Cellular and Molecular Biology Radiation-induced graft polymerization of elastin onto polyvinylpyrrolidone as a possible wound dressing

: The purpose of our study was to obtain new wound dressings in the form of hydrogels that promote wound healing taking advantage of the broad activities of elastin (ELT) in physiological processes. The hydrogel of ELT and polyvinylpyrrolidone (PVP; ELT–PVP) was obtained by cross-linking induced by gamma irradiation at a dose of 25 kGy. The physicochemical changes attributed to cross-linking were analyzed through scanning electron microscopy (SEM), infrared spectroscopy analysis with Fourier transform (FTIR), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). Furthermore, we performed a rheological study to determine the possible changes in the fluidic macroscopic properties produced by the cross-linking method. Finally, we ac complished viability and proliferation analyses of human dermal fibroblasts in the presence of the hydrogel to evaluate its biological characteristics. The hydrogel exhibited a porous morphology, showing interconnected porous with an average pore size of 16 ± 8.42 µm. The analysis of FTIR, DSC, and TGA revealed changes in the chemical structure of the ELT–PVP hydrogel after the irradiation process. Also, the hydrogel exhibited a rheological behavior of a pseudoplastic and thixotropic fluid. The hydrogel was biocompatible, demonstrating high cell viability, whereas ELT presented low biocompatibility at high concentrations. In summary, the hydrogel obtained by gamma irradiation revealed the appropriate morphology to be applied as a wound dressing. Interestingly, the hydrogel exhibited a higher percentage of cell viability compared with ELT, suggesting that the cross-linking of ELT with PVP is a suitable strategy for biological applications of ELT without generating cellular damage.


Introduction
Skin lesions due to burns, pressure ulcers, or diabetes represent a global problem due to the exceptional management required. The significant fluid loss, tissue damage, and the probability of infection exemplify parameters to consider in wound healing. In this regard, the use of dressings as a treatment for wound healing signifies an attractive option because they promote an ideal micro-environment for recovery, preserve the moisture in the wound, provide adequate gas exchange, stimulate the synthesis of growth factors, and protect the wound from pathogens (1,2). In recent years, the use of wound dressings based on biopolymers has generated massive interest because they offer certain advantages over other materials (3)(4)(5), such as biocompatibility, high biodegradability, and the ability to stimulate the tissue regeneration (6). Moreover, these biopolymers can be combined with other synthetic polymers to form reinforced dressings such as hydrogels.
Hydrogels are three-dimensional networks characterized by their high hydrophilicity. These can be obtained by physical or chemical methods of poly-mers cross-linking, such as gamma irradiation (7,8). Cross-linking by gamma irradiation is an efficient method for hydrogels synthesis (9) because the gamma rays possess the required energy to ionize molecules either in the air or in water (10), generating reactive sites along the polymer chain, allowing the combination of these radicals, and leading to the formation of a large number of cross-links (7). Besides, this chemical modification improves the physicochemical and mechanical properties of the combined biopolymers, while their natural biocompatibility, biodegradability, and lack of immunogenicity are retained.
In this respect, elastin (ELT) and polyvinylpyrrolidone (PVP) are polymers widely used for the manufacture of biomedical scaffolds (11). ELT is a structural protein of the extracellular matrix that provides elasticity and strength to various tissues (12). It also modulates cellular behavior, triggering biological responses such as chemotaxis, cell migration, and proliferation (13). Likewise, ELT is part of the architecture that supports cell growth, and its synthesis is involved in the production and maintenance of tissues. Due to these functions, ELT allows the formation of sophisticated biomaterials (14). On the other hand, the PVP is a biocompatible and biodegradable polymer that exhibits interesting biological properties, including low toxicity, the suitable transmission of water vapor, and impermeability to bacteria (8).
This study aimed to obtain a new polymer (ELT-PVP hydrogel) through the simultaneous irradiation of polymers in aqueous solution (15). The hydrogel was characterized through physicochemical and biological techniques to describe its novel chemical structure and biological compatibility as a potential activity for wound dressing in the repair of wounds. The hydrogel presented interconnected porous, which could permit cell internalization and migration during wound healing. It also showed a rheological profile of a pseudoplastic type due to the cross-linking of ELT and PVP. Regarding the biological activity, cell proliferation and viability tests were performed in human dermal fibroblasts, which revealed that the ELT-PVP hydrogel was biocompatible after the chemical cross-linking.

Synthesis of ELT-PVP hydrogel
Alpha fraction hydrolyzed ELT was blended with PVP K-30 maintaining a 15:1 ratio of PVP to ELT (7.45% w/v and 0.5% w/v, respectively) to obtain a final concentration of 7.95% w/v. The mixture was irradiated at 25 kGy with 60 Co in a Gammabeam 651PT deep pool type radiator (Instituto de Ciencias Nucleares, Universidad Nacional Autónoma de México, Mexico City). For dosimetry, Fricke dosimeters, acrylics, and radiochromic dye films were used. ELT and copolymer (ELT-PVP) samples were lyophilized at conditions of -49 °C and 0.06 mBar for 24 h (FreeZone 1, Labconco ® , MO, USA).

Scanning electron microscopy
In order to determine the morphology of the ELT-PVP hydrogel, the lyophilized sample was analyzed by scanning electron microscope (SEM; JCM6000, Jeol, Japan) under high vacuum conditions, and the pore size was determined using the ImageJ software.

Swelling ratio test
Dry and pre-weighed samples of ELT-PVP hydrogel were immersed into PBS 1X at room temperature to evaluate the absorption capacity. The excess of PBS was removed at different time intervals (0, 1, 2, 3, 4, 5, 6, and 7 days), and the weight of the samples was recorded. For each time, three samples were evaluated, and the results were expressed as mean ± SD. The percent of swelling ratio was calculated using the following formula: Where W i refers to the weight of the dry sample, and W f is the weight of the swollen sample.

Spectral analysis
Infrared spectroscopy analysis with Fourier transform (FTIR) was carried out to determine the functional groups present in the samples of the ELT-PVP hydrogel. The evaluation was performed through an infrared spectrometer (Alpha-FT-IR Bruker, USA), and measurements were made in a range of 400 to 4,000 cm -1 , with a resolution of 2 cm -1 .

Thermal properties
The thermogravimetric analysis (TGA) of the lyophilized samples was carried out in a calorimeter Q5000 (TA Instruments, Delaware, USA) to evaluate the thermal properties of the polymers. The analysis was carried out at a heating rate of 10 °C/min, with a temperature range of 0-800 °C, under a nitrogen atmosphere. The weight loss was determined from the experimental data. Differential scanning calorimetry (DSC) was performed on a calorimeter Q2000 (TA Instruments, Delaware, USA) at a heating rate of 10 °C/min, with a temperature range of 20-350 °C. Enthalpy changes were determined from the experimental data. culture conditions (37 ºC, 5% CO 2 ). Images of hydrogel were recorded at 1, 3, and 7 days of culture in a ZEISS Axio Zoom.V16 Microscope.

Morphology of the ELT-PVP hydrogel
The morphology of the ELT-PVP hydrogel was examined by SEM; its microstructure is depicted in Figure 1. The new biopolymer presented an interconnected porous structure, where the average pore size was 16 ± 8.42 µm.

Swelling
The swelling behavior of ELT-PVP is showed in Figure 2. The highest percentage of the swelling ratio (40 %) was observed at 72 h of the experiment. Negative values indicated the dissolution process after reaching the water uptake equilibrium.

Spectral analysis
In order to analyze the possible chemical changes due to gamma radiation, FTIR was performed to ELT, PVP, ELT+PVP, and ELT-PVP ( Figure 3). For ELT spectrum (line a), the signal of 1,646 cm -1 corresponded to the amide I band, while the bands

Rheology
In order to determine the viscosity profiles, the rheology analysis was performed in a viscometer (Brookfield CAP 2000 viscometer, USA), using the No.1 needle, with a constant temperature of 37 °C and controlled humidity. The shear rate experiment was performed from 1,600-8,000 s -1 and vice versa at intervals of 1,600 s -1 , the viscosity records were made after rotating for 20 s at each shear rate (16). Also, experiments at the constant shear rate were carried out at 2,000 s -1 during 1,800 s. The viscosity record was made every 120 s. Each sample was analyzed in triplicate using 250 µL of sample per analysis.

Cell viability assay
Calcein and ethidium homodimer (EthD-1) assays were carried out in human dermal fibroblasts to analyze the hydrogels cytotoxicity. Viability/ cytotoxicity Kit for Mammalian Cells was used according to the manufacturer´s specifications. Fibroblasts were cultured with DMEM/F12 medium supplemented with 10% w/v of FBS and 1% w/v of penicillin/streptomycin. After 10 th pass, 10,000 cells were cultured per well in 48 well culture plates and were treated with the following experimental conditions: PBS (control), ELT (0.1, 0.15, 0.2 and 0.25% w/v), or ELT-PVP (0.1, 0.15, 0.2 and 0.25% w/v). The concentration ratio of ELT-PVP was according to the concentration of ELT in the hydrogel. The cells were incubated with the treatments for 24 hours; then, they were stained with calcein (1 μM) and EthD-1 (2 μM) for one hour. Photographs were taken in an epifluorescent microscope (Zeiss Axiovert), and the number and percentage of positive cells for calcein and EthD-1 were counted using Im-ageJ software.

Cell proliferation assay
The analysis of the promotion of cell proliferation by the hydrogels was performed through a proliferation curve using crystal violet (CV) and human dermal fibroblasts. First, 2,500 fibroblasts were seeded in a 48 well culture plates and incubated with the following experimental conditions: PBS (control), ELT (0.15, 0.2 and 0.25% w/v), or ELT-PVP (0.1, 0.15, 0.2 and 0.25% w/v). The concentration ratio was according to the concentration of ELT in the hydrogel ELT-PVP. Each 24 h during three days, cells were fixed with paraformaldehyde 4% w/v, washed with PBS 1X, and then stained with 5% w/v of CV for 15 min. After, the cells were washed ten times with water, and the wells were dried with filter paper. Finally, 200 μL of methanol was added, and the optical density was measured at 570 nm in a plate reader (Synergy) to obtain the CV concentration. The results were compared with a standard curve to register the number of cells each day of the experiment.

Stability of ELT-PVP in cell culture medium
In order to analyze the ELT-PVP stability and degradation, the samples were immersed in DMEM medium supplemented with FBS, maintaining the  observed at 1,538 cm -1 and 1,222 cm -1 were attributed to amide II and amide III, respectively. These types of signals are commonly presented in proteins spectra (17,18). For the PVP spectrum (line b), the bands located at 1,646 cm -1 , 1,407 cm -1, and 1,276 cm -1 corresponded to the absorption band of the carbonyl group, the stretching vibration of CH bond, and the CN stretching vibration, respectively. These vibrations are characteristic of PVP (19). A notable change in the main bands was noted due to the effect of the induction of gamma radiation in ELT-PVP compared to ELT+PVP.

Thermal properties
In order to evaluate the thermal properties of the hydrogel, TGA, and DSC techniques were carried out. The thermograms obtained by TGA are depicted in Figure 4. The ELT-PVP profile exhibited a significant change in mass loss compared to the ELT+PVP profile, indicating structural changes in the formation of the new polymer.
The thermograms obtained by DSC analysis are shown in Figure 5, whereas the temperature and fusion enthalpy values obtained are presented in Table 1. The shift to the right of the melting peak of ELT-PVP compared to ELT+PVP represented additional evidence of the generation of cross-linking for the formation of ELT-PVP induced by gamma radiation.

Rheology
A rheological study was carried out to determine the fluidic properties of the ELT-PVP hydrogel and the ELT+PVP mixture to demonstrate the changes induced by gamma radiation in obtaining the novel hydrogel. The types of fluids are shown in Figure 6.

Cell viability assay
In order to evaluate the hydrogel biocompatibility, a cell viability test was carried out in human fibroblasts. The novel ELT-PVP hydrogel offered an increase in cell viability from a concentration of 0.15% compared to ELT alone (Figure 7).

Cell proliferation assay
The novel ELT-PVP hydrogel exhibited a considerable effect on cell proliferation from day three at a concentration of 0.15% (Figure 8), revealing a higher degree of protection compared to ELT than from day two at a concentration of 0.10, a significant change is exhibited.

Stability of ELT-PVP in cell culture medium
The biological characterization presented in Figure  9 illustrates the hydrogel's properties when exposed to the cell culture medium. Periodic evaluation of the hydrogel for seven days revealed a gradual decrease in the material's size due to its erosion.

Morphology of the ELT-PVP hydrogel
The porosity and the presence of interconnected porous in biomaterials play a crucial role in cell mi-    gration and proliferation, especially in skin applications. The presence of porous can also facilitate the arrival of nutrients and oxygen to the cells because they allow the regeneration of blood vessels through angiogenesis and the development of new tissue (20). In this regard, Rnjak-Kovacina et al. (21) produced synthetic human ELT scaffolds with pore sizes between 8 and 11 µm, which permitted the infusion and proliferation of dermal fibroblasts on the scaffold surface. Similarly, another study found that ELT hydrogels presenting pores with an average size of around 15 µm were suitable for diffusing nutrients and oxygen (22). On the other side, it has been reported that pore size for cell infiltration of dermal fibroblasts in an ELT scaffold is 11 µm, whereas, for skin regeneration, the adequate size is 20 µm (22,23). Besides, the materials that present high porosity are enabled to the significant release of factors such as protein, genes, or cells, serving as platforms to administer bioactive substances in the wound bed (24). Therefore, the pores in the ELT-PVP hydrogel possesses an appropriate size for a wound dressing.

Swelling
The presence of the pores facilitates the entry of water into the hydrogel, promoting the relaxation of the polymer chains. Additionally, the carboxyl groups of ELT and the carbonyl groups of PVP favor the formation of hydrogen bonds for the uptake of water. The swelling process reaches a maximum at 72 h, and subsequently, a dissolution process was observed with subsequent weight loss. The swelling process could favor cell migration as a tissue scaffold, and it could even act as a possible wound dressing in wound healing processes by capturing exudate from the wound.

Spectral analysis
The ELT+PVP and ELT-PVP spectra indicated the characteristic bands presented in the PVP spectrum (lines c and d). This behavior could be related to that the PVP concentration is higher than the ELT concentration (15:1); thus, the PVP signals predominate. However, a slight decrease in the intensity of the bands was detected in the ELT-PVP spectrum. This decrement could be due to the radicals generated during the ELT-PVP cross-linking, inducing the homolytic cleavage of the C-H and CO-H bonds (25), which suggests that their concentrations in the medium are decreased.

ELT and PVP cross-linking reaction mechanism
The semi-solid ELT-PVP hydrogel presented a remarkable change in physical appearance compared with the un-irradiated liquid mixture. In this regard, due to the cross-linking of the polymer chains, changes in the physicochemical and biochemical properties of the copolymer are expected. It has been reported that radiation at 25 kGy is widely used to sterilize several types of materials    and synthesize biomaterials applied in orthopedic, dental, and pharmaceutical fields (26). Furthermore, it has been reported that gamma radiation is an effective method to obtain protein-based hydrogels. For example, Chen et al. (27) developed a chitosan copolymer by this technique; the obtained scaffold presented a porous morphology, suitable mechanical properties, and high biocompatibility. Likewise, hydrogels of fibroin obtained through cross-linking showed excellent elastic properties, good compatibility, and biodegradability (28). Furthermore, PVP has been cross-linked with structural proteins, such as collagen, obtaining superabsorbent hydrogels (29). These characteristics allow the application of ELT-or PVP-based hydrogels in different fields, such as in cartilage engineering, vascular and skin grafts, among others (30). Figure 10 presents the proposed mechanism for the synthesis of the ELT-PVP hydrogel by the simultaneous irradiation method. First, ELT and PVP are irradiated in the presence of water, under air conditions. Subsequently, the reacting mechanism could occur as follows: a) first, the irradiation step implies the formation of primary radicals; b) second, the initiation step provides the graft chain radicals; and finally, c) termination reactions are performed to yield the ELT-PVP hydrogel (31). The irradiation step includes the radiolysis of water (see compounds 2−8) (32), while different radicals of ELT (compounds 10−23) and PVP (compound 24) are formed (initiation step). Arrows depict the sites where ELT macroradicals can be developed. The termination reaction involves the deactivation of the ELT macroradicals with PVP radicals to yield the ELT-PVP hydrogel (compounds 25-37). Moreover, the complexity of the reaction also includes the possible formation of peroxides and hydroperoxides during the irradiation process (33). However, the decrease in the 3,400 cm -1 bands in FT-IR (Fig. 2) suggests that peroxides do not predominate, as compared to the graft reactions, to yield the hydrogel.

Thermal properties
According to TGA results (Figure 4), three significant weight losses were observed in the case of ELT (line a). The first one occurred from 100 to 207 °C, with a weight loss of 26.76%; this behavior is related to both the evaporation of water and the destabilization of non-covalent interactions, which do not require a large amount of energy. The second loss began at 205 °C and finished at 435 °C, with a total weight loss of 68.32%; in this range, the major denaturation transition occurs, triggering the ELT decomposition (34). The last thermic event was located from 450 to 559 °C, representing a total loss of 86.79%. For PVP analysis (Figure 4, line b), it could be observed the decomposition in a single stage from 375 to 449 °C, with a weight loss of approximately 95%; this deportment coincided with previous reports (35). For ELT+PVP (line c), the maximum decomposition temperature was 458 °C (similar to the PVP), and the weight loss was 88%; this behavior could be attributed to the proportion of PVP concerning the ELT. Finally, the biopolymer ELT-PVP (line d) presented some changes for its components. Three steps of weight loss were observed: the first one occurred between 320 and 390 °C, with 27% weight loss; the second one was found from 410 to 453 °C, losing 72% of the sample; finally, the third event was observed from 490 to 564 °C, showing a 95% of weight loss. The different events can be due to the decomposition of the three-dimensional structure, which changes from a complex three-dimensional network, stabilized by bridges of hydrogen or other weak interactions, to a more straightforward structure, where the covalent bonds predominate, increasing the required time and energy to decompose the molecule (36).
Concerning the DSC study ( Figure 5, Table 1), previous studies showed that ELT presents an endothermic event at approximately 85 °C with an enthalpy of 191 J/g, which is attributed to the interaction between water and proteins, in addition to a second thermal transition at 200 °C (37). On the other hand, only one endothermic event was shown for the PVP at 69.91 °C (curve b), obtaining an enthalpy of fusion of 231 J /g. This value could be related to that PVP is a very hygroscopic material, so the steam from water can affect the calorific capacity. In the literature, this endothermic peak has been reported at around 100 °C and is attributed to the presence of water (38). The comparison between thermograms c) and d) let it observe a slight shift to the right, indicating the formation of new bonds.

Rheology
In order to determine the differences in the types of fluid between ELT+PVP mixture and ELT-PVP hydrogel (after the cross-linking of polymers), a rheological study was carried out. The shear rate test permitted to conclude that the hydrogel is a non-Newtonian fluid because the viscosity varied with the shear rate and time. Additionally, accord- ing to the rheological profiles (Figure 6b), the hydrogel sample is similar to a pseudoplastic fluid with thixotropic behavior. In this regard, in pseudoplastic fluids, decrement in viscosity concerning shear rate is mainly due to the deconstruction of microstructures, allowing easy flow of the material. The reduction of hydrodynamic forces between the fluid and the microstructures cause this response, in addition to the alignment and deformation of these by the action of inertial forces (39). It is noteworthy that pseudo-plasticity is a suitable characteristic in topical products because it ensures uniform distribution of biopolymers on the skin. Likewise, thixotropy is another desired property, since it increases the retention time of the biopolymeric material at the topical application site. Moreover, pseudoplastic and thixotropic behavior can influence the bioadhesion properties of the dressing. Bioadhesion is pivotal because products intended to be in contact with the skin for prolonged periods will be subject to various stresses due to daily activities; thus, it must be ensured that the scaffold remains at the wound site (40)(41)(42).
Besides, when Figure 6a and Figure 6b were compared, the irradiated sample ( Figure 6a) had viscosity values approximately 10-fold higher than the physical mixture (from 1,600 to 8,000 s -1 ). This finding could be attributed to the cross-linking of the polymeric chains of ELT and PVP, which forms a three-dimensional network, increasing the molecular weight and the viscosity (43).
On the other hand, in the physical mixture (Figure 6a), a hysteresis cycle was not observed. However, at 1600s -1 and 4800 s -1, there was a separation of two points where there was no statistically significant difference between them. Contrariwise, in the ELT-PVP hydrogel (Figure 6b), a hysteresis loop, characteristic of thixotropic materials, was observed at 6,399.98 s -1 (44). In fluids with thixotropic behavior, the increase in velocity gradient (blue curve) causes the deconstruction of the structure, while the decrease in gradient (red curve) favors the reorganization (45). In the test of viscosity for the time at a constant shear rate, the physical mixture ( Figure 6c) reached its maximum value at 240 s; after that, it presented a similar behavior to a Newtonian fluid (44). Whereas for the hydrogel (Figure 6d), an increase in viscosity is observed as time progresses, reaching a maximum value at approximately 720 s; after this point, it was found a decrement in viscosity toward constant values due to its thixotropic behavior.

Cell viability assay
Since ELT is an extracellular matrix protein, it was expected that the protein-maintained cell viability; however, surprisingly, only 0.1% w/v of ELT showed a high percentage of cell viability (99.4%); whereas higher concentrations of ELT (0.15, 0.2 and 0.25% w/v) revealed low values of viability (Figure 7a-7b). This finding could be explained by the presence of excessive ELT peptides that could produce an agonist effect in the ELT-laminin receptor. It should be considered that this receptor is dose-dependent; thus, the presence of saturating concentrations of ELT peptides results in chronic overstimulation of the receptor, which leads to sustained production of free radicals and lytic enzymes (46). Likewise, Fulop et al. (47) explained that leukocytes and endothelial cells, which activate the ELT-laminin receptor, trigger the release of nitric oxide and superoxide anion. The combination of both radicals forms the peroxynitrite anion, which is highly toxic. Besides, it has been demonstrated that at high concentrations of ELT, cell proliferation decreases (48).
Interestingly, the adverse effect of ELT was mitigated when it was combined with PVP. In all concentrations of ELT-PVP, the percentage of cell viability was high, although the number of cells decreased when the concentration of ELT and PVP increased (Figure 7c). In this respect, it has been reported that hydrogels combining PVP with other polymers by gamma irradiation do not possess cytotoxic effects (49). Therefore, this result suggests that combining ELT with PVP is an appropriate strategy for the biological applications of ELT because it improves its biocompatibility.

Cell proliferation assay
Similar to the viability test, cell proliferation assays demonstrated that only the concentration of 0.10% w/v of ELT allowed cell culture to proliferate. In contrast, higher concentrations (0.10, 0.15, and 0.20% w/v) of ELT-PVP allowed cell culture progression during the analysis time (3 days). In the proliferation assay may be understandable that high concentrations of ELT do not induce cell proliferation since the percentage of death is high in this condition. However, when fibroblasts were brought into contact with the ELT-PVP combination, they were able to proliferate. Concerning this, it has been established that the PVP of 40 and 360 kDa with a fractional volume occupancy (FVO; the volume occupied by macromolecules) between 18 and 54% may serve as a macromolecular crowder, allowing cell viability, proliferation, and the synthesis of extracellular matrix proteins. Noteworthy, this seems to be linked to the cell line employed, since these characteristics are enhanced more in human dermal fibroblasts than in bone marrow mesenchymal stem cells (50).

Stability of ELT-PVP in cell culture medium
The stability of ELT-PVP based on its appearance was analyzed by microscopy, and the obtained images are presented in Figure 9. After 24 h of the test, the hydrogel exposed the same morphology that the control. In the same way, on the third day of the experiment, the ELT-PVP presented a similar morphology after three days in culture conditions. At seven days, a slight degradation of the hydrogel could be observed. However, the integrity of the hydrogel structure remains in 80% approximately. This behavior demonstrated that the new copolymer is strong and suggests that its application as wound dressing could be adequate, allowing cell proliferation.
In this work, a novel ELT-PVP hydrogel was obtained through gamma irradiation of ELT and PVP in aqueous solution. The ELT-PVP hydrogel showed an ideal morphology to be used as scaffolding in wound healing because it allows cell migration and proliferation. Also, its high porosity could allow the significant release of biologically active substances such as growth factors, genes, or drugs. Likewise, the rheological analysis evidenced that the new biopolymer exhibits a pseudoplastic fluid with thixotropic behavior, which confirms gamma radiation-induced changes in the chemical structure of the polymers. This property is useful since by applying a shear force on the hydrogel, it can flow and be applied easily into the wound, forming a protective film. Finally, the hydrogel exhibited a significantly higher percentage of cell viability concerning ELT, suggesting that the cross-linking of ELT with PVP is a suitable strategy for the biological applications of ELT to preserve the protein and ensure practical therapeutic applications.