Manuka Honey/2-Hydroxyethyl Methacrylate/Gelatin Hybrid Hydrogel Scaffolds for Potential Tissue Regeneration

Scaffolding biomaterials are gaining great importance due to their beneficial properties for medical purposes. Targeted biomaterial engineering strategies through the synergy of different material types can be applied to design hybrid scaffolding biomaterials with advantageous properties for biomedical applications. In our research, a novel combination of the bioactive agent Manuka honey (MHo) with 2-hydroxyethyl methacrylate/gelatin (HG) hydrogel scaffolds was created as an efficient bioactive platform for biomedical applications. The effects of Manuka honey content on structural characteristics, porosity, swelling performance, in vitro degradation, and in vitro biocompatibility (fibroblast and keratinocyte cell lines) of hybrid hydrogel scaffolds were studied using Fourier transform infrared spectroscopy, the gravimetric method, and in vitro MTT biocompatibility assays. The engineered hybrid hydrogel scaffolds show advantageous properties, including porosity in the range of 71.25% to 90.09%, specific pH- and temperature-dependent swelling performance, and convenient absorption capacity. In vitro degradation studies showed scaffold degradability ranging from 6.27% to 27.18% for four weeks. In vitro biocompatibility assays on healthy human fibroblast (MRC5 cells) and keratinocyte (HaCaT cells) cell lines by MTT tests showed that cell viability depends on the Manuka honey content loaded in the HG hydrogel scaffolds. A sample containing the highest Manuka honey content (30%) exhibited the best biocompatible properties. The obtained results reveal that the synergy of the bioactive agent, Manuka honey, with 2-hydroxyethyl methacrylate/gelatin as hybrid hydrogel scaffolds has potential for biomedical purposes. By tuning the Manuka honey content in HG hydrogel scaffolds advantageous properties of hybrid scaffolds can be achieved for biomedical applications.


Introduction
The use of agents derived from nature that possess medicinal properties has always been an intriguing topic. Honey is a bioactive agent, and its healing properties have been recognized for centuries [1][2][3]. Manuka honey (MHo) stands out due to the specific Manuka factor (UMF), which provides antioxidant, antibacterial, and anti-inflammatory effects [4][5][6][7]. MHo is isolated from Leptospermum scoparium nectar species that grow in New Zealand with a pH range of 3.5-4.5. Honey features may influence increased macrophage stimulation, facilitating tissue regeneration and minimizing bacterial infections [4,[8][9][10][11][12][13][14]. Manuka honey has proven to be a very effective agent in the treatment of wounds [1,2].

Hydrogel Scaffold Syntheses
Hybrid hydrogel scaffolds consisting of Manuka honey, 2-hydroxyethyl methacrylate, and gelatin were synthesized using polymerization/crosslinking reactions. An aqueous solution of gelatin was added to 2-hydroxyethyl methacrylate. The mixture was heated (40 • C) with intensive mixing to completely dissolve the gelatin. Honey was added to the homogeneous mixture, and mixing was continued until all the honey was dissolved. Manuka honey content was 10, 20, and 30 wt%. Then, a solution of agents for polymerization/crosslinking reactions (1% solution of APS, TEMED, and EDC) was added with intensive mixing until the mixture became a milky-white color and viscous. The reaction mixtures were transferred to a Petri dish, where the polymerization/crosslinking reactions took place at a temperature of −18 • C for 24 h. When the reactions were finished, the samples were cut into disc form and dried at room temperature to a constant weight. A photograph of the synthesized hybrid hydrogel scaffolds is shown in Figure 1 (the average dimensions of the samples are: diameter, 1 cm; thickness, 0.2 cm; weight, 0.2 g). The samples were placed in a deep freezer (−80 • C) and lyophilized (−55 • C) for further characterization.

Hydrogel Scaffold Syntheses
Hybrid hydrogel scaffolds consisting of Manuka honey, 2-hydroxyethyl methacrylate, and gelatin were synthesized using polymerization/crosslinking reactions. An aqueous solution of gelatin was added to 2-hydroxyethyl methacrylate. The mixture was heated (40 °C) with intensive mixing to completely dissolve the gelatin. Honey was added to the homogeneous mixture, and mixing was continued until all the honey was dissolved. Manuka honey content was 10, 20, and 30 wt%. Then, a solution of agents for polymerization/crosslinking reactions (1% solution of APS, TEMED, and EDC) was added with intensive mixing until the mixture became a milky-white color and viscous. The reaction mixtures were transferred to a Petri dish, where the polymerization/crosslinking reactions took place at a temperature of −18 °C for 24 h. When the reactions were finished, the samples were cut into disc form and dried at room temperature to a constant weight. A photograph of the synthesized hybrid hydrogel scaffolds is shown in Figure 1 (the average dimensions of the samples are: diameter, 1 cm; thickness, 0.2 cm; weight, 0.2 g). The samples were placed in a deep freezer (−80 °C) and lyophilized (−55 °C) for further characterization. The composition and marks of the hybrid hydrogel scaffold samples are presented in Table 1.  The composition and marks of the hybrid hydrogel scaffold samples are presented in Table 1. Hydrogel scaffold structural characteristics were detected by the FTIR spectroscopy method recorded on a Thermo-Scientific Nicolet 6700 FTIR diamond crystal spectrometer using the attenuated total reflectance (ATR) sampling technique. FTIR spectra were recorded over the wavelength range of 700-4000 cm −1 .

Porosity Measurements
The porosity of hydrogel scaffolds was determined by the solvent replacement method. Glycerol (ρ = 1.2038 g/cm 3 ) was used as a wetting medium. Dried hydrogels were submerged in glycerol for 24 h and weighed after removing excess glycerol from the surface: where m i is the initial weight of the dry hydrogel, m glycerol is the weight of the hydrogel with glycerol, ρ is the density of glycerol, and V is the volume of the hydrogel sample. All experiments were performed in triplicate.

In Vitro pH-and Temperature-Dependent Swelling Studies
In vitro swelling studies were performed in a milieu with buffers mimicking biological fluids in a pH range of 2.20-8.0 at 37 • C and a temperature range of 25-39 • C in buffers of pH 7.40 and 5.50 to reveal swelling behavior important for biomedical applications. The amount of fluid absorbed as a function of time was measured gravimetrically. Swollen hydrogels were removed from the swelling medium at regular intervals and dried superficially with filter paper. They were weighed and placed in the same bath until a constant weight was reached for each sample in an equilibrium swelling state. The equilibrium degree of swelling (q e ) is calculated using the following formula: where m e is the weight of a swollen hydrogel at equilibrium, and m 0 is the weight of a dry gel [51,52]. All swelling experiments were performed in triplicate.

In Vitro Degradation Study
The in vitro degradation process was tested by immersion of the dry gel samples in a buffer of pH 7.40 at 37 • C for four weeks. Samples were removed from the buffer at the estimated time, dried at 40 • C until a constant mass was reached, and weighed. Degradation is expressed by weight loss (%) calculated as the residual hydrogel weight percentage: where m i is the initial weight of the dry gel, and m t is the weight of the dried gel sample at the measurement time. Scaffold degradation is expressed as a function of time. All degradation experiments were performed in triplicate.

In Vitro Biocompatibility Assay
The in vitro cytocompatibility of hybrid hydrogel scaffolds is evaluated to indicate potential biomedical applications [53]. Cytotoxicity/antiproliferative activity was measured for interaction scaffolding materials with human lung fibroblasts (MRC5 cell line) and keratinocytes (HaCaT cell line) using MTT assays according to a standard established procedure. All assays were performed in triplicate.

Results and Discussion
Hybrid hydrogel scaffolds were designed with synthetic 2-hydroxyethyl methacrylate (H) monomer and natural origin polymer gelatin (G) (in the form of interpenetrating hydrogel networks (IPN)) loaded with bioactive agent Manuka honey (MHo) by polymerization/crosslinking reactions (Scheme 1).

Results and Discussion
Hybrid hydrogel scaffolds were designed with synthetic 2-hydroxyethyl metha late (H) monomer and natural origin polymer gelatin (G) (in the form of interpenetra hydrogel networks (IPN)) loaded with bioactive agent Manuka honey (MHo) by polym ization/crosslinking reactions (Scheme 1). Scheme 1. Schematic representations of the procedure to obtain an interpenetrating network hy gel of 2-hydroxyethyl methacrylate and gelatin (HG IPN) loaded with Manuka honey (MHo scaffolds).

Structural Features of MHo/HG Hybrid Hydrogel Scaffolds
Fourier transform infrared spectroscopy (FTIR) was used to reveal the main dete ble bands for MHo, HG, and Mho/30HG samples ( Figure 2) and to prove the incorpora of Manuka honey. Specific peaks originating from HEMA are O-H vibrations aro 3200-3340 cm -1 , C-H around 2920-2945 cm -1 , and C=O at 1720 cm -1 , which are attribu asymmetric and symmetric stretching vibrations, methylene stretching, and a carbo group [54]. Bands typical of gelatin are C=O stretching around 1700-1600 cm -1 for am I, N-H definition around 1550-1400 cm -1 for amide I, and 1240-670 cm -1 for amide III [ Manuka honey FTIR spectra show five regions. Region 1 around 2800-3000 cm -1 co sponds to C-H stretching of carbohydrates and O-H stretching of carboxylic acid, as w as NH3 stretching of free amino acids. Region 2 around 1660-1700 cm -1 is caused by O stretching/bending of water and C=O stretching, mainly from carbohydrates and N bending of amide I. Region 3 around 1175-1540 cm -1 corresponds to O-H stretching/be ing, C=O and C-H stretching of carbohydrates, and C=O stretching of ketones. Regio around 940-1175 cm -1 corresponds to C-O and C-C stretching of carbohydrates and vibration, mainly from carbohydrates. Region 5 around 700-940 cm -1 is caused by an a meric region of carbohydrate C-H bending and ring vibration of carbohydrates [56-Bands showing the incorporation of Manuka honey are located in the region of 1449-1 cm -1 for the 30MHo/HG sample.

Structural Features of MHo/HG Hybrid Hydrogel Scaffolds
Fourier transform infrared spectroscopy (FTIR) was used to reveal the main detectable bands for MHo, HG, and Mho/30HG samples ( Figure 2) and to prove the incorporation of Manuka honey. Specific peaks originating from HEMA are O-H vibrations around 3200-3340 cm −1 , C-H around 2920-2945 cm −1 , and C=O at 1720 cm −1 , which are attributed asymmetric and symmetric stretching vibrations, methylene stretching, and a carbonyl group [54]. Bands typical of gelatin are C=O stretching around 1700-1600 cm −1 for amide I, N-H definition around 1550-1400 cm −1 for amide I, and 1240-670 cm −1 for amide III [55]. Manuka honey FTIR spectra show five regions. Region 1 around 2800-3000 cm −1 corresponds to C-H stretching of carbohydrates and O-H stretching of carboxylic acid, as well as NH 3 stretching of free amino acids. Region 2 around 1660-1700 cm −1 is caused by O-H stretching/bending of water and C=O stretching, mainly from carbohydrates and N-H bending of amide I. Region 3 around 1175-1540 cm −1 corresponds to O-H stretching/bending, C=O and C-H stretching of carbohydrates, and C=O stretching of ketones. Region 4 around 940-1175 cm −1 corresponds to C-O and C-C stretching of carbohydrates and ring vibration, mainly from carbohydrates. Region 5 around 700-940 cm −1 is caused by an anomeric region of carbohydrate C-H bending and ring vibration of carbohydrates [56][57][58][59]. Bands showing the incorporation of Manuka honey are located in the region of 1449-1023 cm −1 for the 30MHo/HG sample.

Porosity of MHo/HG Hybrid Hydrogel Scaffolds
High porosity is a significant scaffold feature for biomedical purposes. The establishment of a porous scaffold structure has a favorable effect on cell attachment, differentiation, proliferation and vascularization processes, oxygen supply, and nutrient flow for a successful tissue regeneration process [60]. Cells need free space to settle, which they get inside pores that are interconnected; in that space, the regeneration process takes place, i.e., tissue growth takes place undisturbed. Our results show that Manuka honey content influences the porosity of prepared hybrid scaffolds. The obtained porosity data are in the range of 71.25-90.09% for the synthesized hybrid hydrogel scaffolds ( Figure 3). The sample without honey (HG) has the highest porosity value of 90.09%. The porosity of the hybrid hydrogel scaffolds decreased as the proportion of Manuka honey increased. This phenomenon can be explained by honey filling the free space in the scaffold pores, resulting in decreased porosity. Analysis of the obtained values emphasizes that the synthesized hybrid scaffolds are highly porous, which is adequate for biomedical applications. Because Manuka honey is water-soluble and during dissolution (at the site of application as a scaffold for tissue regeneration, where there is always the presence of body fluids), it frees up space inside the pores due to dissolution, allowing access for cells to populate and spread. According to this hypothesis, Manuka honey dissolves upon interaction with fluid (body fluids), freeing up space within the polymeric network for cells to colonize and spread within the scaffold. Therefore, the sample with 30% Manuka honey is the most favorable for tissue regeneration application. Manuka honey is a very convenient agent for healing, especially for dermal treatments.

Porosity of MHo/HG Hybrid Hydrogel Scaffolds
High porosity is a significant scaffold feature for biomedical purposes. The establishment of a porous scaffold structure has a favorable effect on cell attachment, differentiation, proliferation and vascularization processes, oxygen supply, and nutrient flow for a successful tissue regeneration process [60]. Cells need free space to settle, which they get inside pores that are interconnected; in that space, the regeneration process takes place, i.e., tissue growth takes place undisturbed. Our results show that Manuka honey content influences the porosity of prepared hybrid scaffolds. The obtained porosity data are in the range of 71.25-90.09% for the synthesized hybrid hydrogel scaffolds (Figure 3). The sample without honey (HG) has the highest porosity value of 90.09%. The porosity of the hybrid hydrogel scaffolds decreased as the proportion of Manuka honey increased. This phenomenon can be explained by honey filling the free space in the scaffold pores, resulting in decreased porosity. Analysis of the obtained values emphasizes that the synthesized hybrid scaffolds are highly porous, which is adequate for biomedical applications. Because Manuka honey is water-soluble and during dissolution (at the site of application as a scaffold for tissue regeneration, where there is always the presence of body fluids), it frees up space inside the pores due to dissolution, allowing access for cells to populate and spread. According to this hypothesis, Manuka honey dissolves upon interaction with fluid (body fluids), freeing up space within the polymeric network for cells to colonize and spread within the scaffold. Therefore, the sample with 30% Manuka honey is the most favorable for tissue regeneration application. Manuka honey is a very convenient agent for healing, especially for dermal treatments.

Swelling Features of MHo/HG Hybrid Hydrogel Scaffolds
Swelling performance is a very important property of hydrogel scaffolds, providing information about fluid absorption capacity. For biomedical applications, the equilibrium fluid amount of a hydrogel is connected with its ability to absorb body fluid and transfer nutrients to cells and metabolism components through the scaffold [61]. The equilibrium degree of swelling is a key parameter that represents the swelling process. Swelling tests were carried out in buffers with a pH range of 2.20-8.00 at 37 • C and in a temperature range of 25−39 • C in buffers with a pH in the range of 7.40-5.50. Figure 4a-c represents the data for pH and temperature swelling profiles of the designed HG hydrogel scaffold and MHo/HG hybrid hydrogel scaffold samples. Samples show pH-and temperaturedependent swelling behavior. Considering pH influence, the swelling results indicate that the net 2-hydroxyethyl methacrylate/gelatin hydrogel scaffold recorded the lowest equilibrium degree of swelling (q e ), with a linearly decreasing trend for q e −pH dependence in the range of 2.20−8.00 at 37 • C, i.e., pH-dependent behavior was not detected for HG hydrogel scaffolds. When Manuka honey was loaded into the HG scaffold, it imparted specific q e -pH dependence of hybrid hydrogel scaffolds. q e values for MHo/HG samples are higher than those of the net HG sample. The same q e −pH dependence profile was observed for all MHo/HG samples. q e increases from a pH of 2.20, reaches a maximum value at 5.50, then decreases at a pH of 7.40, with another slight decrease at a pH of 8.00 (q e values are in the range of 0.80−1.60). As the honey content increases, so does the q e . Sample 30MHo/HG shows the highest q e value at a pH of 5.50. These types of hybrid hydrogel scaffolds contain hydrophilic groups (-OH, -COOH, and -NH 2 ), which enhance the permeability and hydrophilic capacity of the hydrogel network. An increase in hydrophilicity increases the tendency of water molecules to infiltrate the hydrogel network, which results in higher swelling capacity. Honey consists mostly of highly hydrophilic moieties, so its loading on hydrogel scaffolds increases the hydrophilic character of the scaffold, leading to more intense water absorption. The q e values of MHo/HG samples are higher than those of the HG sample and increase with increasing MHo content [62]. The loading of bioactive Manuka honey into the 2-hydroxyethyl methacrylate/gelatin hydrogel scaffold achieved specific pH-dependent swelling behavior of the MHo/HG.

Swelling Features of MHo/HG Hybrid Hydrogel Scaffolds
Swelling performance is a very important property of hydrogel scaffolds, providing information about fluid absorption capacity. For biomedical applications, the equilibrium fluid amount of a hydrogel is connected with its ability to absorb body fluid and transfer nutrients to cells and metabolism components through the scaffold [61]. The equilibrium degree of swelling is a key parameter that represents the swelling process. Swelling tests were carried out in buffers with a pH range of 2.20-8.00 at 37 °C and in a temperature range of 25−39 °C in buffers with a pH in the range of 7.40-5.50. Figure 4a-c represents the data for pH and temperature swelling profiles of the designed HG hydrogel scaffold and MHo/HG hybrid hydrogel scaffold samples. Samples show pH-and temperaturedependent swelling behavior. Considering pH influence, the swelling results indicate that the net 2-hydroxyethyl methacrylate/gelatin hydrogel scaffold recorded the lowest equilibrium degree of swelling (qe), with a linearly decreasing trend for qe−pH dependence in the range of 2.20−8.00 at 37 °C , i.e., pH-dependent behavior was not detected for HG hydrogel scaffolds. When Manuka honey was loaded into the HG scaffold, it imparted specific qe-pH dependence of hybrid hydrogel scaffolds. qe values for MHo/HG samples are higher than those of the net HG sample. The same qe−pH dependence profile was observed for all MHo/HG samples. qe increases from a pH of 2.20, reaches a maximum value at 5.50, then decreases at a pH of 7.40, with another slight decrease at a pH of 8.00 (qe The influence of the temperature interval from 25 • C to 39 • C on the swelling properties of HG and MHo/HG is shown in Figure 4b,c. All hydrogel scaffolds show specific temperature-dependent swelling behavior (q e values are in the range of 0.654−1.753). At the beginning of the temperature interval from 25 • C to 35 • C, an almost linear (slightly decreasing) dependence of q e on temperature can be observed. With an increase in temperature to 35 • C and up to 37 • C, a significant increase in q e occurs. As the honey content in the samples increases, there is also a significant increase in q e in this temperature interval. Furthermore, with increasing temperature from 37 • C to 38 • C, q e values decrease. For the last temperature interval from 38 • C to 39 • C, the highest increase in q e was noticed. It is observed that q e values in a buffer with a pH of 5.50 are higher compared to those in a buffer with a pH of 7.40, depending on the honey fraction. The swelling profiles are the same for all MHo/HG samples in terms of temperature dependence. The hydrophilic-hydrophobic balance is a phenomenon that can explain the swelling behavior of the scaffolds in this temperature interval. At lower temperatures, hydrophobic interactions are more dominant, so the scaffolds show lower swelling. With an increase in temperature, that balance changes, and hydrophilic interactions are more dominant, so swelling increases. In the interval of 37-39 • C, the presence of the gelatin network comes to the fore, which results in decreased swelling, followed by an increase in swelling, which is caused by the segmental mobility of parts of the network originating from the presence of gelatin. This behavior leads to the formation of free space between the network polymeric chains, and water molecules can enter the hybrid scaffolds, causing swelling capacity changes, i.e., specific temperature-dependent swelling occurs [63]. According to our earlier studies, the HEMA component in hydrogels does not contribute to temperature-dependent swelling [64]. The samples containing honey compared with the HG sample show different values of q e as a function of pH and T. It should be emphasized that the q e value is 1.2 for 10MHo/HG at a pH of 5.50, but q e is 0.8 for the same sample at a pH of 7.40; a difference of 0.4 is enough to cause different behavior of 10MHo/HG at different pH values. A sufficient difference in swelling was also shown in the dependence of q e on T. Changes in pH and temperature are the most important stimuli for various biomedical applications (including wound healing). The correlations of swelling with pH and temperature are important properties of hydrogel scaffolds. The sample with the highest content of Manuka honey (30MHo/HG) shows the highest q e value in a solution with a pH of 5.50 and at a temperature of 39 • C, which are pathophysiological conditions indicating infections and diseases. Therefore, this sample is expected to show the best therapeutic properties for the treatment of diseased tissues and is recommended for biomedical applications [4][5][6][7].  The influence of the temperature interval from 25 °C to 39 °C on the swelling properties of HG and MHo/HG is shown in Figure 4b,c. All hydrogel scaffolds show specific temperature-dependent swelling behavior (qe values are in the range of 0.654−1.753). At the beginning of the temperature interval from 25 °C to 35 °C , an almost linear (slightly decreasing) dependence of qe on temperature can be observed. With an increase in temperature to 35 °C and up to 37 °C , a significant increase in qe occurs. As the honey content in the samples increases, there is also a significant increase in qe in this temperature interval. Furthermore, with increasing temperature from 37 °C to 38 °C , qe values decrease. For

In Vitro Degradation Behavior of MHo/HG Hybrid Hydrogel Scaffolds
Degradability is an important feature to validate the suitability of biomaterial for biomedical applications. The scaffold degradation process provides new space for tissue Polymers 2023, 15, 589 9 of 14 regrowth and induces the regeneration process. Tunable degradation is possible with synthetic hydrogels depending on chemical composition and the ratio of components. Hydrogels of natural origin are intrinsically degradable and also impact degradability. These combinations are the beneficial synergy that makes it possible to adjust the degradable properties of hybrid hydrogel scaffolds. Because a scaffold provides mechanical support for cell growth, the degradation process should be time-dependent with a controlled rate that guarantees mechanical support during the regeneration process [65]. In vitro degradability of HG and MHo/HG was tested in a phosphate buffer for 4 weeks. The obtained data are presented in Figure 5. It is obvious that composition influences degradation behavior. The net HG scaffold shows the lowest weight loss value. Loading MHo into the HG scaffold accelerates the degradation process. As the honey fraction increases (10−30%), so does MHo/HG weight loss (6.27% to 27.18%). The 30MHo/HG sample shows the highest degradation. This behavior indicates that honey is a component of the hybrid scaffolds that affects the degradation process due to its highly hydrophilic character, making it easier for water molecules to penetrate the hydrogel network and accelerate degradation. honey (30MHo/HG) shows the highest qe value in a solution with a pH of 5.50 and at a temperature of 39 °C , which are pathophysiological conditions indicating infections and diseases. Therefore, this sample is expected to show the best therapeutic properties for the treatment of diseased tissues and is recommended for biomedical applications [4][5][6][7].

In Vitro Degradation Behavior of MHo/HG Hybrid Hydrogel Scaffolds
Degradability is an important feature to validate the suitability of biomaterial for biomedical applications. The scaffold degradation process provides new space for tissue regrowth and induces the regeneration process. Tunable degradation is possible with synthetic hydrogels depending on chemical composition and the ratio of components. Hydrogels of natural origin are intrinsically degradable and also impact degradability. These combinations are the beneficial synergy that makes it possible to adjust the degradable properties of hybrid hydrogel scaffolds. Because a scaffold provides mechanical support for cell growth, the degradation process should be time-dependent with a controlled rate that guarantees mechanical support during the regeneration process [65]. In vitro degradability of HG and MHo/HG was tested in a phosphate buffer for 4 weeks. The obtained data are presented in Figure 5. It is obvious that composition influences degradation behavior. The net HG scaffold shows the lowest weight loss value. Loading MHo into the HG scaffold accelerates the degradation process. As the honey fraction increases (10−30%), so does MHo/HG weight loss (6.27% to 27.18%). The 30MHo/HG sample shows the highest degradation. This behavior indicates that honey is a component of the hybrid scaffolds that affects the degradation process due to its highly hydrophilic character, making it easier for water molecules to penetrate the hydrogel network and accelerate degradation.

Biocompatibility Assays of MHo/HG Hybrid Hydrogel Scaffolds
Biocompatibility assessment is an essential procedure for preclinical probes of biomaterials intended for biomedical purposes [47,[66][67][68][69][70][71][72]. In vitro biocompatibility tests provide data on whether the tested biomaterial or any components that did not react (leachables) in cell interaction assays cause cell death or interfere with cell functions. The biocompatibility of MHo/HG was evaluated for interaction with normal human fibroblasts (MRC5) and normal human keratinocytes (HaCaT). Data from these assays are shown in Figures 6 and 7. The obtained data indicate that Manuka honey content influences cell viability for both cell lines. Better cell viability was achieved for scaffolds interacting with the fibroblast cell line, whereas keratinocytes showed lower cell viability. The sample with the highest honey content (30%) shows the best biocompatible properties for both tested cell lines. Therefore, this sample is the most suitable for applications as a scaffolding biomaterial for tissue regeneration. Interestingly, material extracts containing no Manuka honey inhibited keratinocyte proliferation quite efficiently (Figure 7), and dose dependence was observed in the case of MRC5 cells ( Figure 6). Human HaCaT keratinocyte cells are immortal epithelial cells that are susceptible to malignant transformation [73] and have been previously shown to be more sensitive than MRC-5 cells in the case of compounds including hexamidine, synthalin, para-guanidino ethylphenol [74]. Nevertheless, the MHo component showed a beneficial effect on the proliferation of both cell lines. In comparison, the selection of honey samples tested directly on MRC5 cells showed IC 50 values of 10-50 mg/mL [75]. Previous studies have shown that the MHo component improves wound healing through the stimulation of cytokine induction [76,77]. material for tissue regeneration. Interestingly, material extracts containing no Man honey inhibited keratinocyte proliferation quite efficiently (Figure 7), and dose dep ence was observed in the case of MRC5 cells ( Figure 6). Human HaCaT keratinocyte are immortal epithelial cells that are susceptible to malignant transformation [73] have been previously shown to be more sensitive than MRC-5 cells in the case of pounds including hexamidine, synthalin, para-guanidino ethylphenol [74]. Neverthe the MHo component showed a beneficial effect on the proliferation of both cell line comparison, the selection of honey samples tested directly on MRC5 cells showed values of 10-50 mg/mL [75]. Previous studies have shown that the MHo componen proves wound healing through the stimulation of cytokine induction [76,77].

Conclusions
Our research revealed interesting and advantageous features of newly designed active Manuka honey/2-hydroxyethyl methacrylate/gelatin hybrid hydrogel scaff Manuka honey content determines all tested scaffolding properties. Structural chara istics confirmed the loading of Manuka honey into 2-hydroxyethyl methacrylate/ge hydrogel scaffolds, with porosity values in the range of 71.25% to 90.09 %. It is known

Conclusions
Our research revealed interesting and advantageous features of newly designed bioactive Manuka honey/2-hydroxyethyl methacrylate/gelatin hybrid hydrogel scaffolds.
Manuka honey content determines all tested scaffolding properties. Structural characteristics confirmed the loading of Manuka honey into 2-hydroxyethyl methacrylate/gelatin hydrogel scaffolds, with porosity values in the range of 71.25% to 90.09 %. It is known that Manuka honey has appropriate properties for treatments, especially for dermal purposes. Manuka honey is water-soluble, and during dissolution (at the site of application as a scaffold for tissue regeneration, where there is always the presence of body fluids), it frees up space inside the pores due to dissolution, allowing access for cells to populate and spread. Hybrid hydrogel scaffolds showed pH-and temperature-dependent swelling performances and favorable absorption capacity, with q e values proportional to the MHo amount, showing the highest q e values for sample 30MHo/HG. In vitro degradation studies exhibited scaffold weight loss in the range of 6.27% to 27.18% during four weeks. In vitro biocompatibility probes on healthy human fibroblast (MRC5 cells) and keratinocyte (HaCaT cells) cell lines by MTT test indicated that cell viability depends on the Manuka honey content and that Manuka honey improves cell viability. The results indicate that the use of 2-hydroxyethyl methacrylate/gelatin hybrid hydrogel scaffolds loaded with Manuka honey has the potential for medical use. The advantageous properties of MHo/HG hybrid hydrogel scaffolds for biomedical applications can be realized by modulating Manuka honey content. The simplified design strategy and easy creation of MHo/HG hybrid scaffolds have significant implications for possible clinical probes. This research shows the medical potential for the obtained hybrid scaffolds. Therefore, additional research on these novel scaffolds in the form of in vivo assays and clinical studies is required. The goal of all research considering the design of hydrogel scaffolds is to synthesize ideal scaffolds that meet all requirements when it comes to specific medical applications (regeneration and healing of different tissue types). The use of Manuka honey in treatment is an inexhaustible research topic, as shown by the latest published papers.