Pirfenidone-loaded hyaluronic acid methacryloyl hydrogel for preventing epidural adhesions after laminectomy

It is inevitable that scar formation occurs between the spinal dura and surrounding tissues after laminectomy. While extensive epidural fibrosis, which results in limited nerve root activity and severe pain, is the main cause of postoperative failed-back surgery syndrome. Novel biomaterial loading effective drugs based on reasonable design are eagerly needed for the safe and effective prevention of epidural adhesions. We filtrated a suitable dose of pirfenidone (PFD) to load hyaluronic acid methacryloyl (HAMA) hydrogel in vitro. And then, we compare PFD-loaded HAMA hydrogel with only using PFD or HAMA hydrogels after laminectomy by in vivo studies in rats. We describe a safe and efficient anti-adhesive PFD-loaded HAMA hydrogel that prevents epidural fibrosis through the stable and sustained release of PFD. It was shown that the PFD-loaded HAMA hydrogel effectively inhibited cell penetration and suppressed collagen I/III expression. Thus, it effectively prevented the formation of adhesions through pharmacological and physical processes. The PFD-loaded HAMA hydrogel can effectively prevent adhesion formation in both pharmacological and physical barrier effects.


Introduction
Laminectomy, usually used to provide the spinal cord or nerve root decompression, is an important procedure in posterior spinal operation [1]. The surgery is effective, but it does have some complications, such as persistent low back or leg pain. We called these complications, which result from the dense epidural fibrosis or surgical scar made by excessive fibroblast activation excessive extracellular matrix (ECM) results in adhesion between nerve roots and dura mater, failed back surgery syndrome (FBSS), and approximately 40% of patients suffering from this syndrome [1][2][3][4].
Hence, it is extremely important to inhibit fibroblast activation, migration, proliferation, and expression of excess collagen [5].
Pirfenidone (oral medicine, Marnac, Inc., PFD), a pyridinone derivative, is an anti-inflammatory and anti-fibrotic drug that was previously approved for the treatment of idiopathic pulmonary fibrosis (IPF) in Europe in 2011 and in the US in 2014 [6,7]. PFD was one of the sole two novel drugs conditionally recommended for the treatment of IPF [8]. Studies have revealed that PFD has anti-fibrosis effects by preventing collagen formation in vitro and in vivo [9]. However, long-term oral PFD has many common side effects, including nausea, diarrhea, rash, and liver damage [7,10]. To protect against these common side effects, we sought to explore whether local slow-release application of PFD could be used in a safe and effective prophylactic and therapeutic way.
In recent years, injectable hydrogels, a hydrophilic polymer network that can hold large amounts of water while maintaining a three-dimensional structure, have been extensively studied for local drug delivery and low-release [11,12], wound repair tissue regeneration [13], hemostasis [14], adhesion prevention [15], and facilitate surgery [16]. Hyaluronic acid (HA), a natural polysaccharide generally found in Jiawei Ji and Jiaqi Cheng equally contributed to this study. the extracellular matrix of connective tissues, has been used as an anti-adhesion material in surgery [17][18][19][20]. Based on these key characteristics, we selected HAMA hydrogel as a drug sustained release carrier [21].
In the present study, HAMA hydrogel loading with PFD was fabricated to prevent scar tissue and adhesion formation after laminectomy. We studied the surface morphology, drug release characteristics, in vitro cell biological behavior, and in vivo anti-adhesion effects of HAMA hydrogels loaded with PFD. We found that the application of PFD-loaded HAMA hydrogel was more effective in preventing epidural adhesion formation.

Preparation of HAMA hydrogel
The HAMA hydrogel used in the experiments was purchased from the Suzhou Institute of Intelligent Manufacturing. In order to obtain the finished HAMA hydrogel, 1 mL of hydrogel was weighed as 0.005 gram (g) of hyaluronic acid methacryloyl solid, 1 mL of photoinitiator was added, stirred at room temperature for 30 min, rotated at 5000 revolutions per minute (rmp) for 5 min to remove air bubbles, and sterilized with 0.22 μm needle filter. Keep away from light. If curing is needed, put under blue light irradiation for 30 s.

In vitro drug release profiles
The HAMA hydrogel (EFL,EFL-HAMA-400K, Suzhou, China) loading with PFD (Beijing Konruns Pharmaceutical Co., Ltd., Beijing, China) was poured out 1 mL and cured (Fig. 1A, B), and gently shaken with 10 mL of PBS (pH = 7.4) as release medium at 40 rmp in a constant temperature shaking incubator (Blue Pard, Hong Kong, China) at 37 °C. Remove the supernatant liquid from the centrifuge tube and replace the fresh solution at the same time every day. The quantity of released PFD in the supernatant was measured by ultraviolet-visible spectrophotometry at 314 nm; meanwhile, plot a standard curve according to the absorbance of the standard product and, finally, calculate the PFD concentration of each time point.

Observed by scanning electron microscopy (SEM)
The hydrogels were observed by scanning electron microscopy (SEM) by first placing the hydrogels in a -80 °C refrigerator for 24 h and then freeze-drying them in a freezedryer (FD-1D-50, Jiangsu Tianling Co., Ltd., China) at -49 °C under 15 Pa vacuum. A little sample was taken and glued to the conductive gel, tested by gold spray evacuation at a voltage of 20 kV, and the morphology was observed by SEM (QUANTA FEG 45, FEI, USA) at a working distance of 10.1 mm.

In vitro cell studies
Cell culture L929 fibroblasts were purchased from the University of California, San Francisco (USA). The cells were cultured by Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco, Billings, MT, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (HyClone) at 37 °C under a relative saturation humidity (95%) atmosphere of 5% CO 2 .

Barrier function observation
The ability of the anti-adhesive hydrogel to act as a barrier was determined in an in vitro cell permeation assay using a cell strainer device. The cell strainer was pinned into a sieve-like hole, and the HAMA hydrogel was spread on the surface. The fixed hydrogel was first sterilized with UV radiation for 30 min and then placed into a 6-well plate. L929 fibroblasts were then resuspended in DMEM supplemented with 10% FBS and seeded on the hydrogel at a density of 3.0 × 10 4 cells/mL. After 1, 4, 7, and 14 days of incubation at 37 °C and 5% CO 2 , the bottom surface of the six-well plate was observed with an Olympus inverted microscope, and the hydrogel was removed to observe the reverse side of the hydrogel with SEM. The results showed that no cells were present. It indicates that fibroblasts themselves do not migrate through the hydrogel.

Observation of cell morphology after treatment
Observation of cell morphology after PFD treatment was detected using Olympus Inverted Microscope (Olympus Corporation, Japan). HAMA hydrogel and with three different concentrations of PFD (40 mg/mL, 80 mg/mL, and 120 mg/mL) were dissolved and mixed, cured by blue light for 30 s. Then sterilized by UV light (EFL, Suzhou, China) for 30 min and soaked in a DMEM medium containing 10% fetal bovine serum and 1% penicillin/streptomycin for 72 h. The medium was collected and sterile filtered to become the specific medium for all subsequent cell experiments. Fibroblasts were inoculated on 6-well plates with 3 replicate wells and incubated for 24 h. The medium was removed from the 6-well plates and replaced with specific medium for 24 h. The cells were observed and photographed using an Olympus inverted microscope and analyzed.

Cell viability assay
Cell viability after PFD treatment was detected using Cell Counting Kit-8 (CCK-8) (Shanghai Biyuntian Bio-Technology Co., Ltd). First, fibroblasts were seeded on 96-well plates, repeated 6 times, and cultured for 24 h. Remove the culture media from 96-well plates and replace specific medium as explained above. Then, they were cultured for 12 h and replaced by DMEM containing 10% CCK-8 in the plate for 2 h to determine the absorbance at 450 nm for calculating by a microplate reader (Elx800, Bio-Tek, USA).

Edu assay
The effects of HAMA hydrogel loading with PFD on fibroblast proliferation were evaluated using the Click-iT™ EdU Cell Proliferation Kit (Beyotime, Shanghai, China). The fibroblasts were seeded on 24-well plates for 24 h and then treated with specific media, which was explained above at different concentrations for another 24 h. The cells were then incubated in 10 mM EdU working solution at 37 °C for 1 h, fixed in 4% paraformaldehyde at room temperature for 15 min, and permeated with PBS containing 0.3% Triton X-100 at room temperature darkness for 15 min. After incubating in the click reaction mixture for 30 min, cell nuclei were stained with DAPI at room temperature darkness for 2 min. Finally, the cells were observed and photographed under an Olympus fluorescence microscope, and the positive EDU cells were calculated by Image J software. Green was considered a positive signal for cell proliferation, and the nucleus is sapphire blue.

Animals
Fifty healthy female Sprague-Dawley (SD) rats (200-250 g; 6-8 weeks) were proved from the Laboratory Animal Center of Nantong University. The animals received care in compliance with the principles of the guidelines of the Chinese Society of Laboratory Animals on animal welfare, and the experimental protocol was approved by the Standard Operating Procedures for Laboratory Animal Center of Nantong University (no. S20220103-921). Forty rats were randomly divided into four groups (ten rats in each group): the control (saline) group; the HAMA hydrogel group; the PFD group; the PFD-loading HAMA hydrogel group. Ten rats were used in the degradation of HAMA hydrogels in vivo.

The degradation of HAMA hydrogels in vivo
Rats were anesthetized using sodium pentobarbital by intraperitoneal injection and fixed in a prone position on a special plate. The back hair was shaved, and the exposed skin was disinfected three times with povidone-iodine. A median dorsal incision was removed, the skin was freed, and HAMA hydrogel was placed 1 cm from the edge of the incision. The hydrogel was removed and observed and weighed after the rats were anesthetized at weeks 1, 4, and 8 to assess the degradation of the hydrogel in vivo.

Laminectomy model
The rats were anesthetized by peritoneal injection of pentobarbital sodium and fixed on a special plate in the prone position. Shave all hair around L1 and L2 and disinfect exposed skin with iodophor three times. A dorsal midline incision was made and the paraspinal muscles were isolated at the level of the L1-L2 spine [22]. The spinous process, lamina, and ligamentum flavum were excised to expose the dural mater at L1-L2 level by the bonebiter. After hemostasis, different formulations (0.1 mL per sample) were applied directly and gently to the top of the dura mater, which was cured as a roof structure ( Fig. 2A-D). Finally, the rat incision was closed by suturing the muscle, fascia, and skin layer by layer, and the external skin was exposed skin was sterilized with iodophor again. After that, the rats were resuscitated in an incubator and administered penicillin (4 × 106 u/ kg/day, i.m.) to prevent infection. After 8 weeks, epidural fibrosis was assessed after euthanasia [23].

Evaluation of gross adhesion
After 8 weeks post-operation, the experimental rats were sacrificed, and the surgical sites were explored again. From each group, 4 rats were selected for evaluation of the gross adhesion. To prevent bias, investigators were blinded to the animal information. Four rats in each group were randomly selected for evaluation of the gross adhesion according to Rydell's standard grading [24,25]. The researchers were blinded to the animal groups to reduce bias.

Histological evaluation
After 8 weeks post-surgery, rats were deeply anesthetized with an intraperitoneal injection of sodium pentobarbital and intracardiac perfusion of 500 mL of PBS and 500 ml of 4% paraformaldehyde. L1-L2 vertebrae were taken and fixed in 4% paraformaldehyde for 7 days. Subsequently, samples were decalcified with 10% EDTA (pH 7.4) for 2 weeks at 37 °C. Then, after dehydration in a gradient concentration of sucrose, they were embedded in optimal cutting temperature compound (OCT) and sectioned on a frozen sectioning machine at -22 °C on the freezing table and -20 °C in the box at a thickness of 4 μm on the horizontal plane. Slices with 4-μm thickness were randomly picked from all four groups and stained by hematoxylin and eosin (H&E) and Masson's Trichrome following the standard protocols for observation. Histological morphometric analyses were evaluated in the epidural fibrosis degree and collagen synthesis capability by Image J software (version number 2.5.0).

Western blot analysis
The tissues of spinal cord epidural adhesions were collected from different groups and homogenized with RIPA buffer (Biosharp, Hefei, China). The mixture was incubated on ice for 30 min, and then the supernatant was extracted by centrifugation at 12,000 rpm for 15 min in a refrigerated centrifuge at 4 °C. Protein content was detected by bicinchoninic acid protein determination kit (Beyotime Institute of Biotechnology, Shanghai, China) and heated at 95 °C for 10 min. Total proteins were separated by 12% tris-glycine gels (Epizyme, Shanghai, China) electrophoresis and transferred to polyvinylidene fluoride (PVDF) membrane (0.45 μm; Millipore, Bedford, MA, USA). Then, the membranes were blocked with 5% skimmed milk (Sangon Biotech, Shanghai, China) and incubated with primary antibodies overnight at 4 °C. Afterward, the PVDF membranes were washed with TBST buffer and incubated with secondary antibodies for 1 h at room temperature. After washing, Use chemiluminescence detection kits on blots according to the manufacturer's instructions (Millipore). Images were collected with the Chem-iDoc™ XRS+ system (Bio-Rad Laboratories, Hercules, CA, USA) and quantified by Image J software.

Statistical analysis
All statistical analyses were performed using Prism 8 (Graph-Pad, San Diego, CA, USA), and the data were expressed as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) was conducted to compare differences between groups. p < 0.05 was considered significant.

Characterization of HAMA hydrogels
HAMA hydrogel we chose was offered by the Suzhou Intelligent Manufacturing Research Institute. While HAMA hydrogel is a colorless and transparent liquid before the 405 nm light source hits, obvious sol-gel transformation and hydrogel formation were observed under 405 nm light source (Fig. 3A). HAMA hydrogel loading with PFD is yellow after the 405 nm light source (Fig. 3B). The surface of lyophilized hydrogel showed porous network structure in Scanning electron microscope (QUANTA FEG 45, SEM) (Fig. 3C). HAMA hydrogel loading with PFD is the same as the HAMA hydrogels in the scanning electron microscopy (SEM).

The sustained release effect of HAMA hydrogel
The daily drug release from HAMA hydrogel exhibits a typical three-stage release pattern: a burst release on the first day, followed by a stable and sustained release on the next 7 days, and a terminal release on the last 3 days. The initial burst release is dominated by internal diffusion of the drug, which coincides with the onset of the inflammatory cascade in the surgical area after surgery. Then, the initial release ends and sustained release begins, with the sustained release phase being mainly due to internal diffusion of the drug and slow degradation of the hydrogel due to the interaction of the pericellular microenvironment and the polymer matrix. Until the end of terminal release. The sustained and steady release of the drug during the first week inhibits the migration and proliferation of fibroblasts, thus directly and effectively preventing the formation of adhesions. Calculations show that the release efficiency in HAMA hydrogels is on average 81% (Fig. 3D, E).

HAMA hydrogel degrades slowly in vivo
An important feature of a successful anti-adhesive hydrogel is the rate of degradation in vivo, which directly affects its ability to act as a physical barrier to fibroblasts. Evaluation of changes in shape and quality of hydrogels at weeks 1, 4, and 8 after transplantation into the body using macroscopic observation and mass weighing methods. After 4 weeks of implantation, we observed that the edges of HAMA hydrogel became thinner and irregular in shape, and the outer layer of HAMA hydrogel still maintained its unique hydrogel morphology without significant changes. After 8 weeks of implantation, the edges of HAMA hydrogel became thinner and more irregular in shape as the degradation continued (Fig. 4A). The line graph also shows that the mass of HAMA hydrogel gradually decreases with time; the mass decreases from an average of 0.81 g before implantation to an average of 0.25 g after 8 weeks (Fig. 4B).

Barrier function toward fibroblast cells
The ability of the anti-adhesive hydrogel to act as a barrier was determined in an in vitro cell permeation test using a cell strainer device ( Figure 5A, B). After 14 days of seeding fibroblasts on the top layer of HAMA hydrogel, we observed the surface of the six-well plate with an Olympus inverted microscope and removed the hydrogel to observe the bottom surface of the hydrogel with SEM. We observed that no cell attachment was found on the surface of the six-well plate,

HAMA hydrogel loading with PFD inhibited fibroblast proliferation
After treatment with a special medium, the fibroblasts were observed to shrink to an irregular oval shape under light microscopy, instead of the original spindle-shaped feature. In the 120 mg/ml PFD-treated group, more than half of the fibroblasts showed an irregular oval shape and more deformed cells compared to other dose groups. Meanwhile, the number of fibroblasts showed a significant dose-dependent decrease (Fig. 6A-D). The CCK-8 assay similarly showed a decrease in fibroblast proliferation and viability with increasing doses of PFD ( Figure 6E). The results of the EdU assay were the same as those of the two tests mentioned above. The percentage of EdUpositive cells in the control group exceeded 35% (35.2%) after 24 h, while the percentage of EdU-positive cells in the high-dose PFD-treated group was only 3.8% (Fig. 7A,  B). Therefore, we chose 120 mg/mL PFD as the dose for animal experiments.

HAMA hydrogel loading with PFD downregulated fibrosis in epidural areas
At 8 weeks postoperatively, the surgical area was surgically exposed, and the tissue adhesions were observed visually and graded using the Rydell score (Fig. 8C). The rats in the control group showed severe adhesion of scar tissue to the spinal cord tissue. The Rydell score was significantly lower in the HAMA hydrogel group loaded with PFD compared with the blank control group, the drug-only group, and the hydrogel-only group. The Rydell score was significantly lower in the PFD-loaded HAMA hydrogel group compared with the drug-only group, suggesting that local drug slow release would be more effective than direct drug action.
In addition, histological observations and quantitative analysis were performed. H&E staining at week 8 showed that the epidural area of the control group was filled with a large number of collagen and fibroblasts. In contrast, the HAMA PFD-loaded hydrogel group had the lowest percentage of fibrous tissue, sparse distribution of dense collagen tissue, and a significant reduction of epidural adhesions in the group. Masson staining showed densely distributed collagen tissue visible in the epidural area of the control group. The rats in the drug-only group and the hydrogel-only group had less epidural fibrous tissue adhesions, with some fibroblasts and a moderate number of collagen fibers. The PFD-loaded HAMA hydrogel group had the least epidural fibrous tissue adhesions, with few fibroblasts and fewer collagen fibers (Fig. 8A, B). The above results suggest that HAMA hydrogel combined with PFD can effectively reduce the formation of fibrous scars after laminectomy.
Western blot was performed to detect the effect of PFDloaded HAMA hydrogel on adhesion-related protein inhibition and adhesion formation in vivo. The expression levels of type I collagen, type III collagen, and α-SMA protein in the adhesion tissues at the surgical site were measured 8 weeks after surgery (Fig. 9). The expression levels of type I collagen, type III collagen, and α-SMA in the control group were significantly other experimental groups. Notably, in all three experimental groups, the expression levels of all three adhesion-related proteins in the HAMA hydrogel group loaded with PFD were significantly lower than those in the drug-only group and the hydrogel-only group. This again indicates that the combined anti-adhesive effect of PFD-loaded HAMA hydrogel resulted in a significant improvement in epidural fibrosis compared with the drugonly group and the hydrogel-only group ( Figure 6A-D).

PFD and HAMA hydrogel are not harmful to the body
To test whether PFD and HAMA hydrogel has toxic effects on other organs throughout the body. Therefore, we performed hematoxylin and eosin (H&E) staining tests, and no significant physiological abnormalities were found in major organs such as the heart, liver, spleen, lung, and kidney in the PFD-loaded HAMA hydrogel group compared with the control group (Fig. 10).

Discussion
The aim of this study was to use an anti-adhesive injectable hydrogel and to load the hydrogel with a PFD drug. The design objective is to prevent the formation of epidural fibrosis after laminectomy by slow and sustained release of PFD through the slow release action of the hydrogel [26]. In this study, different concentrations of PFD (40 mg/mL, 80 mg/mL, and 120 mg/mL) were loaded into HAMA hydrogels, and the optimal concentration of PFD loading was determined by in vitro cellular assays. C Gross evaluation of the adhesion formation by the Rydell scores. ns: p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 Fig. 9 Western blot assay for collagen I/III and α-SMA expressions in adhesion tissues of different groups after 8 weeks. ns: p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

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Then, the anti-adhesive ability of HAMA hydrogels loaded with optimal concentrations of PFD was compared with PFD only or HAMA hydrogels alone after laminectomy. In addition, in vitro studies were performed to reveal the slow-release effect of HAMA hydrogel. Finally, the in vivo study showed that HAMA hydrogel and PFD have good biocompatibility and no significant toxicological problems [27,28].
Injectable hydrogel, as a particularly powerful means of improving drug delivery, has attracted a lot of attention in the field of sustained-release drug delivery systems in recent years [29]. The HAMA hydrogel loading with PFD exhibits early burst release after implantation, followed by a slow sustained release, with approximately 80% of the loaded drug release occurring within approximately 9 days. Although the initial burst release is usually considered a drawback of the drug delivery system, the timing of drug release in this experiment corresponds to the first postoperative week, which coincides with the time of inflammatory response and fibroblast invasion and proliferation. This will be followed by the anti-epidural fibrosis effect exerted by the physical barrier effect of HAMA hydrogel [30]. Thus, slow release during the first week after laminectomy helps to reduce the harmful inflammatory response and effectively inhibits the pathological process of adhesion formation [31]. We found that the viability of fibroblasts gradually decreased with increasing PFD doses when only special medium interventions with increasing PFD doses were available, while the proliferation of fibroblasts was inhibited. Since fibroblasts reach a steady state and scar tissue formation within 4 weeks, we infer that the anti-adhesive hydrogel should maintain structural integrity for 4 weeks after laminectomy, followed by gradual degradation and absorption [32]. Macroscopic observations and mass weighing results from in vivo tests showed that the structural integrity of the anti-adhesive HAMA hydrogel used in this study could be maintained for at least 4 weeks and could meet the requirement of a physical barrier for the prevention of epidural fibrosis after laminectomy. Therefore, considering the cytotoxicity and other potential side effects and the results of this trial, 120 mg/ml PFD-loaded HAMA hydrogel is the most suitable anti-adhesive product for further clinical application exploration at this stage.
In the animal model, the large amount of collagen and scar formation was consistent with the clinical presentation and the modeling was successful. Therefore, the aggregation of fibroblasts in the epidural area indicates the important role of fibroblasts in epidural fibrosis [33]. The blank control group confirmed that laminectomy does cause epidural fibrosis. By detecting the epidural collagen distribution and content, PFD and HAMA hydrogel were found to be effective in inhibiting postoperative epidural fibrosis. Furthermore, HAMA hydrogel loading with PFD was found to have the best inhibitory effect on postoperative epidural fibrosis.
However, in this study, we did not explore the exact local concentration of PFD, nor did we specifically distinguish the effects of local application of hyaluronic acid and PFD, which still needs further study. After treatment with a special medium, the fibroblasts were observed to shrink to an irregular oval shape under light microscopy, instead of the original spindle-shaped feature. The principle of this is not very clear to us, whether this involves the effect of pirfenidone on the cytoskeleton as well as on the cell linkage aspect; whether there is some pathway mechanism of intrinsic regulation, which will be part of what we will study next. The drug is weighed on a balance at the beginning and measured and calculated by a spectrophotometer during the release process; the two weighing methods are different and there are certain errors. And when the concentration of the drug in the solution is below a certain range, the spectrophotometer cannot measure the difference, but the drug is really present in the solution. We calculated the release efficiency to be only 80%. Therefore, we still have some problems in drug mass weighing, and we need to improve it in future research. The exploration of the optimal concentration of PDF is still not very precise, especially in the fraction where the loading is greater than 120 mg/mL. This is to be further investigated next.

Conclusion
In this study, we have described a novel and efficient antiadhesion PFD-loaded HAMA hydrogel after laminectomy, which can release PFD in a controlled and sustained manner. In this study, we describe a novel and highly effective anti-adhesive PFD-loaded HAMA hydrogel that can be covered with an epidural after a laminectomy to prevent epidural fibrosis by releasing PFD in a stable and sustained manner. The tests showed that the PFD-loaded HAMA hydrogel not only effectively inhibited cell penetration but also inhibited collagen I/III expression. These results suggest that PFD-loaded HAMA hydrogel can effectively prevent the formation of adhesions through pharmacological and physical processes. Therefore, we believe that these PFD-loaded HAMA hydrogels have great potential as antiadhesive nanomaterials for postoperative repair in clinical applications. Finally, further clinical trials are pending regarding the biosafety of PFD-loaded HAMA hydrogel.