of the two groups reaches its maximum value at around 30°C, after which the average particle size rapidly decreases with increasing temperature. This shows that both sample solutions can strongly aggregate and form fibrillated collagen at around 30°C. At a certain temperature, the molecules in the collagen solution can be reattached, which is known as the re-fibrillation of collagen, whereas denatured collagen lacks this unique re-fibrillation.

As shown in the graph above, the molecules in both sets of solutions tend to aggregate with each other as the temperature increases within a certain range, and the strongest aggregation ability is observed around 30°C. It is evident that neither group has been denatured, and that both groups can aggregate to form a three-stranded helical and complex spatial structure of fibrous collagen under appropriate temperature conditions.

3.2.2 Tensile strength analysis

The elongation at break indicates the tearing resistance of materials. For polymer materials, the higher the elongation at break, the stronger the tearing resistance, the better the toughness, and the lower the strength and hardness. Figure 8(a) shows the tensile strength distribution of the two sample groups. As shown above, the tensile strength of the 40 sample groups was between 15 and 60MPa, which is good and adheres to clinical medical dressing requirements. Figure 8(b) shows elongation distribution at the break of the two groups of samples; the elongation at the break of the 40 sample groups was concentrated in the range of 3.2%-8.3%, and both groups of materials met the requirements.

3.2.3 In vitro degradation capacity

The degree of influence of type I collagenase on the degradation rate and rate of the two groups of membrane samples was analyzed, as shown in Fig. 9. The degradation rate of both groups in the degradation solution increased with time, and the overall degradation rate of the mechanical group was slow unlike that of the laser group. The degradation rate of the laser group was 69.77% after 48h, whereas the degradation rate of the mechanical group was 55.34%. Both groups had been degraded by more than 50%. The reason for the shorter degradation time of the laser group may be that the laser processing loosens the three-strand spiral structure of collagen edges and weakens the hydrogen bond, thereby making degradation easy.

3.2.4 Hydroscopicity test

Bioremediation membranes with good water absorption can prevent further wound infection, and water absorption tests were performed on both groups of membranes. Figure 10 shows mass changes of the two membrane groups measured after water absorption. The bioremediation membrane, whether mechanically processed or laser processed, is much larger than the requirement of a 35% water absorption rate, clinically meeting the need for rapid absorption of exudate from the wound in the pre-treatment period.

3.2.5 pH value determination

Table 4

pH values of dissolved solutions of the two groups
The sample	The pH of the leached solution
Deionized water	5.50
Mechanical group deionized water solution	6.84 ± 0.09
Laser group deionized water solution	6.63 ± 0.14
PBS	7.40
Mechanical group PBS degradation solution	7.54 ± 0.09
Laser group PBS degradation solution	7.90 ± 0.11

Of note, the pH value of the internal environment of a healthy human ranges between 7.35 and 7.45, which is weakly alkaline. If the pH value of body fluids shifts from the normal range, viruses, and bacteria multiply, damaging normal cells and affecting the normal healing of wounds. Table 4 shows the results of pH measurements.

As shown, the pH value of deionized water for the blank control solution was 5.50, which is weakly acidic; the pH value of deionized water for the bioremediation membrane in the mechanical group was 6.84 ± 0.09; the pH value of deionized water for bioremediation membrane in the laser group was 6.63 ± 0.14, which is within the specified range. In Table 4, the pH value of PBS was 7.40. The pH value of the solution after two months of bioremediation membrane degradation in PBS for the mechanical group was 7.54 ± 0.09, and that for the laser group was 7.90 ± 0.11. The difference between the pH value and that of the PBS solution was not more than 1.5, which adhered to the regulations for medical devices. Both groups of bioremediation membranes met the requirements in terms of the pH value of the lysate and could be used as tissue engineering materials. The pH values of both groups remained weakly alkaline after degradation, which is beneficial to wound healing.

3.3 Biocompatibility analysis

3.3.1 Co-culture of fibroblasts and natural bioremediation membrane

When cells are cultured on the material surface with toxicity, it will impede cell growth. Therefore, the most direct approach to determine whether a material is cytotoxicity is to conduct a compound culture of materials and cells and observe cell growth [23]. Figure 11 (a, c) shows the microscope pictures of fibroblasts co-cultured with sample membranes of the mechanical and laser groups for 12h, respectively. As shown, the cells are small in volume and long in shape under the microscope. Figure 10 (b, d) shows the images of 24h co-culture respectively. When compared to 12h, cell volume increased and the morphology relaxed, (e, g) and (f, h) are co-culture images for 48h and 72h, respectively. At this point, the cell morphology was more spreading, the number of cells increased, cells were mostly long shuttle-shaped, the bulges between cells were connected; the cell morphology became clearer, and the cytoplasm was abundant, all of which were fibroblast-like characteristics.

3.3.2 CCK-8 cell proliferation detection

The degree of cell proliferation reflects whether the material has cytotoxicity and whether it is beneficial to cell adhesion, growth, and proliferation. The degree of cell proliferation was measured by CCK-8. As shown in Fig. 12, the absorbance value of both the mechanical group and the laser group was higher than that of the blank control group within 7 days; this indicates that the number of cells in both groups was higher than that of the blank control group. After the 5th day, there was an increase in the number of cells in the mechanical and laser groups, whereas that in the control group decreased after the 3rd day. The cell number of the mechanical and laser groups started to steadily increase after day 5, and the cell number in the mechanical and laser groups was significantly different from that of the control group on the 7th day (P < 0.05). The results showed that both bioremediation membranes have good biocompatibility and are appropriate for adhesion, growth, and proliferation of human dermal fibroblasts.

3.3.3 Double fluorescence staining with calcein/ethidium homodimer

The number of living cells directly confirms whether the bioremediation membrane has good biocompatibility. Living cells fluoresce green, whereas dead cells fluoresce red by calcein/ethidium homopdimer staining. As shown in Fig. 13, the fibroblast growth on the surface of the three sample groups was in a good state. Most of the cells demonstrated typical characteristics of fibroblasts, with a long spindle shape or irregular triangle shape, apparent nucleus, and round or oval shape. The above figure shows numerous live cells and invisible red dead cells. By comparing the three groups, the laser group has a more positive effect on cell viability because the cells are arranged in a swirling pattern and the number of live cells is higher and denser than the other two groups.

In summary, this paper sought to assess the feasibility of using a femtosecond laser to process natural bioremediation membranes. The final optimized parameters of 18.45 W power, 300 mm/s scanning speed, 200 kHz frequency, 0 defocusing, and 30 or 60 scans were selected to process subsequent samples. The cut surface of the bioremediation membrane with this set of parameters was smooth and burr-free, with no carbonization, resulting in a good-quality surface.

The processing of bioremediation membranes using the femtosecond laser and the physicochemical properties and biocompatibility of the samples were investigated and the following conclusions are drawn: (1) The results of FTIR and CD show that the characteristic absorption of the material at the processed interface is consistent with that of natural collagen at three different wavelengths. It is proved that laser processing does not affect the type and content of elements in the processed interface. (2) Through the analysis of physical and mechanical properties, the laser-processed biological repair membrane meets the requirements of medical-biological dressings in terms of water absorption(much larger than the water absorption rate of 35% requirements), tensile strength (The tensile strength of 40 sample films is between 15-60MPa), thermal performance (The aggregation capacity is strongest near 30℃), and pH value (6.63 ± 0.14). This conclusion provides strong data support for femtosecond laser processing of natural bioremediation membranes. Besides, the enzyme degradation rate of the laser-processed bioremediation membrane was slightly faster than that of the mechanical group. At 48h, the laser group degraded 69.77%, the mechanical group degraded 55.34%, both groups have degraded more than 50%, it is speculated that the two groups will be completely degraded in about 4 days. (3) Biocompatibility study of femtosecond laser processing of the interface cell viability is more conducive to the adhesion, growth, and proliferation of human dermal fibroblasts.

At present, femtosecond laser processing natural bioremediation film only stays in the laboratory stage, is not currently used in industrial production, the future should be its large-scale application and intelligent upgrading of extensive research, to replace the traditional processing methods to improve the purpose of production efficiency, which plays an important role in the medical and industrial progress.

Our findings indicate that laser processing of bioremediation membranes is feasible, solving the problems of slow processing speed, low precision, and heat accumulation that plague conventional processing methods. Moreover, it can completely replace mechanical processing, providing ideas and methods for realizing the processing of natural bioremediation membranes as well as other medical regenerative materials.

Funding information

This work is supported by the Key Research and Development Plan of Shandong Province (2018GGX103006), the National Natural Science Foundation of China (52205480), Yantai High-end Talent Introduction "Double Hundred Program" Special Funding, 2021.

Conflict of interest

The authors declare that they have no competing interests.

Ethics approval

Compliance with ethical standards.

Authors' contributions

All authors contributed to the study's conception and design. Material preparation, data collection, and analysis were performed by Jiahui Zhang, Li Wang, and Yihua Feng. The first draft of the manuscript was written by Zefei Zhang and all authors commented on previous versions of the manuscript. Yujing Sun and Li Wang made final revisions to the manuscript. All authors read and approved the final manuscript.

Data Availability

The datasets supporting the conclusions of this article are included within the article.

Acknowledgements

We are grateful to the Yantai Zhenghai Bio-Tech Co., Ltd for their bioremediation

membranes and professional assistance with the study.

Consent for publication

All authors are consent for publication.

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Performance evaluation of femtosecond laser processed natural bioremediation membranes

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Abstract