{"paper_id":"443e0256-921d-425e-97c0-4827a47eab1a","body_text":"The mammalian uterus is\na complex female reproductive organ that\nplays a vital role in the various stages of reproduction. It is composed\nof three distinct layers: the innermost layer, known as the endometrium;\nthe middle layer, referred to as the myometrium; and the outermost\nlayer, called the perimetrium. 1  This study\nfocuses on the myometrium, a specialized layer primarily composed\nof smooth muscle cells within the uterus. The myometrium plays a crucial\nrole in maintaining pregnancy and initiating childbirth. The myometrium\nundergoes a complex and dynamic physiological process known as uterine\ncontractility, which is observed in both nonpregnant and pregnant\nwomen phases across the menstrual cycle and is placed in the group\nof spontaneously active and readily excitable muscle tissue. 2  Hormones such as estradiol, oxytocin, and prostaglandins\nexert a substantial influence on myometrial function, contributing\nto its growth, contraction, and effective functioning. 3\nVarious complex and intricate interplays of molecules\nand interactions\ntake place within the uterus. Among these biomolecules, carbohydrates\nhold particular importance, serving crucial functions throughout different\nstages of reproduction. The mammalian uterus consists of a high concentration\nof glycoconjugates, primarily engaging implanted embryos, fetuses,\nand protecting them from potential pathogens that may enter the uterine\nenvironment. 4  Studies have confirmed that\nthe surface of the uterus expresses various sugar epitopes that are\ndetected by lectin proteins. These epitopes are regulated during various\nstages of pregnancy, including preimplantation and postimplantation.\nIt has been established that uterine and trophoblast cells express\nboth sugar-binding proteins and cell surface glycoconjugates. For\nsuccessful implantation to occur, the uterine lining must be receptive.\nCarbohydrate recognition has been observed to play a crucial role\nin controlling the implantation of the human embryo during the initial\nstages. 5 , 6  In another study, it was shown that trophoblasts\nhave galactose present in them, which is recognized by them human\nuterus for invasion and implantation. 7\nThe uterus, while possessing remarkable regenerative abilities,\nis susceptible to various diseases and conditions. Notable among these\nare fibroids, also known as leiomyomas, which are noncancerous growths\nthat develop within the uterine wall, potentially causing discomfort\nand complications. 8  Another condition is\nadenomyosis, where endometrial glands migrate into the myometrial\nregion of the uterus, resulting in an enlarged uterus. 9  Furthermore, certain medical procedures, like cesarean\ndeliveries and uterine surgeries, although often necessary, can impact\nthe uterus. These interventions may lead to complications in the form\nof scarring. These scars have the potential to disrupt the normal\nstructure of the myometrium, which can give rise to issues such as\nabnormal placenta placement and uterine rupture. 10\nTraditional approaches such as allografts and transplants,\nwhile\neffective in some cases, are not without their significant drawbacks,\nincluding the risk of rejection, infections, and complex postsurgical\ncomplications. In light of these challenges, it is important to consider\nalternative approaches. One such approach involves tissue engineering\nusing principles to create new scaffolds tailored to specific tissues\nis essential, which has been explored in this study to design a patch\nto treat the region of scar or wound in the myometrium. This includes\ndesigning scaffolds for the uterus, which is a promising way to tackle\nimportant medical issues related to the uterus, pregnancy, and childbirth. 11  In tissue engineering, biomaterials play an\nimportant role by providing a framework similar to the extracellular\nmatrix (ECM) structure. 12  In order to mimic\nthe ECM fibrous structure of tissues, the electrospinning technique\nis used to create nanofibrous scaffolds. 13  Various polymers have been used for electrospinning nanofibers,\nincluding both natural and synthetic polymers. However, it is more\nconvenient to process synthetic polymers through electrospinning,\nwhich aids in better control of the nanofiber morphology compared\nto natural polymers. Natural polymers are primarily water-soluble,\nwhich poses challenges in directly converting them to nanofibers due\nto their inherent instability. Additionally, they are susceptible\nto harsh processing conditions because of their mechanical weakness.\nIn contrast, synthetic polymers offer greater versatility for specific\nbiological functions while exhibiting desirable properties such as\na cost-effective and easily scalable approach to scaffold development,\nensuring excellent mechanical strength and surface integrity. 14 , 15\nPolycaprolactone (PCL), an aliphatic polyester, was utilized\nas\na biomaterial in this study. It has a melting point of 55 °C\nand remarkable load-bearing mechanical characteristics. It is ideal\nfor soft tissue engineering, especially for myometrium. 16  Further, we have compared two different types\nof nanofibers: randomly oriented and aligned nanofibers. A recent\nstudy by Miki et al. examined the orientation of decellularized uterine\nscaffolds (DUS) in rats revealed that scaffold orientation significantly\ninfluences uterine tissue regeneration. Incorrectly oriented DUS led\nto aberrant tissue topology. 17  Building\non this insight, we conducted a comparative analysis of random and\naligned nanofibers for uterine tissue regeneration, considering the\nmyometrium’s complex layers, which are oriented longitudinal,\ncrisscross, and circular. 18\nPCL is\npreferred for regeneration, but it has a hydrophobic nature.\nTo address the issue of the hydrophobic nature of PCL, in this study,\nPCL fibers are modified using chemical methods in order to establish\nimproved cell-scaffold contact and integration. We have used amine\ngroups to conjugate galactose on the surface of PCL nanofibers. This\ndesign is based on a comprehensive understanding of the role of carbohydrates,\nparticularly galactose. In this study, the novelty lies in the development\nof galactose-conjugated PCL nanofibrous scaffolds tailored to enhance\nmyometrial regeneration, concurrently establishing a biomimetic environment\nand simultaneously creating a biomimetic environment. The rationale\nbehind the conjugation of galactose is that by modifying polymeric\nscaffolds with carbohydrate molecules like galactose, we aim to replicate\nthe natural interactions that occur within the uterine wall and blastocyst.\nThe addition of galactose causes the  l -selectin-based interaction\nof uterine cells with fibers. The ECM is remodeled as a result of\nuterine fibroblast activation brought on by  l -selectin–galactose\ninteraction. 19  This modification enhances\nthe scaffold’s ability to encourage cell attachment, growth,\nand tissue repair. The initial step of aminolysis involves the addition\nof amino groups to PCL fibers by breaking ester bonds present on the\nPCL, 20  and the second step is the addition\nof galactose, where galactose is added using lactose, where anomeric\ncarbon of the glucose will covalently attach to the surface and galactose\nis exposed on the surface. 21  The goal of\nthis surface modification is to improve the hydrophilicity of the\nsurface of the polymer without altering its mechanical strength. The\naddition of galactose is used to increase the hydrophilicity of PCL\nwhich further improves cell adhesion on fibers. This patch presents\na remedy for the efficient regeneration and recovery of uterine myometrium\nwounds, with the potential to transform the approach to managing such\ninjuries related to uterine tissue.\n\nPoly(ε-caprolactone)\n(Mn = 80,000) pellets were obtained from Sigma-Aldrich. The solvents\nchloroform and methanol to dissolve PCL were obtained from Himedia\nCo. For the preparation of the 10% (w/v) solution, the first PCL pellets\nwere added to chloroform and stirred for 15 min. Later, to the same\nsolution, methanol was added in the ratio of 4:1 and kept for 12 h\nstirring at room temperature until the pellets were dissolved completely. 16\nNanofibers were produced\nusing an electrospinning unit (Model-HO-NFES-040) with a set of optimized\nparameters. The electrospinning parameters were optimized and described\nin a prior study by our group. 16  A 10 mL\nsyringe containing polymer was set, and a voltage of 18 kV was applied.\nA needle with a gauge of 24G was used, and a constant distance of\n20 cm was maintained between the needle tip and the collector, while\nthe flow rate was accurately held at 0.002 mL/min. A rotating mandrel\nwith a speed of 2000 rpm was used for aligned fibers. The electrospinning\nwas carried out for 24 h to obtain a single mat of random or aligned\nfibers. The random fibers and aligned fibers collected on the stationary\nand rotating mandrels were stored in a vacuum desiccator for further\ncharacterization.\nThe surface\nmodification of PCL nanofibers was done by conjugating the surface\nof the nanofibers with galactose in two steps.\nPCL nanofibrous\nmats obtained are cut into pieces measuring 1 × 1 cm. To eliminate\noil or other dirt on scaffolds, it was immersed in an alcohol–water\n(1:1, v/v) solution, washed with deionized water, and dried. Fibers\nwere then immersed in a 10% 1,6-hexane diamine/2-propanol solution\novernight at 37 °C, rinsed with deionized water to remove any\nunattached or excess 1,6-hexane diamine, and dried. 16\nTo conjugate\ngalactose on the aminated scaffolds, the aminated discs were soaked\nin 100 mL of citrate buffer solution overnight containing 1.88 g of\nsodium cyanoborohydride (NaBH3CN) and 21.61 g of galactose, with a\npH of 6.1, following the neo glycosylation protocol from ref ( 22 ).\nMorphology and post\nsurface modification structure of electrospun fibers were analyzed\nusing scanning electron microscopy (SEM, Carl Zeiss Ultra 55, CeNSE,\nIISc Bangalore). Prior to analysis, samples were desiccated for 24\nh in a desiccator to remove any solvents present and then gold sputter-coated\nto apply a conductive layer before mounting. Analysis was conducted\nat magnifications of 75K, 25K, and 5K X at an accelerating voltage\nof 5 kV. SEM images were utilized for fiber diameter and orientation\nanalyses using ImageJ software.\nPost-aminolysis,\na ninhydrin assay was performed to quantify the presence of amine\ngroups. Ninhydrin reagent (1 M) prepared in 10 mL of ethanol was added\nto the scaffold discs. Scaffolds were incubated in 100 μL of\nninhydrin solution in a hot water bath at 70 °C for 15 min. Subsequently,\nthe tubes were allowed to cool to room temperature. To dissolve the\nscaffolds, 500 μL of chloroform and isopropyl alcohol were added\nto the tubes. From this solution, 100 μL was transferred to\n96-well plates, and the intensity was measured at a wavelength of\n562 nm using a spectrophotometer [PerkinElmer (Ensight) multimode\nplate reader HH34000000].\nTo quantitatively and qualitatively assess the amount of galactose\nconjugated on the nanofiber’s surface, two types of ELLA assays\nwere conducted. In the first assay, a lectin with FITC fluorescence\nwas used (FITC-ELLA). In the second assay, a lectin conjugated with\nhorseradish peroxidase (HRP) was employed. For the FITC-ELLA assay,\nPCL and galactose-conjugated PCL samples were suspended in a phosphate-buffered\nsaline (PBS) solution containing FITC-conjugated lectin from  Arachis hypogaea  (peanut lectin) (Sigma-Aldrich L7381)\nat a concentration of 40 μg/mL. The samples were then stirred\nin the dark for 2 h. After this incubation, they were washed with\nPBS. Subsequently, the samples were examined for their fluorescence\nusing a fluorescence microscope (Nikon Eclipse-TE2000-U).\nThe\nHRP-ELLA assay involved treating PCL and galactose-conjugated PCL\nsamples with a 2% BSA solution in PBS (100 μL) and shaking them\nat 5 °C for 14 h. Subsequently, the samples were incubated at\nroom temperature with a solution of peanut lectin conjugated to HRP\n(Sigma-Aldrich, L7759) (0.01 mg/mL, 200 μL) in PBS (200 μL)\nfor 2 h with shaking. After incubation, excess unbound lectin was\nremoved by thorough washing with PBS. Next, the samples were treated\nwith a solution of OPD ( o -phenylenediamine dihydrochloride)\n(SIGMAFASTM OPD Sigma-Aldrich, catalog no. P9187) for 1 h. The absorbance\nof a 200 μL aliquot of this solution was then measured at 450\nnm using a spectrophotometer [PerkinElmer (Ensight) multimode plate\nreader HH34000000].\nThe hydrophobicity\nand hydrophilicity of the nanofiber surface were confirmed with a\ncontact angle using a goniometer. Each scaffold ( n  = 3) was considered for the study, and using a water droplet, the\nangle between the water droplet and surface was used to study the\nsurface energy of the scaffolds at room temperature 23–25 °C.\nThe mechanical\ncharacteristics of unmodified and modified PCL nanofibrous scaffolds\nin both random and aligned configurations were evaluated using a Shimadzu\nuniversal texture analyzer (EZ-SX) device, manufactured by Shimadzu\nCorporation, Japan. Electrospun mats were trimmed to produce samples\nmeasuring approximately 100 mm in length and 20 mm in width. These\nsamples were securely clamped at both ends and subjected to a constant\nstretching rate of 10 mm/min until they reached the point of fracture.\nThe collected data was subsequently transformed into stress–strain\ncurves, and tensile strength as well as the percentage of elongation\nat break were determined based on the sample’s width and thickness.\nThe results are presented as the mean value ± the standard deviation\nbased on three separate measurements.\nNanofiber scaffolds, cut into 1 cm 2  pieces, were immersed\nin PBS (pH = 7.4) and incubated in vitro at 37 °C for 7, 14,\nand 21 days. At these intervals, water uptake and degradation were\nassessed. Water uptake was determined by measuring the wet weight\nof the scaffolds after blotting excess surface water. Subsequently,\nthe scaffolds were washed, dried for 24 h at room temperature, and\nweighed to assess degradation. Additionally, morphological changes\nwere observed using SEM analysis. 23\nHuman uterine\nfibroblast cells (HUF) (PCS-460-010) were maintained using fibroblast\nbasal medium (ATCC-PCS-201-030) supplemented with the Fibroblast Growth\nKit-low Serum—(ATCC PCS-201-0410) and 1% penicillin–streptomycin.\nThe cultures were incubated at 37 °C in 5% carbon dioxide. Subsequently,\nconfluent HUF was seeded onto scaffolds for further investigation.\nThe scaffolds were cut\nto fit the size of a 96 well plate and then sterilized under UV in\nthe laminar hood for 24 h before cell seeding. HUF were seeded onto\nthe scaffolds at a density of 5000 cells per well. Scaffolds were\ncut into 5 mm diameters each and placed in the 96 well plate, and\ntissue culture polystyrene (TCPS) was kept as a control. The plate\nwas then placed in a CO 2  incubator at 37 °C. Readings\nwere taken at three-time intervals: 1, 7, and 14 days after culturing.\nThe plate was incubated for 3–4 h with an MTT reagent (0.5\nmg/mL). Afterward, 100 μL of DMSO reagent was added to each\nwell and left for 1 h to dissolve the formazan crystals, resulting\nin a color change. Finally, the absorbance of the formazan solution\nwas measured at 570 nm using a spectrophotometer [PerkinElmer (Ensight)\nmultimode plate reader HH34000000].\nCell viability of\nthe seeded cells on the scaffolds was evaluated at two different time\npoints, day 1 and day 3, using a live/dead assay kit (L3224). To prepare\nthe live/dead reagent, a stock solution was created by combining 4\nμL of EthD-1 and 1 μL of Calcein-AM in 2 mL of PBS. Subsequently,\n100–150 μL of the live/dead reagent was added to the\nscaffolds and incubated at room temperature for 1 h. The cells were\nthen examined by using a fluorescence microscope (Nikon Eclipse-TE2000-U).\nCells were\nfixed with 4% paraformaldehyde for 1 h at room temperature, after\nfixing, additional PBS washes were performed, followed by permeabilization\nusing 0.5% Triton X-100 for 30 min at room temperature. The cells\nwere then blocked with 5% BSA for 1 h at room temperature. The cells\nwere later incubated with the appropriate dilution of primary antibody-Versican\n(1:100 dilution, NBP2-22408 Novus Biologicals) overnight at 4 °C.\nWashes were performed gently, and secondary antibody Rabbit anti-Mouse\nIgG1 fluorescein (NBP1-73636 Novus Biologicals) was added at a final\nconcentration of a 1:1000 for 1 h at room temperature. It was counterstained\nwith rhodamine-phalloidin stain (R415, Invitrogen) for 45 min and\nwashed with PBS. The cells were stained with 1:1000 diluted DAPI solution,\nand they were visualized under the fluorescence microscope (NikonTE2000U)\nwith filters.\nTotal RNA\nis extracted from the HUF cultured on the scaffolds for 7 and 14 days\nusing the standard Trizol RNA (RNA iso Takara) isolation protocol.\nThe obtained RNA is later subjected to cDNA synthesis using the kit\n(RDRT Sigma-Aldrich ReadyScript cDNA Synthesis Mix) according to the\nmanufacturer’s instruction. Then, the mRNA expression is carried\nout by real-time PCR using the SYBR Green master mix (BioRad iTaq\nUniversal SYBR Green Supermix). The experimental procedure was conducted\nwith a total volume of 10 μL, comprising 0.5 μL of each\nprimer, as listed in  Table  1 , 5 μL of SYBR green master mix, 3.5 μL of diethylpyrocarbonate-treated\nwater, and 0.5 μL of cDNA template. Subsequently, the samples\nwere subjected to 100 cycles using the Qiagen Rotar Q series machine,\nand the obtained results were analyzed using the accompanying software,\nRotorgene Qiagen software for the instrument.\nWistar rats ( Rattus norvegicus ) were used for in vivo experiments.\nAll animals were provided with care in strict adherence to the guidelines\nestablished by the Kasturba Medical College, Manipal, MAHE, for Animal\nCare. Furthermore, the Institute’s Ethical Review Committee\ngranted approval for the experimental protocols under reference number\nIAEC/KMC/76/2022. For each group, 3 rats were assigned randomly. Each\nanimal weighed between 250 and 300 g. Intraperitoneal injections of\nketamine and xylazine were administered to anesthetize animals based\non their weight. The dorsal area of the animals was shaved and sterilized\nwith 70% ethanol. Using a sterile surgical blade, an incision of about\n1 cm was made on the dorsal lobes of animals. A subcutaneous pouch\nwas created on the incision. Scaffolds were UV sterilized before using\nfor implantation, and an implant was inserted into each pocket. Upon\nimplantation of the polymer into the pouch, the cut was sutured. The\nsutures were removed 7 days after surgery. After 3 weeks, the tissue\nsurrounding the implant was excised to study and understand the inflammatory\nresponse by using hematoxylin and eosin (H&E) staining and Masson’s\ntrichome staining.\nHaematoxylin stain’s\nacidic part, stains mainly the nucleus, while eosin acts as an acidic\nstain and binds the basic part, i.e., the cytoplasm. The samples were\nfixed with methanol for 30 min, and after drying in air, different\nranges of alcohols such as 100, 70, and 40% are added for a few seconds\neach for hydration. The samples were further stained in hematoxylin\nfor 20 min and washed with 1% acetic acid for 10 min until the nuclei\nappeared blue; eosin was added for 30 s, washed with distilled water,\nand further treated with a different range of alcohol for dehydration.\nTissue samples were fixed in 4% paraformaldehyde at 4 °C for\n24 h and then paraffin embedded. After fixation, slides were stained\nwith Weigert’s iron hematoxylin for 10–15 min, which\nstained the nuclei blue–black, and rinsed in distilled water.\nSlides were immersed in Biebrich scarlet acid fuchsin for 5–10\nmin, which stained the muscle fibers and cytoplasm red, and then rinsed.\nTissue sections were differentiated with phosphomolybdic–phosphotungstic\nacid for a few minutes, which removes excess stain from collagen,\nand rinsed. Slides were finally immersed in aniline blue for 5–10\nmin, which stained collagen fibers blue green. Stained sections were\ndehydrated in alcohol (70, 95, and 100% ethanol), cleared in xylene,\nand mounted using Permount. After drying, Masson’s trichrome-stained\ntissue sections were ready for microscopic examination, aiding collagen,\nnuclei, and muscle fiber visualization for tissue analysis. Subsequently,\nthe image intensities were calculated by using ImageJ software.\nThe data were\ncollected with replication schemes, and mean values were calculated\nfor each set. Statistical analysis was performed using GraphPad Prism\nsoftware with ANOVA. Significance levels were represented as follows:\n* p  < 0.05; ** p  < 0.01; *** p  < 0.001; and **** p  < 0.0001.\n\nIn this study, the electrospinning technique was utilized to fabricate\nPCL nanofibers to replicate the intricate ECM present in the uterus\nmyometrium. This method yielded nanofibers that closely mimic the\ntissue’s ECM structure. Subsequently, the PCL nanofibers were\nsubjected to surface modification with galactose. The primary focus\nof the SEM analysis was to investigate four distinct characteristics:\nmorphology, diameter, porosity, and orientation. Beginning with morphology,\nin  Figure  1  the nanofibers\ndisplayed a smooth, bead-free structure in both random and aligned\nconfigurations. Post modification, no significant roughness was observed.\nSimilarly to the previous study, post maltose conjugation using a\nsimilar technique made the nanofiber’s surface smooth. 24  The treatment time plays a crucial role, as\nthe morphology will be damaged and the fibers will break if they are\ntreated for a longer time. 25  Regarding\ndiameter, the nanofiber measurements fell within the nanorange, i.e.,\n1–1000 nm. The diameter of random PCL fibers was found to be\n360.9 ± 151.3 nm, and aligned PCL fibers had 570.7 ± 219.53\nnm. The diameter of the nanofibers is affected by the type of collector;\nin aligned nanofibers, in order to obtain less deviation in alignment\nangle, the rotating mandrel speed was set to 2000 rpm, which resulted\nin the diameter variation, whereas random fibers resulted in a consistent\nnanometer range. 26  Notably, after modification,\nthe morphology and diameter of the fibers changed slightly, random\nfiber diameter was 395.6 ± 81.4 nm, and aligned fibers diameter\nwas 591.4 ± 304.1 nm.\nSEM images of (A) PCL random (PCL-R), (B) PCL\naligned (PCL-A),\n(C) GPCL-R, and (D) GPCL-A. (E–H) Histograms of fiber diameter\n(minimum 100 fibers were measured) (E) PCL-R, (F) PCL-A, (G) GPCL-R,\nand (H) GPCL-A. (I,J) Histograms showing the alignment of nanofibers.\nThrough image analysis software (ImageJ), the porosity\nof both\naligned and random fibers was quantified. As shown in  Table  2 , it is noteworthy that aligned\nfibers exhibited lower porosity compared with random fibers. Specifically,\nrandom fibers demonstrated a porosity of 13.8%, while aligned fibers\ndisplayed a porosity of 4.5%. After surface modification, galactose\nPCL random (GPCL-R) has 11.6% and galactose PCL aligned (GPCL-A) has\n7% of porosity, respectively. Aligned fibers are densely packed when\ncompared to random fibers; therefore, their porosity is reduced. 27 Figure  1 I,J shows that the angle of alignment for the fibers is approximately\n0.30 ± 7.24° for aligned fibers, and for random fibers,\nthe alignment is not in a single direction, but most of the fibers\nwere around 36.90 ± 11.90°. The above results indicate that\naminolysis and galactose treatment had no remarkable effect on morphology,\nwhereas diameter increased slightly when compared to unmodified PCL.\nThis slight increase in diameter may be attributed to the tendency\nof nanofibers to form fused structures during surface modification,\nwhich consequently led to a slight increase in diameter and porosity. 28  These findings align with similar studies; for\ninstance, in our previous study 24  we reported\nthat aminolysis and sugar conjugation did not affect the surface morphology\nof fibers, and the diameter did not change. In addition, in a study\nby Amoures de Sousa et al., no alteration was found in the morphology\nof PCL nanofibers following the modification. 68\nThe process of aminolysis\ninvolves breaking ester bonds present on PCL, leading to the generation\nof amide bonds. This study utilizes 1,6-hexanediamine to perform this\nreaction on PCL. This reaction results in one amino group, reacting\nwith the –COO– group to establish a covalent –CONH–\nbond, while the second amino group remains unreacted and free. This\navailable free amino group is subsequently used to conjugate galactose;\nthis reaction is an intermediate step for galactose conjugation. 28 − 31\nThe confirmation test for the presence of a free amino group\non the surface was achieved through the ninhydrin test. The ninhydrin\n(triketohydrindene hydrate) test, a simple and sensitive assay, works\non the basis of reacting with the primary amino group to produce a\ncolored product known as diketohydrindylidene-diketohydrindamine (Ruhemann’s\nPurple), as shown in  Figure  2 B. 20  As represented in  Figure  2 A, the amine concentration\nof the samples was calculated using the standard curve obtained using\n1,6 hexanediamine. Aminolyzed random fibers showed 42.6 ± 0.6\nμg/mL and aligned fibers showed 28.6 ± 1.2 μg/mL\nof amine concentration. Since random fibers are more porous, and aminolysis\noccurs in depth in the  Z  direction; the amount of\namine groups is higher on random fibers when compared to aligned fibers. 31  On GPCL-R fibers, it decreased to 7.7 ±\n0.4 μg/mL and in GPCL-A, it decreased to 8.4 ± 0.4 μg/mL,\nwhich is similar to unmodified random and aligned PCL fibers, which\nhad 7.2 ± 0.2 and 6.3 ± 0.2 μg/mL of amine concentration,\nrespectively. A similar decrease in the percentage of amine groups\nafter conjugation with galactose and maltose was also observed in\na few other studies. 21 , 22  The NH 2  group participates\nin the conjugation of galactose; therefore, free amine groups are\nnot available to react with the ninhydrin reagent. 32\n(A) Quantification of amine groups using the ninhydrin assay on\nPCL-R, APCL-R, GPCL-R, PCL-A, APCL-A, and GPCL-A (*** p  < 0.001). (B) Macroscopic picture of ninhydrin reaction color\ntransition on the surface-modified scaffold. Scale bar = 100 μm.\n(C) ELLA-HRP-conjugated assay quantification of the galactose moiety\n(**** p  < 0.0001). (D) ELLA-FITC conjugated assay-quantitative\nanalysis of the galactose moiety (**** p  < 0.0001).\n(E) Fluorescent images of the ELLA-FITC assay on (i) PCL-R, (ii) PCL-A,\n(iii) GPCL-R, and (iv) GPCL-A nanofibers.\nCarbohydrates can only perform\ntheir biochemical role if they are exposed to the appropriate receptors. 33  Galactose is conjugated on the surface by using\nlactose sugar. Lactose sugar has two components, glucose and galactose.\nGlucose attaches to free amine groups, and galactose is present on\ntop. Therefore, to confirm the galactose moiety on the top of the\nsurface, an ELLA assay is performed. Both quantitatively and qualitatively,\nthe galactose moiety present is calculated using HRP and FITC-conjugated\npeanut lectin from  A. hypogaea . To\nquantitatively measure the quantity of galactose, we used a standard\ncurve of lectin. As shown in  Figure  2 C, the galactose content is determined to be 106.2\n± 0.12 μg/mL on random nanofibers and 110.9 ± 0.4\nμg/mL on aligned nanofibers. To qualitatively see the spread\nof galactose on the surface, the FITC-conjugated ELLA assay was done.\nWe can clearly differentiate between galactose-conjugated intensity\nin  Figure  2 E(iii,iv)\non the scaffolds conjugated with galactose compared to the unmodified\nscaffolds in  Figure  2 E(i,ii), and the intensity of the fluorescence is calculated and\ndepicted in graph  Figure  2 D. This confirms the presence of a galactose moiety on top\nof the fibers surface.\nSurface wettability,\nreferred to as hydrophobicity or hydrophilicity, stands as a critical\nfactor influencing various cellular behaviors. The degree of wettability\nof the scaffolds was determined through water contact angle measurements,\na reliable parameter that measures how readily water droplets spread\non the nanofibrous surface. 34  The results\nof the contact angle shown in  Table  3  show that unmodified PCL-R fibers exhibited hydrophobic\ntraits, with a left contact angle of 134.94 ± 3.31° and\nright contact angle of 135.52 ± 3.09°. On the other hand,\ngalactose-conjugated PCL surfaces display reduced contact angles,\nwith a left angle of 77.85 ± 8.0° and a right angle of 78.14\n± 3.9°. Similarly, aligned PCL fibers show a right angle\nof 127.63 ± 3.09° and a left angle of 128.4 ± 3.2°,\nwhile galactose-conjugated aligned PCL surfaces exhibit even further\nreduction to 64.63 ± 8.0 and 65.14 ± 3.9° of left and\nright angles, respectively. The surface modification involving galactose\nresults in the introduction of hydroxyl groups and an increase in\nsurface energy, consequently enhancing hydrophilicity. 35  Additionally, it was observed in  Figure  3 iB,D that water droplets spread\nmore rapidly on galactose-grafted PCL surfaces and were quickly absorbed\nupon contact. This phenomenon promotes the attachment of negatively\ncharged cells to the surface of the modified PCL fibers. 36  Furthermore, scaffold alignment revealed a different\nrange of wettability. Aligned scaffolds demonstrated increased hydrophobicity\ncompared with randomly oriented scaffolds. The aligned fibers, being\ncompactly packed, yield lower porosity in contrast to the loosely\narranged, highly porous random fibers, which influence the wettability. 37\n(i) Contact angle of (A) PCL-R, (B) GPCL-R, and (C) PCL-A, (D)\nGPCL-A nanofibers. (ii) Mechanical properties of unmodified PCL-R,\nPCL-A, and modified GPCL-R and GPCL-A scaffolds. (A) Representative\nstress–strain curves. (B) Tensile strength and elongation at\nbreak. Data are presented as average ± standard deviation ( n  = 3). (iii) Degradation rate of nanofibers: (A) PCL-R,\n(B) PCL-A, (C) GPCL-R, (D) GPCL-A, and (iv) Water uptake kinetics\nof nanofibers (A) PCL-R, (B) PCL-A, (C) GPCL-R, and (D) GPCL-A.\nThe mechanical\nproperties of random and aligned nanofibers with modifications were\ncompared using a universal tensile testing machine. As observed in  Figure  3 iiA, galactose-modified\nscaffolds have demonstrated good elasticity along with moderate tensile\nstrength. Random galactose-modified PCL showed the highest tensile\nstrength of 0.041 ± 0.01 MPa when compared to random unmodified\nPCL with 0.026 ± 0.01 MPa, aligned unmodified PCL with 0.011\n± 0.001 MPa and aligned modified PCL with 0.016 ± 0.002\nMPa of tensile strength, respectively. Together, the galactose-conjugated\nscaffolds, both random and aligned fibers’ tensile strengths\nwere improved when compared to unmodified fibers, indicating that\nthe chemical treatment did not make the scaffolds brittle. Due to\nrandom nanofiber orientation, the nanofibers could be drawn out or\nstretched relatively easily via deformation under the applied stress. 38\nThe analysis, as seen in  Figure  3 iiB, also reveals that random\nPCL unmodified showed 28 ± 13.3% elongation at break when compared\nto modified PCL, which shows 49.5 ± 11.6%. Similarly, in aligned\nunmodified, an elongation at break of 65.3 ± 14.1%, and in aligned\nmodified, 91.3 ± 15.4% elongation at break was observed. In general,\nmaterials with higher tensile strength tend to have lower elongation\nat break, and vice versa. This is because materials that are very\nstrong and rigid (high tensile strength) are less likely to deform\nor elongate significantly before breaking, whereas materials that\nare more flexible and ductile (low tensile strength) can stretch and\ndeform more before reaching their breaking point. 39\nScaffold\ndegradation is essential in tissue engineering as it facilitates the\ngradual breakdown of the scaffold over time, promoting the growth\nand regeneration of new tissue. 40  Under\nphysiological conditions, PCL undergoes degradation via ester bond\ncleavage through hydrolysis. 41  Therefore,\nwater uptake influences the degradation and mechanical properties\nof the polymer. 42  The aim of this experiment\nis to compare the water uptake and degradation behavior of electrospun\nPCL nanofibers with and without surface modification, as degradation\nrates are influenced by the structure, geometry of the polymers, and\nsurface area. 43 Figure  3 iii,iv presents the weight loss percentage\nand water absorption of electrospun PCL nanofiber surface-modified\nand unmodified, respectively. Both modified and unmodified scaffolds\nremained almost unchanged during the degradation period, with both\nabsorption and weight loss remaining very low after 21 days. PCL-R\nfibers showed 8% uptake at 21 days, while surface-modified scaffolds\nshowed 12% uptake. Similarly, PCL-A fibers showed 7% uptake, whereas\nsurface-modified scaffolds showed 13% uptake, as depicted in  Figure  3 iii. The higher the\nwater uptake, the higher the degradation that was observed, particularly\nin GPCL-A. However, there was not much difference in the morphology\nof fibers observed, as shown in Supporting  Figure S1 .\nThe proliferation of primary\nHUF on galactose-conjugated nanofibers was evaluated after days 1,\n7, and 14 time points. The number of live cells on PCL unmodified\nand modified was similar at the first day time point. The cell viability\non the surface of modified scaffolds increased significantly as compared\nwith unmodified scaffolds after all the time points. In  Figure  4 A, it is evident that the absorbance\nof PCL-R and PCL-A on days 7 and 14 is lower than that on day 1. The\ndecrease in absorbance of PCL-R and PCL-A over time may be attributed\nto several factors, including potential cell confluence, nutrient\ndepletion, or metabolic changes. MTT results also suggest that the\nmodified scaffolds are not toxic to cells and, therefore, are cytocompatible.\nMoreover, these results imply that enhanced cell proliferation contributes\nto improved regenerative potential. 22  While\ngalactose-grafted scaffolds exhibited superior proliferation rates\nof HUF cells compared with the unmodified PCL scaffolds, this outcome\naligns with our expectations. The lack of significant changes in the\nabsorbance of GPCL-A and GPCL-R could be indicative of the enhanced\nperformance of the galactose-grafted scaffolds in maintaining cell\nviability and supporting prolonged cell proliferation. The interaction\nbetween the galactose moiety and cell receptors promoted enhanced\ncell attachment, resulting in significantly higher proliferation rates\nat all time points. These findings affirm the preference of HUF cells\nfor a hydrophilic surface. 44  This is because\ngalactose acts as a cellular matrix adhesive component and triggers\nthe cellular response. Galectin receptors present on fibroblasts are\nactivated, and cell proliferation has increased. 45 , 46\n(A)\nMTT assay using human uterine fibroblasts on TCPS, PCL-R, PCL-A,\nGPCL-R, and GPCL-A nanofibers on days 1, 7, and 14 of culture (* p  < 0.05), (*** p  < 0.0001). (B) Live/dead\nassay with human uterine fibroblasts seeded on TCPS, PCL-R, GPCL-R,\nPCL-A, and GPCL-A scaffolds. (C) Quantification of live cells (green)\nin live dead assay for viability percentage.\nThe live-dead test\nwas performed on two-day points, day 1 and day 3. All the scaffolds\nhad very few dead cells, and the difference in morphology on each\nscaffold could be visualized in  Figure  4 B. Similar to the TCPS control, most of the cells on\nthe scaffolds were not stressed or dead. It was also noted that the\ncells seeded on aligned nanofibers exhibited elongation and alignment\nsimilar to nanofiber orientation, closely resembling the underlying\nscaffold morphology. These results in  Figure  4 B clearly indicate that the topology of the\nPCL scaffold has the ability to influence the orientation of cell\ngrowth and spreading. 47  On random unmodified\nand galactose-modified PCL scaffolds, cells tend to spread randomly\nin all directions. Using the ImageJ software directionality plugin,\nthe degree of direction was calculated, and it was observed that on\nPCL-A and GPCL-A scaffolds, on day 3, cells are observed to align\nthemselves at an angle of approximately 5 and 16°, respectively.\nThis study suggests that after 3 days of culture, cells exhibit elongation\nalong the direction of the nanofibers, possibly in response to the\ndetected convex curvature of the cylindrical nanofiber structures.\nGiven that aligned fibers mimic the ECM structure of the uterine myometrial\nlayer, they may be a preferred choice over random fibers for certain\napplications.\nPhalloidin\nstaining is used for staining F-actin filaments, which are an important\ncomponent of the cytoskeleton. The F-actin protein plays a major role\nin cells as a structural and translocation protein. Numerous signals,\nsuch as growth factors, ECM, and chemokines, cause cytoskeletal rearrangement. 48  In this study, the scaffold properties might\nhave caused a change within the cells and caused the rearrangement\nof f-actin. Therefore, to study the change in the cytoskeleton, this\nstaining was done.\nTo assess the cellular morphology and the\narrangement of their actin cytoskeleton on the scaffolds, cells were\nstained with phalloidin and examined using a fluorescence microscope.\nThe findings, depicted in  Figure  5 , revealed that the number of cells on unmodified PCL\nappeared comparatively lower than on the modified counterpart. This\nobservation depicts the impact of the scaffold’s hydrophobic\nproperties on cellular adhesion and distribution. In various recent\ninvestigations, it has been shown that cell attachment and spreading\nare more pronounced on hydrophilic surfaces with positive amine modifications\ncompared to hydrophobic surfaces, under conditions with or without\nthe presence of serum. 49\n(A) Immunofluorescence\nassay using DAPI (blue), Rhodamine Phalloidin\n(red), and Versican (green) for the human uterine fibroblast cells\non the modified and unmodified surfaces of the scaffolds after 2 days\nof culture. Scale bar = 100 μm. (B) Quantification of fluorescence\nintensity of Versican (green).\nVersican is one of the major proteoglycans expressed\nby cultured\nfibroblasts and present in the ECM of smooth muscle tissue. 50  It helps in the binding of hyaluronan via the\namino terminal. It also has several other domains to bind to, including\nlectin, epidermal growth factor, and complement regulatory proteins.\nThe major role of Versican is that it helps in cell adhesion and modulation\nof the ECM. 51  To visualize the distribution\nof Versican in HUFs since it plays a major role in cell phenotype\nand cell migration, this staining was performed. In  Figure  5 , it was observed that the\nmorphology and expression of Versican were observed on scaffolds similar\nto the TCPS. Surface-modified scaffolds exhibited improved expression\nof Versican by providing amino-terminal groups.\nTo investigate\nthe impact of galactose conjugation and lectin-based cell–fiber\ninteractions on mimicking trophoblast invasion, cell growth, and potential\nECM remodeling, we conducted gene expression experiments. Our hypothesis\nrevolved around the concept of  l -selectin-based interactions\nand potential ECM remodeling. RNA was isolated after 7 and 14 days\nof culture of HUFs on the scaffolds, and CDNA was prepared. Versican,\ncollagen, laminin, and galectin genes were studied using primers mentioned\nin  Table  1 . Galectin\n3, a member of the lectin family, possesses unique characteristics\nfor glycan binding. It serves as a versatile regulator of crucial\nbiological processes, including cell adhesion, growth, proliferation,\nand differentiation. 52  They also play a\nrole in mediating cell-to-ECM heterotypic adhesion processes. Existing\nliterature suggests that modulating galectin-3 functions can either\nenhance or diminish cell adhesion to ECM protein ligands like laminin,\ncollagen type IV, and fibronectin. 53  Additionally,\ngalectin-3 contributes to wound healing and cell re-epithelialization\nand plays a critical role in modulating interactions between cells\nand the ECM during wound re-epithelialization. 54 , 55  Studies by Bevan et al. confirmed the presence of beta- d -galactoside-binding lectins in the uterine wall, while experiments\nby Vicovac et al. indicated that galectin-1 and galectin-3 are predominantly\nfound in the placental bed of the uterus, where trophoblast attachment\noccurs. Galectins also play a role in organizing the ECM and presenting\nECM ligands to surface receptors on migrating cells, whether of trophoblastic\nor bone marrow origin. 56 , 57  Our qPCR results in  Figure  6 A revealed that galectin\n3 genes are expressed and upregulated after day 7, peaking at day\n14 in galactose-modified scaffolds, particularly in GPCL-R compared\nto GPCL-A. This upregulation of galectin 3 corresponds to phases of\ncell proliferation and differentiation. In fibroblast culture, exogenous\ngalectin-3 has been reported to stimulate cell proliferation. 58  Inohara et al. reported that galectin-3 acts\nas a mitogen, capable of stimulating fibroblast cell proliferation\nin a paracrine manner through interactions with cell surface glycoconjugates. 59\nQuantitative real-time RT-PCR gene expression analysis\nof ECM protein\ngenes. (A) Galectin 3, (B) Versican, (C) laminin, (D) collagen I,\nand (E) collagen III (* p  < 0.05) (**** p  < 0.0001).\nIn  Figure  6 B, Versican,\na substantial chondroitin sulfate proteoglycan known for binding hyaluronan\nand forming extensive ECM aggregates, can influence critical physiological\nprocesses such as cell proliferation, adhesion, and migration in the\nendometrium. It also plays a role in embryo attachment. 60  We observed significant changes in the expression\nof Versican on both day 7 and day 14 in galactose-conjugated random\nand aligned PCL scaffolds. In  Figure  6 C, laminin, another ECM protein present in the uterus,\nis typically found in the myometrium and endometrium as part of the\nbasement membranes, particularly in nonpregnant uteri. During embryo\nattachment and invasion, laminin interacts with trophoblasts. 61  Therefore, laminin expression is associated\nwith cellular differentiation, adhesion, and growth. 62  Our study indicated increased laminin expression on day\n14 in both modified random and aligned PCL scaffolds, particularly\nwhen compared to unmodified scaffolds. 54 − 57 , 59\nThe ECM of the uterus is characterized as a fiber-reinforced\ncomposite\nviscoelastic material primarily composed of fibrillar collagen, with\napproximately two-thirds being type I collagen and one-third being\ntype III collagen. 63  It is important to\nnote that the relative composition of collagen types significantly\ninfluences the mechanical properties of the tissues. Maintaining an\nappropriate balance between collagen types I and III is crucial for\npreserving the functional integrity of various tissues. An increase\nin the collagen I/III ratio results in heightened tissue rigidity,\nwhereas a decrease enhances tissue elasticity. 64  In our study, as shown in  Figure  6 D,E, we observed significant expression of\ncollagen I in treated scaffolds when compared to collagen III.\nIn conclusion, our study highlights the profound impact of lectin\nand galactose conjugation on galactose-conjugated scaffolds. These\nmodifications have shown remarkable improvements in the expression\nof key ECM components, including galectin, Versican, and laminin.\nThese enhancements in ECM expression signify the potential of these\nmodified scaffolds to mimic trophoblast invasion, promote cell growth,\nand potentially contribute to ECM remodeling.\nSurface-modified nanofibrous scaffolds (1 × 1 cm 2 ) were implanted into adult Wistar rats to evaluate their in vivo\nbiocompatibility. Rats ( n  = 3) were subcutaneously\nimplanted with the materials, as shown in  Figure  7  and assessed 21 days postimplantation. Continuous\nmonitoring for local inflammation was conducted, including macroscopic\nand histological evaluations for signs of inflammation and foreign\nbody responses. Throughout the observation period, the rats exhibited\nnormal behavior, and no indications of local inflammation, implant\nexposure, extrusion, or mortality were observed. Upon macroscopic\nexamination at the time of retrieval, the implants were surrounded\nby healthy, unaffected tissue devoid of inflammation markers such\nas redness, swelling, or any adverse tissue reactions that could compromise\nthe integrity of the implanted area.\n(i) Illustration of steps involved in\nimplanting both modified\nand unmodified scaffolds in the subcutaneous region of Wistar rats.\n(ii) Evaluation of tissue integration for the modified and unmodified\nscaffolds after a 21 day subcutaneous implantation in Wistar rats\n( n  = 3) using H&E staining, with the following\ncategories: (A) control, (B) PCL-R, (C) PCL-A, (D) GPCL-R, and (E)\nGPCL-A. The arrow indicates the muscle layer damage region. (iii)\nMasson’s trichome staining to assess tissue samples for collagen\nand scar formation on (A) control, (B) PCL-R, (C) PCL-A, (D) GPCL-R,\nand (E) GPCL-A. (iv) Quantitative analysis of collagen expression\nintensity on the scaffolds after Masson’s trichome staining.\nH&E staining\nis a commonly used technique to visualize the morphology of cells\nand tissues. In the current study, the region of tissue was evaluated,\nand the degree of damage is classified into three groups: intact,\nmild, and severe, as shown in  Table  4 .  Table  5  illustrates the degree of damage or intactness in the subcutaneous\ntissue at the implanted location. 65  In  Figure  7 iiB,D, the muscle\nlayer of the PCL-R nanofibers and PCL-R galactose-conjugated nanofibers\nimplanted site showed mild damage. Mild damage to tissue may be characterized\nby the presence of inflammatory cells, such as neutrophils or lymphocytes,\nin the affected area. Additionally, there may be changes in the arrangement\nof cells or the ECM, such as disorganization or increased fibrosis.\nHowever, mild damage may not necessarily cause significant changes\nin tissue morphology or function, and the tissue may still be capable\nof repair and regeneration. Whereas in  Figure  7 iiC, PCL-A showed severe damage at the site,\nwhich is due to extensive changes in the tissue architecture and cellular\nmorphology. Severe tissue damage may be characterized by the presence\nof large areas of necrosis, the loss of normal tissue structure, and\nthe infiltration of immune cells, such as neutrophils, macrophages,\nand lymphocytes. In contrast, PCL-A scaffolds conjugated with galactose  Figure  7 iiE exhibited significantly\nless damage, indicating that surface modification mitigates adverse\neffects and enhances scaffold biocompatibility.\nCollagen is a major component of the ECM in connective tissues, including\nsubcutaneous tissue, and is produced by fibroblasts in response to\ninjury or foreign material. 66  The presence\nof high levels of collagen in the subcutaneous tissue surrounding\nthe nanofiber implant indicates that the tissue is undergoing a healing\nresponse and attempting to isolate the foreign material by forming\na fibrous capsule around it. This is a common response to implantable\nmaterials and is known as the foreign body response in the first few\ndays after implantation. Whereas in PCL-A unmodified nanofiber samples,\na notably higher level of collagen content was observed and quantified\nfrom the stained images. The observation of increased collagen content\nin tissue post implantation of polymer nanofibers suggests that these\nnanofibers have incited a fibrotic reaction within the tissue, as\nvisually represented in  Figure  7 iiib,c for the unmodified samples. Additionally, the quantification\nof collagen intensity revealed higher levels in unmodified PCL-A,\nas shown in  Figure  7 iv. While some degree of fibrosis is a normal and expected response\nto biomaterial implants, excessive fibrosis can impede the integration\nof the implant with the surrounding tissue, leading to decreased functionality\nand potential complications. 67  The modified\nscaffolds did not cause an excessive degree of fibrosis; therefore,\nthe amount of collagen is similar to the control.\n\nIn conclusion, this study\nsuccessfully optimized PCL nanofibers\nvia electrospinning, followed by surface modification through a wet\nchemistry consisting of aminolysis and a galactose conjugation process.\nThe incorporation of sugar groups onto the PCL nanofiber surface was\nconfirmed through a comprehensive ELLA assay, contact angle measurements,\nand ninhydrin assays. Surface modification enhanced the elasticity\nof scaffolds, a crucial requirement for myometrium tissue engineering,\nas demonstrated through mechanical characterization. The biocompatibility\nof these PCL scaffolds was rigorously assessed through cell culture\nexperiments using HUF cells and subcutaneous implantation in Wistar\nrats. The results obtained from the MTT assay, live/dead assay, gene\nexpression, and immunofluorescence assays clearly demonstrated the\ncytocompatibility of the modified PCL scaffolds in terms of cell adhesion,\nproliferation, and viability. Moreover, these scaffolds exhibited\na remarkable enhancement in HUF proliferation compared with pristine\nPCL scaffolds, signifying their potential for fostering cell growth\nand tissue regeneration. Additionally, the galactose-conjugated PCL\nscaffolds induced superior cytoskeletal morphology and upregulated\nfibroblast ECM marker expression compared to their unmodified counterparts.\nThese findings strongly support the suitability of galactose-conjugated\nPCL fibers as a versatile platform which activates HUFs to upregulate\nthe regeneration process in myometrium. In vivo subcutaneous implantation\nresults showed that the degree of inflammation and damage in the muscle\nlayer was less in modified scaffolds when compared to unmodified scaffolds.\nFuture investigations could explore the feasibility of using these\nmodified scaffolds with human uterine smooth muscle cells and could\nprovide valuable insights about behavior and the potential for supporting\nthe repair of the uterine myometrium. Additionally, in vivo animal\nstudies in a uterine myometrial injury model will provide better understanding\nof scaffold’s efficiency and integration in complex host tissues.\nInsights from these studies could lay the foundation for clinical\napplications aimed at reducing uterine scarring after C-sections or\nfibroid surgeries, addressing a significant clinical concern and offering\na potential solution for improving patient outcomes in reproductive\nhealth. In this study, while both random and aligned PCL fibers were\nstudied, PCL-A fibers conjugated with galactose were preferred over\nrandom galactose conjugated fibers because they exhibited significantly\nless damage in animal studies when compared to unmodified fibers.\nAlso, due to their better mimicry with the uterine myometrial layer,\nit makes them more advantageous for myometrial tissue engineering\napplications. In summary, the successful surface modification of PCL\nnanofibers with galactose conjugation holds immense promise for the\ndevelopment of advanced scaffolds in uterine tissue engineering. This\ninnovative approach opens new horizons for the integration of bioactive\ncomponents, potentially revolutionizing the field of regenerative\nmedicine for uterine repair and beyond.","source_license":"CC-BY-4.0","license_restricted":false}