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Article

Fibrous Polycaprolactone-Based Composite Materials with the Addition of Hardystonite: Haemostatic Potential, Antioxidant Activity, and Biocompatibility Assessment

by
Anna Kaczmarek
1,*,
Marcin H. Kudzin
1,
Michał Juszczak
2,
Katarzyna Woźniak
2,
Paulina Król
1,
César I. Hernández Vázquez
1,
Zdzisława Mrozińska
1 and
Jerzy J. Chruściel
1,*
1
Łukasiewicz Research Network—Lodz Institute of Technology, 19/27 Marii Sklodowskiej-Curie Str., 90-570 Lodz, Poland
2
Department of Molecular Genetics, Faculty of Biology and Environmental Protection, University of Lodz, 90-236 Lodz, Poland
*
Authors to whom correspondence should be addressed.
Macromol 2026, 6(1), 5; https://doi.org/10.3390/macromol6010005
Submission received: 5 August 2025 / Revised: 1 December 2025 / Accepted: 8 December 2025 / Published: 13 January 2026
Browse Figures
Versions Notes

Abstract

Fibrous polycaprolactone-based composite materials with the addition of hardystonite (1, 3, and 5 wt.%) were developed using the electrospinning method. The obtained PCL and PCL-HT nonwovens were evaluated in terms of their physiochemical properties (SEM, EDS, BET, and zeta potential). Furthermore, the antioxidant potential [measured by thiobarbituric acid reactive substance (TBARS) levels], blood plasma coagulation parameters, and cyto- and genotoxicity towards PBM and Hs68 cells were assessed to determine the biochemical activity of the composites. The conducted experiments confirmed that hardystonite was successfully incorporated into the PCL matrix. No substantial changes in the fibres’ surface morphology and the structure of the composites were observed. Similarly, the specific surface area, total pore volume, and average pore size did not change significantly. The addition of hardystonite to the polymer solution resulted in a shift in zeta potential toward less negative values. With regard to plasma coagulation parameters, no significant changes were observed in the aPTT, PT, or TT, likely due to the counterbalancing effect of Zn2+ and Ca2+ ions. Furthermore, the PCL-HT composites exhibited a lowered TBARS level, suggesting antioxidant properties, which could be attributed to the presence of zinc in hardystonite. The PCL and PCL-HT composites demonstrated no cytotoxic or genotoxic effects on the tested blood or skin cell types, suggesting their safety.

1. Introduction

In recent years, increasing attention has been devoted to tissue engineering, which aims to regenerate tissues and organs damaged by trauma, chronic diseases, or congenital defects [1,2,3]. This field presents a promising alternative to traditional methods, such as organ transplantation and the implantation of biomaterial-based grafts [1,3]. This is because tissue engineering addresses several critical issues, such as the shortage of organ donors, the necessity of using immunosuppressive drugs, and the unsatisfactory biocompatibility of artificial biomaterials [1].
Tissue engineering utilizes three-dimensional scaffolds that support cell growth [1,2,3,4,5]. These cells are harvested either from the patient, in a process known as autologous transplantation, or from a donor, in the case of allogeneic transplantation [1,3,6]. They are then seeded onto three-dimensional substrates and cultured in vitro [1,3,6]. The resulting material–cell composites are implanted into the site of damaged or missing tissue to support its regeneration [1,6]. Through proliferation and self-organization, the cells develop functional tissues with structural and functional properties similar to those of healthy tissues [1]. In the initial (in vitro) phase, scaffolds serve as artificial extracellular matrices that provide a substrate for the cells and stimulate their proliferation and differentiation [1,3]. Once new tissue has formed in vivo, the scaffolds should gradually degrade and resorb, thus avoiding the need for implant removal or reoperation [1,3,6].
Due to their intended function, scaffolds must meet several specific requirements [1,2,3,4,7]. First and foremost, scaffold materials must be biocompatible to ensure proper cell adhesion and proliferation [1,3,7]. Furthermore, they should be biodegradable [1,2,4], meaning that they must break down gradually into non-toxic products through metabolic processes, which are then excreted from the body. It is important that the degradation rate of the scaffold can be controlled and is matched to the regeneration rate of the replaced tissues [1,3,4,7]. Another important feature is suitable mechanical properties [1,2,4,7], including strength, to maintain the spatial arrangement of proliferating and differentiating cells and to support the newly formed tissue, both in vitro and in vivo. The mechanical properties should also be compatible with those of the surrounding tissues [3,4]. Moreover, the structure and spatial architecture of the scaffolds are crucial [1,4]. They must exhibit appropriate porosity and pore size to allow free cell migration and ensure proper transport of nutrients and metabolic waste [1,3,4,8].
Among the most promising materials for tissue engineering applications are nonwoven fabrics [2,3,5,9]. These are composed of thermally, mechanically, or chemically bonded fibres, typically forming a flat spatial structure [3]. Nonwovens are lightweight, have a high specific surface area, and exhibit good porosity and air permeability [9,10,11,12]. They are also versatile and easy to process, allowing customization for specific functions, making them well-suited for tissue regeneration [3]. Due to their three-dimensional fibrous structure, nonwovens effectively mimic the architecture of the extracellular matrix and natural tissues, thus providing an appropriate matrix for regeneration [3,5,8,9,10,13]. Their high porosity facilitates free cell migration into their structure and the flow of nutrients and metabolic products, enabling the growth of new tissue [3]. The literature reports numerous applications of nonwovens as scaffolds for the regeneration of bone, cartilage, epithelial tissue, nerves, and skin [3,8,14,15,16,17,18,19,20,21,22]. Additionally, nonwovens can be used as vascular prostheses and modern wound dressings for hard-to-heal wounds such as burns [13,23].
Tissue engineering primarily employs biodegradable polymers, both natural and synthetic [1,4,10,24]. Among natural polymers, the most commonly used are saccharide and protein compounds, such as chitosan, hyaluronic acid, and collagen [1,24]. Besides supporting cells, these also serve as a source of nutrients [1]. Unfortunately, natural polymers tend to have insufficient mechanical properties and are not resistant to high temperatures or pH changes [1]. This significantly limits the choice of fabrication methods and their subsequent application [1]. In contrast, synthetic polymers possess superior mechanical properties and better resistance to environmental conditions [1], making them widely used as cell scaffolds. The most commonly used synthetic polymers in tissue engineering include polylactic acid (PLA), polyglycolic acid (PGA), and polycaprolactone (PCL) [1,2,5,10]. PCL, in particular, has garnered attention for bone and cartilage tissue regeneration [2,4,6,7,8,25,26,27,28,29,30,31,32,33,34,35], as its mechanical properties closely resemble those of natural cartilage [3,30]. PCL is a non-polar aliphatic polyester characterized by good thermal stability, plasticity, and favourable mechanical properties, including strength [1,2,4,7,25,32,35]. It is also relatively inexpensive and easy to process [25,33], and it can be modified to tailor its properties to specific applications [7]. In the body, PCL undergoes biodegradation through hydrolysis to 6-hydroxycaproic acid, which is then metabolized in the citric acid cycle and excreted [1]. Compared to other biodegradable polymers, PCL has a longer degradation time (over 2 years), which contributes to prolonged mechanical strength [1,4,35]. This ensures sufficient time for tissue regeneration [4]. Furthermore, PCL is widely recognized as biocompatible and has been approved by the U.S. Food and Drug Administration (FDA) [2,6,27,30,35]. It does not trigger adverse immune responses or inflammation. PCL can be used for both load-bearing and non-load-bearing implants. However, its application in tissue engineering is limited by its hydrophobic nature and lack of bioactivity [2,4,6,30,32,36]. Therefore, increasing attention is being paid to composite materials made of polymers and organic/inorganic particles, which exhibit improved mechanical and biological properties [10,24,25,30,32,36]. With the right additives, it is possible to obtain hydrophilic materials with enhanced mechanical strength and bioactivity, as well as osteoconductivity [4,9,10,24,25,32,36]. One of the most promising polymer modifications is doping with bioceramics, including hydroxyapatite (HAp), tricalcium phosphate (TCP), bioactive glass, and silica (SiO2) [2,4,6,8,9,24,25,26,27,28,29,30,31,32,33,34,36,37,38,39,40].
Among these, calcium silicates—especially zinc-doped variants such as Ca2ZnSi2O7 (hardystonite, HT)—have attracted increasing interest [41,42,43,44,45,46,47,48,49,50,51,52,53,54,55]. Zinc is a vital trace element involved in bone homeostasis, promoting mineralization and regulating resorption processes [43,44,49,55,56,57]. It enhances alkaline phosphatase activity, collagen synthesis, and expression of osteogenic markers [58,59]. Consequently, Ca2ZnSi2O7 exhibits favourable bioactivity for bone tissue engineering, stimulating the proliferation and osteogenic differentiation of mesenchymal stem cells, and increasing the expression of markers such as alkaline phosphatase, osteocalcin, and type I collagen [43,45,47,52]. Additionally, it demonstrates superior chemical stability and a slower degradation rate than other calcium silicates [46], along with enhanced flexural strength and fracture resistance compared to hydroxyapatite [43,45,47]. The release of Zn2+ from HT also contributes antibacterial and anti-inflammatory effects [46,48,49,50].
Beyond bone regeneration, HT has been shown to aid wound healing. Zhang et al. [60] reported the fabrication of a Janus-type bilayer membrane incorporating hydrophilic HT particles within hydrophobic PLA fibres, designed for treating burn injuries in rats. The bilayer structure facilitated unidirectional fluid movement toward the ceramic layer, preventing direct ceramic–tissue contact and enabling exudate absorption. This configuration significantly accelerated wound healing, enhanced epithelial regeneration, and promoted dermal papilla cell activity. It also increased the expression of stem cell markers such as KGF, HGF, VEGF, and BMP-6, with therapeutic effects attributed to the synergistic ionic release of Zn2+ and SiO32−. In another study, HT was incorporated into electrospun fibres composed of PCL and tetrafluoroethylene (TPE) [61]. HT addition improved cell proliferation, migration, and differentiation, and influenced fibre morphology through changes in conductivity and viscosity. Another study developed an injectable hydrogel based on sodium alginate and hardystonite to evaluate its effect on HUVEC and HDF cell lines [62]. HT content up to 2 wt.% improved compressive strength and gelation rate. HT-containing hydrogels significantly promoted cell growth, viability, and migration, and exhibited potent antibacterial activity in vitro, with complete inhibition of bacterial growth in both extract and direct-contact assays.
According to a review of the literature, composites containing hardystonite have been primarily studied in the context of bone regeneration. Although the potential of HT-containing materials has also been explored with respect to wound healing, there is a lack of comprehensive studies addressing their haemostatic potential, including blood coagulation parameters, as well as antioxidant activity and biocompatibility with blood and skin cells. Therefore, in the present study, electrospinning was employed to create PCL nonwoven scaffolds incorporating hardystonite. The electrospinning parameters were selected based on the authors’ previous experience. The obtained composites were evaluated in terms of their physicochemical traits, antioxidant capacity, and effects on blood coagulation. Biocompatibility was assessed by measuring cell viability and DNA damage in peripheral blood mononuclear (PBM) cells and Hs68 skin fibroblasts, serving as models of healthy human cells. This approach enabled a comprehensive biological evaluation of the composites in relation to their potential use in wound treatment and blood-contacting applications.

2. Materials and Methods

2.1. Materials

Polycaprolactone (M.W. 80,000–85,000 g/mol; CAS: 24980-41-4) was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Hardystonite (Ca2ZnSi2O7) in powder form (particle size: 0–80 μm) was obtained from Matexcel (Shirley, NY, USA). Solvents, i.e., dichloromethane (DCM) and N, N-dimethylformamide (DMF) were purchased from Chempur (Piekary Śląskie, Poland) and POCH S.A. (Gliwice, Poland), respectively. Human blood plasma lyophilizates (Dia-CONT I), along with the reagents Dia-PTT, Dia-PT, Dia-TT, and a 0.025 M calcium chloride solution (Dia-CaCl2), were purchased from Diagon Kft. (Budapest, Hungary). The human foreskin fibroblast cell line Hs68 (ATCC® CRL-1635™) was obtained from the American Type Culture Collection (ATCC™, Manassas, VA, USA).

2.2. Methods

2.2.1. Sample Fabrication

Polymer solutions with a constant PCL concentration of 10% w/w were prepared using a solvent mixture of DCM and DMF in a 1:1 mass ratio. Solutions enriched with hardystonite [Ca2ZnSi2O7] nanoparticles (HT) were prepared at three distinct concentrations: 1%, 3%, and 5% w/w, relative to the polymer mass (Figure 1).
For PCL solutions, the polymer was gradually added to the pre-mixed DCM and DMF solvent blend under slow stirring. After complete addition of the polymer, the solution was vigorously stirred using a mechanical stirrer for approximately 4 h until homogeneity was achieved.
For HT-modified solutions, an appropriate amount of hardystonite nanoparticles was first dispersed in the DCM and DMF solvent blend using ultrasonic homogenization for approximately 20 min. Following this step, PCL was gradually added to the dispersion under continuous slow stirring. The mixture was then subjected to vigorous stirring for approximately 4 h using a mechanical stirrer to ensure uniform dispersion and polymer dissolution.
All prepared polymer solutions were allowed to degas by standing undisturbed at room temperature for approximately 12 h prior to electrospinning.
The electrospinning method was employed to fabricate ultrafine polymer fibres with nano- and microscale diameters. The setup used is shown in Figure 2. The fibres were deposited on a rotating metallic drum collector to produce randomly oriented fibrous mats. The electrospinning parameters were as follows: ambient temperature: 25.0–25.5 °C; needle-to-collector distance: 15 cm; needle gauge: 23 G (0.33 mm inner diameter); solution flow rate: 18.5 mL/h; voltage: 14–15 kV.
The electrospinning technique and parameters were selected based on the authors’ previous experience.
The resulting electrospun fibre mats were collected for subsequent morphological and physicochemical analyses.

2.2.2. Surface Morphology Using Scanning Electron Microscopy and Energy-Dispersive X-Ray Spectroscopy

The surface morphology of the samples was investigated using scanning electron microscopy (SEM). SEM analysis was conducted using a Phenom ProX G6 system (Thermo Fisher Scientific, Waltham, MA, USA) under low vacuum conditions (60 Pa) and with an accelerating voltage of 15 keV. Imaging was conducted with a back-scattered electron detector. In addition, qualitative elemental composition was determined via energy-dispersive X-ray spectroscopy (EDS) using a microanalyzer from Oxford Instruments (Abingdon, UK).

2.2.3. Specific Surface Area, Total Pore Volume, and Average Pore Size

The Brunauer–Emmett–Teller (BET) method was used to determine the specific surface area, total pore volume, and average pore size of the samples. Measurements were conducted with an Autosorb-1 analyser (Quantachrome Instruments, Boynton Beach, FL, USA), using nitrogen gas at 77 K as the adsorbate. Before analysis, the samples were dried at 105 °C for 24 h and subsequently degassed at room temperature. Approximately 0.4–0.9 g of material was used for each measurement. For each sample, both 5-point and 39-point BET isotherms were recorded. The presented mean value of the specific surface area is the average of the values obtained from the 5-point and 39-point isotherms, while the total pore volume and average pore size were calculated based on the 39-point isotherms.

2.2.4. Zeta Potential

Zeta potential measurements were performed using the SurPASS 3 streaming potential analyser (Anton Paar, Graz, Austria). The experiments were conducted at room temperature, with phosphate-buffered saline (PBS, pH ~7.4) serving as the electrolyte. The applied pressure ranged from 200 to 600 mbar, with the zeta potential calculated based on data collected up to 400 mbar. A cylindrical measuring cell was used. Measurements were performed on two independent samples of each type, with five repetitions per sample.

2.2.5. Blood Plasma Coagulation: Activated Partial Thromboplastin Time, Prothrombin Time, and Thrombin Time

The influence of the developed composites on blood plasma coagulation was assessed by measuring activated partial thromboplastin time (aPTT) and prothrombin time (PT). These parameters were evaluated using a Coag4D coagulometer (Diagon Kft., Budapest, Hungary). For each test, samples (~100 mg) were incubated at 37 °C for 1 h in 250 µL of plasma prepared from commercially available lyophilized human plasma (Dia-CONT I, Diagon Kft., Budapest, Hungary). The aPTT assays were performed using Dia-PTT reagent and a 0.025 M calcium chloride solution (Dia-CaCl2), both supplied by Diagon Kft., following the manufacturer’s instructions. PT testing was conducted using the Dia-PT reagent (Diagon Kft., Budapest, Hungary), while TT was assessed using Dia-TT reagent (Diagon Kft., Budapest, Hungary). All tests were conducted according to the manufacturer’s instructions. The aPTT measurements were performed in triplicate (using three independent samples), while the PT and TT tests were performed in duplicate (using two independent samples).

2.2.6. Thiobarbituric Acid Reactive Substances Assay

Lipid peroxidation in blood plasma was evaluated using the thiobarbituric acid reactive substances (TBARS) assay in the presence of 0.1 M hydrogen peroxide. This assay was employed to determine whether the developed composites promote oxidative stress via the Fenton reaction or exhibit antioxidant properties. Lipid peroxidation, which involves the oxidation of unsaturated fatty acids, results in the formation of byproducts such as peroxides and malondialdehyde (MDA). MDA reacts with thiobarbituric acid (TBA) to form a coloured complex, which is detected spectrophotometrically at 535 nm. In the experiment, 0.5 mL of plasma was incubated with 2.5 mg of the test material and 0.1 M H2O2 for 30 min. The TBARS reaction was then conducted using trichloroacetic acid (TCA) and TBA. Following heating and centrifugation, absorbance was recorded at 535 nm. TBARS concentrations were calculated in micromolar (µM) units using a molar extinction coefficient of ε = 156,000 M−1cm−1, with samples diluted threefold prior to measurement. The measurements were performed in duplicate (using two independent samples).

2.2.7. Preparation of Samples for the Assessment of Biological Properties

To analyse the influence of PCL and PCL-HT nonwoven composites incorporating hardystonite on cells, 1 cm2 samples of modified and unmodified PCL nonwovens were incubated in 3 mL of RPMI or DMEM medium, depending on the cell line, at 37 °C in 5% CO2 for 24 h. Subsequently, post-incubation mixtures were filtered using a 0.2 µm filter to maintain aseptic conditions. Then, post-incubation mixtures were added to cells in a ratio of 1:1 to assess the effect of the extracts on cell viability and DNA damage.

2.2.8. Cell Culture

Peripheral blood mononuclear cells (PBM cells) were isolated from a leucocyte buffy coat collected from the blood of healthy non-smoking donors from the Blood Bank in Lodz, Poland, as described previously [63]. The first step in PBM cell isolation involved mixing the buffy coat with PBS at a ratio of 1:1. In the next step, the mixture was centrifuged using a Lymphosep (Cytogen, Zgierz, Poland) density gradient at 2200 RPM for 20 min, with minimal acceleration and deceleration settings. PBM cells were subsequently washed three times via centrifugation in 1% PBS. After isolation, the cells were suspended in RPMI 1640 medium. The study was approved by the University of Lodz Research Ethics Committee (approval number: 12/KEBN-UŁ/I/2024-2025).
The human foreskin fibroblast cell line Hs68 (ATCC® CRL-1635™) was obtained from the American Type Culture Collection (ATCC™, Manassas, VA, USA). Hs68 cells were cultured in high-glucose DMEM supplemented with 100 units/mL of potassium penicillin, 100 μg/mL of streptomycin sulphate, and 10% (v/v) fetal bovine serum (FBS). Cultures were maintained at 37 °C in a humidified atmosphere with 5% CO2.

2.2.9. Cell Viability Resazurin Assay

The cell viability resazurin assay was performed according to the method described by O’Brien et al. [64]. Resazurin salt was dissolved in sterile PBS. Post-incubation mixtures were added to PBM cells at a density of 5 × 104 cells and Hs68 cells at 1 × 104 cells, followed by incubation for 24 and 48 h at 37 °C in 5% CO2 atmosphere. The control consisted of RPMI1640 (for PBM cells) and DMEM (for Hs68 cells), both prepared in the same manner as the post-incubation mixtures.
Subsequently, 10 µL of resazurin solution was added to each well, and the plates were incubated again for 2 h at 37 °C in 5% CO2. Fluorescence was then measured using a HT microplate reader (BioTek Synergy HT, Agilent Technologies Inc., Santa Clara, CA, USA) with excitation/emission wavelengths of λex  =  530/25 and an λem  =  590/35 nm. The effects of the post-incubation mixtures were expressed as a percentage relative to the control fluorescence. The experiment was performed in six replicates.

2.2.10. DNA Damage

PCL and PCL-HT post-incubation mixtures were added to PBM cells at a density of 1 × 105 and to Hs68 cells at 5 × 104, followed by incubation for 24 and 48 h at 37 °C in 5% CO2 atmosphere. The negative control consisted of RPMI1640 (for PBM cells) and DMEM (for Hs68 cells), both prepared in the same manner as the post-incubation mixtures. The experiment also included a positive control: a cell sample incubated with 25 μM hydrogen peroxide (H2O2) for 15 min on ice.
The comet assay was performed under alkaline conditions according to the procedure of Tokarz et al. [65]. A freshly prepared cell suspension in 0.75% low-melting-point (LMP) agarose dissolved in PBS was layered onto microscope slides, pre-coated with 0.5% normal-melting-point (NMP) agarose. The cells were lysed for 1 h at 4 °C in a buffer containing 2.5 M NaCl, 0.1 M EDTA, 10 mM Tris, 1% Triton X-100, at pH = 10. After lysis, the slides were placed in an electrophoresis unit, and DNA was allowed to unwind for 20 min in an alkaline solution (300 mM NaOH and 1 mM EDTA, pH > 13). Electrophoresis was carried out in a buffer containing 30 mM NaOH and 1 mM EDTA, pH > 13, at 4 °C (with the running buffer temperature not exceeding 12 °C) for 20 min at an electric field strength of 0.73 V/cm (28 mA). Subsequently, the slides were washed with distilled water, drained, stained with 2 µg/mL DAPI, and covered with coverslips. All steps were performed under limited light conditions to minimize additional DNA damage.
The comets were observed at 200× magnification using an Eclipse fluorescence microscope (Nikon, Tokyo, Japan) equipped with a COHU 4910 video camera (Cohu Inc., San Diego, CA, USA), a UV-1 A filter block, and the personal computer-based image analysis system Lucia-Comet v. 7.0 (Laboratory Imaging, Prague, Czech Republic). One hundred comets were randomly selected per sample, and the mean percentage of DNA in the comet tail was used as an indicator of DNA damage.

3. Results and Discussion

3.1. Surface Morphology Using Scanning Electron Microscopy and Energy-Dispersive X-Ray Spectroscopy

The surface morphology of the developed PCL and PCL-HT nonwovens was investigated using scanning electron microscopy. The SEM analysis revealed no significant alterations in fibre morphology or overall nonwoven structure after hardystonite incorporation. The results are presented in Figure 3. All samples exhibited a three-dimensional porous structure with interconnected pores. The fibres were randomly oriented, with most having diameters less than 1 µm. In PCL nonwovens modified with hardystonite, randomly distributed HT particles and agglomerates of various sizes were visible between the fibres, confirming the successful incorporation of hardystonite into the PCL matrix. However, the fibres themselves did not show any noticeable morphological changes resulting from the addition of hardystonite to the electrospinning solution. In both PCL and PCL-HT nonwovens, the fibres exhibited a smooth and uniform surface. Similar findings were reported by Jaiswal et al. [43], who observed no changes in fibre surface morphology upon embedding HT particles in the polymer matrix.
The qualitative composition of the fibrous composites was assessed by means of energy-dispersive X-ray spectroscopy. The recorded spectra of unmodified PCL and PCL-HT nonwovens are shown in Figure 4. In the case of the PCL sample, only peaks corresponding to carbon and oxygen were present in the EDS spectrum. This is consistent with the fact that polycaprolactone is composed of carbon, oxygen, and hydrogen. In the case of the PCL-HT composites, additional peaks attributed to calcium, zinc, and silicone were observed, confirming the presence of hardystonite within the polymer composites.

3.2. Specific Surface, Total Pore Volume, and Average Pore Size

The specific surface area, total pore volume, and average pore size were investigated by means of BET analysis. The resulting values obtained for unmodified PCL nonwoven and PCL-HT composites with varying amounts of hardystonite are summarized in Table 1. The acquired N2 adsorption–desorption isotherms are displayed in Figure 5. BET analysis demonstrated no substantial differences in specific surface area, total pore volume, or average pore diameter between the modified and unmodified samples.
The data presented in Table 1 show that there was no direct correlation between the specific surface area, total pore volume, average pore size, and the amount of hardystonite added to the polymer solution. All the measured parameters were comparable. The high standard deviation obtained for the PCL-HT-1 sample in the measured specific surface area, as well as higher total pore volume and average pore size, may be related to the fact that the samples had to be removed from the substrate, i.e., aluminium foil, prior to analysis. In the case of the PCL-HT-1 sample, disruption of the fibrous structure of the composite was observed during mechanical removal, which could have affected the results. In summary, it may be concluded that the addition of hardystonite to the polymer solution used for electrospinning in the range of 1–5 wt.% had no significant impact on the specific surface area, total pore volume, or average pore size. This is in agreement with the results of the SEM analysis, which did not reveal any substantial changes in the surface morphology of PCL fibres and the structure of the nonwoven samples due to the addition of HT. According to the obtained results, the observed HT particles within the fibrous structure did not affect the porosity of the composites.
As illustrated in Figure 5, the recorded isotherm curves resemble an S-shape, which aligns with type II isotherms, according to the IUPAC classification. This type of isotherm is characteristic of monolayer adsorption followed by multilayer adsorption processes [66,67]. For all investigated samples, the transition point, known as Point B, where the curve shifts to a nearly linear middle section, was not clearly defined. This indicates the overlap between monolayer adsorption and the onset of multilayer formation [66,67]. The overall profile of the isotherms implies a predominance of mesoporous and macroporous structures, with micropores being less significant in the material [68]. The steep rise in nitrogen adsorption with increasing relative pressure supports this conclusion, suggesting that nitrogen first enters the micropores at low pressures, followed by monolayer adsorption, and then multilayer buildup as pressure increases [68].
All samples displayed a hysteresis loop in their isotherms. According to the literature, this is indicative of mesoporous structures and the occurrence of capillary condensation within mesopores [66,67]. The shape of the loops corresponds to type H3, typically linked to slit-shaped pores [68]. This type of hysteresis often arises in systems containing aggregates of plate-like particles or when larger pores, especially macropores, are not fully filled during adsorption [67]. The size of the hysteresis loops was similar for all samples.

3.3. Zeta Potential

Evaluating the zeta potential at physiological pH is essential, as it significantly affects interactions with proteins and blood components, thereby impacting both plasma coagulation and the material’s overall biological activity. The surface charge and potential play a critical role in regulating the quantity, type, and conformational changes in proteins that adsorb onto the material’s surface, which subsequently affect cell attachment, integrin interactions, and the development of focal adhesions [69,70].
Therefore, in this study, the zeta potential of the developed PCL and PCL-HT samples was examined at physiological pH (~7.4), and the results are presented in Table 2. For the unmodified PCL nonwoven, a negative zeta potential of −34.05 ± 0.95 mV was recorded. The addition of HT to the polymer solution resulted in a shift toward less negative values, i.e., −30.08 ± 0.27, −27.54 ± 0.48, and −23.50 ± 1.89 mV for PCL-HT-1, PCL-HT-3, and PCL-HT-5, respectively.
The negative zeta potential of PCL may be explained by the deprotonation of the carboxylic acid groups present on the surface [71]. The shift toward less negative zeta potential values of the modified PCL nonwovens with increasing concentration of hardystonite in the polymer solution may result from the presence of positively charged calcium [72] and zinc ions in hardystonite. This observation is also consistent with the less negative zeta potential for HT particles in the literature [43].

3.4. Blood Plasma Coagulation: Activated Partial Thromboplastin Time, Prothrombin Time, and Thrombin Time

Activated partial thromboplastin time (aPTT), prothrombin time (PT), and thrombin time (TT) are commonly used screening tests that provide rapid but non-specific insights into haemostatic abnormalities [73]. aPTT assesses the intrinsic coagulation pathway, serving as a key indicator of coagulation efficiency [73]. PT evaluates the extrinsic pathway by measuring the time to clot formation following the addition of tissue factor and calcium, which triggers the conversion of prothrombin into thrombin [73]. TT reflects the final stage of coagulation, measuring how quickly a fibrin clot forms after the addition of exogenous thrombin [73]. Therefore, to assess the potential haemostatic effects of the developed PCL composites incorporating hardystonite, aPTT, PT, and TT were measured, with results shown in Figure 6, Figure 7 and Figure 8, respectively.
For aPTT, a slight shortening was observed for unmodified PCL, suggesting enhanced intrinsic pathway activation, which may be related to its relatively high negative zeta potential. This aligns with the existing literature, where negatively charged surfaces are known to initiate the intrinsic coagulation pathway [74]. Factor XII interacts with negatively charged surfaces via positively charged amino acid residues located in its heavy chain [75,76]. This interaction induces conformational changes in factor XII, promoting its conversion into the active form (XIIa) through autocavitation [75,76]. The activation of factor XIIa is a critical step in the coagulation cascade, leading to fibrin formation [75,76]. Consequently, negatively charged surfaces are considered important triggers for the initiation of the intrinsic coagulation pathway [75,76]. Nevertheless, it should be noted that the observed change in aPTT was minor, and the measured value remained within the reference range.
The addition of hardystonite to the polymer solution resulted in the normalization of the aPTT time, restoring it to the level of the control. At the same time, there was no significant difference in aPTT among the PCL-HT composites with varying HT concentrations between 1 and 5 wt.%. This normalization of coagulation time may be associated with the less negative zeta potential observed for the PCL-HT composites. Surfaces with a negative charge generally lead to reduced protein adsorption compared to positively charged ones, as most proteins carry a net negative charge at physiological pH, resulting in repulsive interaction [77]. In contrast, positively charged materials tend to enhance protein adsorption significantly and can also induce changes in protein conformation [77]. Consequently, materials with better blood compatibility usually have surfaces with low interfacial energy [77]. As previously mentioned, although negative surface charges can reduce protein adsorption, a high charge density may enhance activation of the contact system. Therefore, it is crucial to precisely control the surface charge density of biomaterials [77]. Based on these findings, it can be assumed that materials with a moderately negative surface charge exhibit optimal hemocompatibility, which may explain the lack of disruption in the coagulation cascade observed for the PCL-HT composites. Furthermore, this effect may also be explained by the presence of both Ca2+ and Zn2+ ions, which can have opposite effects. Calcium ions are widely recognized as essential cofactors in the blood coagulation process [78,79,80]. Therefore, their presence in hardystonite would theoretically enhance clot formation by activating the intrinsic coagulation pathway, leading to a shortened aPTT. In contrast, certain transition metals, including copper, cobalt, nickel, and zinc, have been shown to interact with contact factors XI, XII, and HK in human plasma, thereby interfering with the intrinsic pathway and leading to a prolonged activated partial thromboplastin time [81]. Hence, it may be assumed that in the case of the PCL-HT composites, the above-described phenomena counterbalanced each other, and as a result, no notable change in aPTT was observed.
Similarly, there was no evident effect of either unmodified PCL or hardystonite addition on PT and TT, as all measured times remained within the normal reference ranges. Therefore, it can be concluded that neither unmodified PCL nor PCL-HT composites affect PT or TT.
The absence of any disruption to the blood coagulation cascade is critically important for materials intended for blood-contacting applications. This is because suppression of the coagulation process may lead to prolonged clotting times, which, in the case of wound dressings, can result in excessive bleeding. In contrast, excessive activation of the coagulation cascade may elevate fibrinogen and platelet levels, increasing the risk of thrombus formation [82,83]. High concentrations of D-dimers, which are markers of fibrin degradation, may indicate ongoing thrombotic or inflammatory processes and are associated with severe complications, including disseminated intravascular coagulation (DIC) and deep vein thrombosis (DVT) [82,83].
In summary, the presented results indicate that the fibrous composites based on PCL and hardystonite do not affect blood plasma coagulation, which supports their potential safety for use in blood-contacting applications.

3.5. Thiobarbituric Acid Reactive Substances Assay

To assess the potential oxidative stress triggered by the manufactured PCL and PCL-HT composites, lipid peroxidation in blood plasma was measured using the TBARS assay. The collected data are summarized in Table 3.
It can be observed the TBARS levels measured for the control sample (i.e., blood plasma) and the unmodified PCL nonwoven are comparable. This confirms the biocompatibility and bioinertness of PCL, suggesting no induction of oxidative stress. At the same time, the TBARS levels obtained for the PCL-HT samples were lower in comparison to those of both the control and the unmodified PCL. However, no direct correlation between the amount of hardystonite added to the polymer solution and the TBARS level was observed. Nevertheless, the presented findings indicate that the PCL-HT composites may exhibit antioxidative properties.
In general, according to the literature, materials with a positive surface charge may promote ROS formation and oxidative stress [84]. Therefore, it can be assumed that surfaces with a negative surface charge, such as the investigated nonwovens, are less likely to induce oxidative stress. This assumption aligns with the obtained results.
The observed antioxidative effect of PCL-HT composites may be attributed to the presence of zinc in hardystonite, which is well known for its antioxidant potential [85]. According to the literature, zinc serves as a cofactor for key enzymes essential for the proper functioning of the cellular antioxidant defence mechanisms [85]. Furthermore, it contributes to cellular protection against oxidative damage by stabilizing cell membranes and inhibiting the activity of nicotinamide adenine dinucleotide phosphate oxidase [85]. Zinc also promotes the expression of metallothioneins, i.e., proteins that effectively neutralize hydroxyl radicals and bind reactive oxygen species (ROS) [85].
Studies have shown that the presence of antioxidant agents that regulate reactive oxygen species at safe levels in wound tissues can enhance the healing process [86,87]. As a result, the use of antioxidant agents in wound treatment is gaining attention, with numerous biomaterials being developed and tested for this purpose [86]. Therefore, the revealed antioxidant activity of the PCL-HT composites highlights their potential suitability for wound management, including use in wound dressing applications.

3.6. Effect on the Viability of PBM and Hs68 Cells

The resazurin reduction assay was employed to determine cell viability following incubation with post-incubation mixtures derived from unmodified PCL and PCL-HT composites. This assay relies on the use of an indicator dye to detect oxidation–reduction reactions, which predominantly occur in the mitochondria of metabolically active cells. The non-fluorescent, dark blue dye (resazurin) is reduced by viable cells to form resorufin, a fluorescent compound that exhibits pink fluorescence at 570 nm and red fluorescence at neutral pH. Our results demonstrate that incubation of PBM cells with PCL and PCL-HT post-incubation mixtures led to an increase in cell viability after 24 and 48 h of incubation (Figure 9). This suggests a stimulatory effect on PBM cell metabolism. In the case of Hs68 cells (Figure 10), we observed an increase in cell viability after 24 h and no evidence of cytotoxicity after 48 h.
Hardystonite has previously shown promise in bone regeneration, as evidenced by its stimulatory effects on MG-63 and MC3T3-E1 cell lines [88,89]. Furthermore, studies on silicate platelets supporting Ag and ZnO demonstrated low cytotoxicity toward mammalian cells [90]. The observed lack of cytotoxicity in PCL-HT composites may be attributed to their negative zeta potential. According to Shao et al. [91], polymeric nanoparticles with a negative surface charge exhibited lower cytotoxicity compared to those that were positively charged, and a less negative charge was associated with increased cell viability. The authors suggested that stronger interactions between positively charged surfaces and cell membranes contribute to higher cytotoxicity. In addition, the absence of cytotoxic effects may be linked to the antioxidant properties of PCL-HT composites. Materials with a negative surface charge have been reported to produce fewer ROS than neutral and positively charged materials [84], which could further contribute to their biocompatibility.
In summary, the obtained results indicate that both PCL and PCL-HT composites are non-cytotoxic to PBM and Hs68 cells, confirming their biocompatibility and supporting their potential for use in blood-contacting applications, such as wound dressings and wound healing therapies.

3.7. Effects on DNA Damage in PBM Cells and Hs68 Cells

The comet assay performed under alkaline conditions is a sensitive and straightforward method for detecting DNA damage, including single- and double-strand breaks, as well as alkali-labile sites in living cells [92]. In our study, extensive DNA damage was observed in PBM and Hs68 cells incubated with 25 µM H2O2, which served as the positive control. In contrast, no increase in DNA damage was detected following 24 or 48 h of incubation with post-mixtures derived from either unmodified PCL or PCL-HT composites (Figure 11 and Figure 12). Additionally, representative comet images are shown in Figure 13 and Figure 14, confirming the absence of DNA damage in samples treated with post-incubation mixtures, while severe damage is clearly visible in the positive control.Previous research on mE-ASCs cells, which possess osteogenic differentiation potential, demonstrated that hardystonite does not exert cytotoxic or genotoxic effects [43]. Similarly, a review on calcium silicate-based dental sealers reported their genotoxicity to be lower than that of certain conventional sealers [93]. Our findings confirm that the tested PCL-HT composites do not induce DNA damage, supporting their safety in wound healing and blood-contacting biomedical devices. The absence of genotoxic effects may be associated with the negative zeta potential of the composites. As reported by Shah et al. [94], nanoparticle genotoxicity can be influenced by surface charge, with positively charged nanoparticles inducing greater genotoxicity, which is related to the formation of micronuclei.

4. Conclusions

Fibrous composites based on polycaprolactone (PCL) containing 1–5 wt.% hardystonite were successfully fabricated by electrospinning and comprehensively characterized. The incorporation of hardystonite did not significantly affect fibre morphology, porosity, or surface structure while inducing a shift in the zeta potential toward less negative values, which was attributed to the presence of Ca2+ and Zn2+ cations. Coagulation assays (aPTT, PT, and TT) showed no significant deviations from control values, most likely due to the counterbalancing effects of calcium and zinc ions on the intrinsic coagulation pathway. The PCL–hardystonite composites exhibited reduced TBARS levels, indicating moderate antioxidant activity, which could be associated with the zinc content of hardystonite. Extracts from both PCL and PCL–hardystonite materials showed no cytotoxic or genotoxic effects in PBM and Hs68 cell models, confirming good biocompatibility. Overall, the results indicate that PCL–hardystonite fibrous composites are biologically safe and demonstrate promising potential for blood-contacting and wound-healing applications, without disturbing the haemostatic balance. Further studies should focus on ion release kinetics and the evaluation of antimicrobial and mechanical properties to optimize the material for clinical use.

Author Contributions

A.K. developed the concept and designed the experiments, performed the experiments, analysed the data, and wrote the paper; M.H.K. developed the concept and analysed the data; M.J. performed the experiments and analysed the data; K.W. analysed the data and wrote the paper; P.K. performed the experiments; C.I.H.V. performed the experiments; Z.M. performed the experiments; J.J.C. analysed the data. All authors have read and agreed to the published version of the manuscript.

Funding

This research was partly conducted within the National Science Centre (Poland), project Miniatura-8, No. 2024/08/X/ST5/00404.

Institutional Review Board Statement

This study was conducted in accordance with the Declaration of Helsinki and approved by the University of Lodz Research Ethics Committee (12/KEBN-UŁ/I/2024-2025), approved on 17 December 2024.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data are included within the text.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The sequence of steps in composite manufacturing.
Figure 1. The sequence of steps in composite manufacturing.
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Figure 2. TL-Pro-BM_TONG LI TECH—PRC device.
Figure 2. TL-Pro-BM_TONG LI TECH—PRC device.
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Figure 3. SEM images of unmodified and modified PCL nonwovens: (a) PCL; (b) PCL-HT-1; (c) PCL-HT-3; (d) PCL-HT-5 (magnification 30,000×; scale bar = 5 µm).
Figure 3. SEM images of unmodified and modified PCL nonwovens: (a) PCL; (b) PCL-HT-1; (c) PCL-HT-3; (d) PCL-HT-5 (magnification 30,000×; scale bar = 5 µm).
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Figure 4. EDS spectra for unmodified and modified PCL nonwovens: (a) PCL; (b) PCL-HT-1; (c) PCL-HT-3; (d) PCL-HT-5 (spot analysis).
Figure 4. EDS spectra for unmodified and modified PCL nonwovens: (a) PCL; (b) PCL-HT-1; (c) PCL-HT-3; (d) PCL-HT-5 (spot analysis).
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Figure 5. Adsorption and desorption isotherms of unmodified and modified PCL nonwovens: (a) PCL; (b) PCL-HT-1; (c) PCL-HT-3; (d) PCL-HT-5.
Figure 5. Adsorption and desorption isotherms of unmodified and modified PCL nonwovens: (a) PCL; (b) PCL-HT-1; (c) PCL-HT-3; (d) PCL-HT-5.
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Figure 6. Activated partial thromboplastin time measured for the unmodified and modified PCL nonwovens after 1 h of incubation at 37 °C.
Figure 6. Activated partial thromboplastin time measured for the unmodified and modified PCL nonwovens after 1 h of incubation at 37 °C.
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Figure 7. Prothrombin time measured for the unmodified and modified PCL nonwovens after 1 h of incubation at 37 °C.
Figure 7. Prothrombin time measured for the unmodified and modified PCL nonwovens after 1 h of incubation at 37 °C.
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Figure 8. Thrombin time measured for the unmodified and modified PCL nonwovens after 1 h of incubation at 37 °C.
Figure 8. Thrombin time measured for the unmodified and modified PCL nonwovens after 1 h of incubation at 37 °C.
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Figure 9. Effects of unmodified PCL and PCL-HT post-incubation mixtures on PBM cell viability after 24 and 48 h of incubation. The results are presented as the mean of 6 repeats. The error bars denote SD; *** p < 0.001.
Figure 9. Effects of unmodified PCL and PCL-HT post-incubation mixtures on PBM cell viability after 24 and 48 h of incubation. The results are presented as the mean of 6 repeats. The error bars denote SD; *** p < 0.001.
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Figure 10. Effects of unmodified PCL and PCL-HT post-incubation mixtures on Hs68 cell viability after 24 and 48 h of incubation. The results are presented as the mean of 6 repeats. The error bars denote SD; *** p < 0.001.
Figure 10. Effects of unmodified PCL and PCL-HT post-incubation mixtures on Hs68 cell viability after 24 and 48 h of incubation. The results are presented as the mean of 6 repeats. The error bars denote SD; *** p < 0.001.
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Figure 11. Effects of unmodified PCL and PCL-HT post-incubation mixtures on DNA damage in PBM cells after 24 and 48 h of incubation. The results are presented as the mean of 100 comets. The error bars denote SEM; *** p < 0.001.
Figure 11. Effects of unmodified PCL and PCL-HT post-incubation mixtures on DNA damage in PBM cells after 24 and 48 h of incubation. The results are presented as the mean of 100 comets. The error bars denote SEM; *** p < 0.001.
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Figure 12. Effects of unmodified PCL and PCL-HT post-incubation mixtures on DNA damage in Hs68 cells after 24 and 48 h of incubation. The results are presented as the mean of 100 comets. The error bars denote SEM; *** p < 0.001.
Figure 12. Effects of unmodified PCL and PCL-HT post-incubation mixtures on DNA damage in Hs68 cells after 24 and 48 h of incubation. The results are presented as the mean of 100 comets. The error bars denote SEM; *** p < 0.001.
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Figure 13. Effects of medium (A), 25 µM hydrogen peroxide (B), PCL (C), PCL-HT-1 (D), PCL-HT-3 (E), and PCL-HT-5 (F) post-incubation mixtures on DNA damage in PBM cells after 24 h of incubation.
Figure 13. Effects of medium (A), 25 µM hydrogen peroxide (B), PCL (C), PCL-HT-1 (D), PCL-HT-3 (E), and PCL-HT-5 (F) post-incubation mixtures on DNA damage in PBM cells after 24 h of incubation.
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Figure 14. Effects of medium (A), 25 µM hydrogen peroxide (B), PCL (C), PCL-HT-1 (D), PCL-HT-3 (E), and PCL-HT-5 (F) post-incubation mixtures on DNA damage in Hs68 cells after 24 h of incubation.
Figure 14. Effects of medium (A), 25 µM hydrogen peroxide (B), PCL (C), PCL-HT-1 (D), PCL-HT-3 (E), and PCL-HT-5 (F) post-incubation mixtures on DNA damage in Hs68 cells after 24 h of incubation.
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Table 1. Specific surface area, total pore volume, and average pore size of the unmodified and modified PCL nonwovens.
Table 1. Specific surface area, total pore volume, and average pore size of the unmodified and modified PCL nonwovens.
Sample NameSpecific Surface Area [m2/g]Total Pore Volume [cm3/g]Average Pore Size [nm]
PCL1.586 ± 0.0387.357 × 10−319.01
PCL-HT-11.135 ± 0.9369.592 × 10−3193.1
PCL-HT-31.443 ± 0.0297.321 × 10−319.9
PCL-HT-51.600 ± 0.0718.390 × 10−321.95
Table 2. Zeta potential of unmodified and modified PCL nonwovens (pH ~7.4).
Table 2. Zeta potential of unmodified and modified PCL nonwovens (pH ~7.4).
Sample NamePCLPCL-HT-1PCL-HT-3PCL-HT-5
Zeta potential [mV]−34.05 ± 0.95−30.08 ± 0.27−27.54 ± 0.48−23.50 ± 1.89
Table 3. Lipid peroxidation in blood plasma: TBARS concentrations in the investigated samples.
Table 3. Lipid peroxidation in blood plasma: TBARS concentrations in the investigated samples.
Sample NameControlPCLPCL-HT-1PCL-HT-3PCL-HT-5
TBARS [nM]1.30 ± 0.151.42 ± 0.140.96 ± 0.060.59 ± 0.121.17 ± 0.34
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Kaczmarek, A.; Kudzin, M.H.; Juszczak, M.; Woźniak, K.; Król, P.; Vázquez, C.I.H.; Mrozińska, Z.; Chruściel, J.J. Fibrous Polycaprolactone-Based Composite Materials with the Addition of Hardystonite: Haemostatic Potential, Antioxidant Activity, and Biocompatibility Assessment. Macromol 2026, 6, 5. https://doi.org/10.3390/macromol6010005

AMA Style

Kaczmarek A, Kudzin MH, Juszczak M, Woźniak K, Król P, Vázquez CIH, Mrozińska Z, Chruściel JJ. Fibrous Polycaprolactone-Based Composite Materials with the Addition of Hardystonite: Haemostatic Potential, Antioxidant Activity, and Biocompatibility Assessment. Macromol. 2026; 6(1):5. https://doi.org/10.3390/macromol6010005

Chicago/Turabian Style

Kaczmarek, Anna, Marcin H. Kudzin, Michał Juszczak, Katarzyna Woźniak, Paulina Król, César I. Hernández Vázquez, Zdzisława Mrozińska, and Jerzy J. Chruściel. 2026. "Fibrous Polycaprolactone-Based Composite Materials with the Addition of Hardystonite: Haemostatic Potential, Antioxidant Activity, and Biocompatibility Assessment" Macromol 6, no. 1: 5. https://doi.org/10.3390/macromol6010005

APA Style

Kaczmarek, A., Kudzin, M. H., Juszczak, M., Woźniak, K., Król, P., Vázquez, C. I. H., Mrozińska, Z., & Chruściel, J. J. (2026). Fibrous Polycaprolactone-Based Composite Materials with the Addition of Hardystonite: Haemostatic Potential, Antioxidant Activity, and Biocompatibility Assessment. Macromol, 6(1), 5. https://doi.org/10.3390/macromol6010005

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