Lizardfish (Saurida tumbil) skin collagen hydrolysates accelerate wound healing: integrated in vitro and in vivo evidence

Hidrolisados de colágeno da pele de peixe-lagarto (Saurida tumbil) aceleram a cicatrização de feridas: evidências integradas in vitro e in vivo

Abstract

Collagen hydrolysates derived from tropical marine by-products are emerging as promising bioactive ingredients for tissue repair, although evidence for lizardfish (Saurida tumbil) skin remains limited. This study investigated the wound-healing potential of pepsin-soluble collagen hydrolysed with papain (SCHP), alkaline protease (SCHA), and a combination of both enzymes (SCHC) using in vitro and in vivo models. The bioactivity of the resulting collagen hydrolysates was evaluated using the BALB/3T3 clone A31 mouse fibroblast cell line for in vitro cell viability and scratch wound assays, followed by in vivo wound-healing assessment in BALB/c mice. In BALB/3T3 fibroblasts, all lizardfish hydrolysates increased cell viability above 100% after 24 and 48 h. At 24 h, viability was significantly higher than the negative control at all tested concentrations (0, 6.25, 12.5, 25, 50, and 100 µg/mL), whereas the commercial hydrolysate showed significance only at 50-100 ug/mL (p < 0.05). SCHA and SCHC produced the strongest proliferative effects and were not significantly different from the 10% FBS positive control at concentrations ≥12.5 ug/mL (p ≥ 0.05). In the scratch assay, SCHA significantly enhanced cell migration at 25-100 ug/mL after 24 h (p < 0.05), achieving 54-57% closure, and reached the highest migration rate after 48 h (94% at 25 ug/mL). In BALB/c mice, oral administration of SCHA accelerated wound repair more effectively than other treatments, producing a smaller wound area than the control on day 4 (77.50% vs 91.25%; p < 0.001) and near-complete closure by day 14 (0.62%). Histological analysis further confirmed superior re-epithelialisation, epidermal thickness, and collagen deposition in the SCHA group. Overall, alkaline protease-derived lizardfish collagen hydrolysate demonstrated strong safety and wound-healing efficacy.

Keywords:
collagen hydrolysate; tropical marine by-products; wound healing; in vitro assessment; in vivo evaluation

Resumo

Hidrolisados de colágeno derivados de subprodutos marinhos tropicais estão emergindo como ingredientes bioativos promissores para a reparação tecidual, embora as evidências para a pele de peixe-lagarto (Saurida tumbil) ainda sejam limitadas. Este estudo investigou o potencial de cicatrização de feridas do colágeno solúvel em pepsina hidrolisado com papaína (SCHP), protease alcalina (SCHA) e uma combinação de ambas as enzimas (SCHC), utilizando modelos in vitro e in vivo. A bioatividade dos hidrolisados de colágeno obtidos foi avaliada utilizando a linhagem de fibroblastos murinos BALB/3T3 clone A31 para os ensaios in vitro de viabilidade celular e migração por risco (scratch wound), seguida da avaliação in vivo da cicatrização de feridas em camundongos BALB/c. Em fibroblastos BALB/3T3, todos os hidrolisados de peixe-lagarto aumentaram a viabilidade celular acima de 100% após 24 e 48 h. Após 24 h, a viabilidade foi significativamente maior do que a do controle negativo em todas as concentrações testadas (0, 6,25, 12,5, 25, 50 e 100 µg/mL), enquanto o hidrolisado comercial apresentou diferença significativa apenas em 50–100 µg/mL (p < 0,05). SCHA e SCHC apresentaram os efeitos proliferativos mais intensos e não diferiram significativamente do controle positivo com 10% de FBS em concentrações ≥12,5 µg/mL (p ≥ 0,05). No ensaio de “scratch”, SCHA aumentou significativamente a migração celular em 25–100 µg/mL após 24 h (p < 0,05), atingindo 54–57% de fechamento da área da ferida, e apresentou a maior taxa de migração após 48 h (94% em 25 µg/mL). Em camundongos BALB/c, a administração oral de SCHA acelerou a cicatrização de feridas de forma mais eficaz do que os outros tratamentos, resultando em menor área da ferida no dia 4 (77,50% vs. 91,25%; p < 0,001) e fechamento quase completo no dia 14 (0,62%). A análise histológica confirmou ainda melhor reepitelização, maior espessura epidérmica e maior deposição de colágeno no grupo SCHA. De modo geral, o hidrolisado de colágeno de peixe-lagarto obtido por protease alcalina demonstrou elevada segurança e eficácia na cicatrização de feridas.

Palavras-chave:
hidrolisado de colágeno; subprodutos marinhos tropicais; cicatrização de feridas; avaliação in vitro; avaliação in vivo

1. Introduction

Skin wounds arise when tissue integrity is disrupted by physical, chemical, or biological insults, thereby impairing the barrier function of the skin and compromising host defence mechanisms when repair is inadequate. Whereas acute wounds generally resolve within a relatively short period and restore normal tissue function, chronic wounds are characterized by prolonged inflammation and delayed healing that may persist for months or even years (Landén et al., 2016). These chronic lesions impose a substantial clinical and socioeconomic burden because they are frequently associated with pain, reduced mobility, diminished quality of life, and high treatment costs. In the United States alone, approximately 5.7 million people experience wound lesions annually, generating healthcare expenditures of around USD 20 billion (Childs and Murthy, 2017). In Malaysia, chronic wounds such as diabetic foot ulcers likewise represent a serious healthcare challenge, with more than 260,000 diabetic patients estimated to suffer from foot ulcers at any given time (Nair et al., 2022). Physiologically, wound healing is a highly coordinated process involving haemostasis, inflammation, proliferation, and remodelling, which must proceed in balance to achieve effective tissue restoration (Askari et al., 2022).

Collagen is the major structural protein of the extracellular matrix and plays a pivotal role in maintaining the integrity and mechanical stability of connective tissues. Owing to its tensile strength and structural rigidity, collagen is widely distributed in skin, tendon, bone, and ligament, accounting for nearly 30% of the total protein mass in animals (León-López et al., 2019). To date, about 29 collagen types have been identified, each distinguished by specific molecular structures and amino acid sequences. Among them, type I collagen is the most abundant form in skin and bone and is extensively utilized in food, cosmetic, pharmaceutical, and healthcare applications (Senadheera et al., 2020). The industrial relevance of type I collagen is reflected in its growing global market, which was estimated at 936.5 tons in 2020 and is projected to expand further in response to increasing consumer interest in health-promoting products (Pulidindi and Ahuja, 2024). Traditionally, collagen has been obtained from terrestrial animal sources such as bovine, porcine, and poultry skin and bone. However, the use of mammalian collagen has become less desirable because of concerns related to zoonotic disease transmission, including bovine spongiform encephalopathy, foot-and-mouth disease, and avian influenza, as well as religious restrictions associated with bovine- and porcine-derived materials (Jaziri et al., 2025). In this context, fish collagen has emerged as an attractive alternative, with studies indicating that its functional performance can be comparable or even superior to terrestrial collagen following structural modification (Zhang et al., 2020).

Collagen hydrolysates and collagen-derived peptides have gained increasing attention because of their broad applicability in functional foods, pharmaceuticals, and medical products (Shenoy et al., 2022). Compared with conventional acid- or alkali-mediated hydrolysis, enzymatic hydrolysis is considered more advantageous because it offers higher conversion efficiency, minimizes residual by-products, reduces equipment corrosion, and lowers energy consumption (Amândio et al., 2023). Through enzymatic cleavage, large collagen molecules are converted into smaller peptides, typically containing 2-20 amino acid residues, which improves their solubility, bioavailability, and biological functionality (León-López et al., 2019). Importantly, enzymatically hydrolysed fish collagen can yield bioactive peptides with diverse physiological activities, including antioxidant, antihypertensive, and wound-healing effects (Yang et al., 2018). In the present study, papain and alkaline protease were selected because they are effective proteases for collagen hydrolysis and can generate low-molecular-weight peptides with enhanced bioactivity. Papain has broad substrate specificity, while alkaline protease exhibits strong proteolytic efficiency under mild conditions, allowing comparison of different enzymatic cleavage patterns. Their reported ability to produce collagen peptides with antioxidant, proliferative, and wound-healing potential makes them suitable for evaluating the bioactivity of lizardfish collagen hydrolysates (Felician et al., 2019; Chotphruethipong et al., 2021).

In recent years, collagen and collagen-based composites have been extensively investigated for wound management. Collagen-containing biomaterials, including hydrogels, sponges, and dressings, have shown considerable promise in supporting wound repair (Deng et al., 2022). Nevertheless, the therapeutic potential of orally administered collagen has received comparatively less attention. Unlike intact collagen, collagen hydrolysates are readily digested and absorbed, allowing their peptide fractions to enter systemic circulation and stimulate fibroblasts to synthesize new collagen by mimicking collagen degradation signals (Asserin et al., 2015). These peptides may also enhance fibroblast migration and proliferation, both of which are essential for tissue regeneration (Yang et al., 2018). Several studies have demonstrated the wound-healing efficacy of enzymatically hydrolysed collagen and collagen-derived peptides from marine by-products. For example, collagen hydrolysates from Atlantic salmon (Salmo salar) skin promoted wound healing in rodents through modulation of the cutaneous microbiota (Mei et al., 2020), while oral supplementation with Alaska pollock (Theragra chalcogramma) skin collagen hydrolysates improved cutaneous wound healing and re-epithelialisation in rat excision models (Yang et al., 2018). Similarly, tilapia collagen peptide administered via drinking water enhanced wound healing by increasing collagen deposition, hydroxyproline levels, growth factor expression, nitric oxide production, antioxidant enzyme activities, and regulating serum cytokines (Xiong et al., 2020). Hou and Chen (2023) further reported that sturgeon fish (Acipenser baerii × Huso huso) skin collagen peptide improved wound repair in mice. Despite these encouraging findings, most available studies have focused on temperate marine species or aquaculture-derived resources, whereas collagen hydrolysates from tropical marine by-products remain insufficiently explored.

Lizardfish (Saurida tumbil), a tropical marine species belonging to the family Synodontidae, is characterized by an elongated cylindrical body and a lizard-like head morphology. Adults generally range from 19 to 35 cm in body length, with a brown dorsal region and a silvery ventral surface marked by faint dark crossbands (Fishbase, 2023). This species is economically important in Malaysia because of its favourable gel-forming properties, desirable flavour, pale flesh colour, and relatively high muscle yield, making it suitable for surimi-based products (Guo et al., 2019). Moreover, lizardfish is affordable and widely available. Data from the Department of Fisheries Malaysia indicate that the average production of lizardfish between 2015 and 2019 reached approximately 48,153 metric tons, most of which was utilized as raw material for surimi manufacture under the trade name eso (Malaysia, 2023). Such large-scale processing generates substantial quantities of by-products, yet their valorisation remains limited, despite preliminary evidence indicating that their nutritional value is comparable to that of the edible portion (Jaziri et al., 2021).

Given this context, the conversion of lizardfish processing by-products into collagen hydrolysates represents a promising and sustainable approach. Although marine collagen hydrolysates have been widely reported to possess antioxidant and wound-healing properties, studies specifically addressing lizardfish-derived collagen hydrolysates are still scarce. In particular, their wound-healing efficacy has not been comprehensively elucidated. Therefore, the present study aimed to investigate collagen hydrolysates derived from lizardfish (S. tumbil) skin by examining the effects of different enzymatic treatments on wound-healing activities through in vitro assays using BALB/3T3 fibroblast cells and in vivo testing in BALB/c mice. The results are expected to provide insight into the optimal hydrolysis strategy, identify the most effective protease treatment, and clarify the contribution of lizardfish collagen hydrolysates to cutaneous wound repair. More broadly, this work may support the development of collagen-based functional foods, nutraceuticals, and pharmaceutical products from underutilized tropical marine by-products.

2. Materials and Method

2.1. Materials

The pepsin-soluble collagen (PSC) derived from lizardfish (S. tumbil) skin used in this study was prepared according to our previous work (Jaziri et al., 2025). Materials employed for the wound-healing experiments included BALB/3T3 clone A31 fibroblast cells (CL-0477) and Dulbecco’s modified Eagle’s medium (DMEM; PM150278), both purchased from Elabscience Biotechnology Inc. (Texas, USA). Fetal bovine serum (FBS; 16629525) and trypsin-EDTA solution (11677104) were obtained from Gibco (Grand Island, USA). In addition, 1× phosphate-buffered saline (PBS; 27575-31), penicillin-streptomycin (P/S; 09367-34), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; CAS 298-93-1), and dimethyl sulfoxide (DMSO; CAS 67-68-5) were supplied by Nacalai Tesque (Kyoto, Japan). BALB/c mice used for the in vivo wound-healing study were provided by the Biotechnology Research Institute, Universiti Malaysia Sabah, Kota Kinabalu, Malaysia. Chemicals used for collagen extraction and hydrolysate preparation included commercial fish collagen hydrolysate (type I collagen), which was purchased from SRB Suria Trading (Kedah, Malaysia), and bovine pepsin (1:10,000; CAS 9001-75-6), which was procured from Himedia (Maharashtra, India). Papain from papaya (800 kU/g; G8430) and alkaline protease (200 U/mg; B8360) were obtained from Solarbio (Beijing, China). All other chemicals and reagents used throughout the study were of analytical grade.

2.2. Enzymatic hydrolysis process

The optimized pepsin-soluble collagen (PSC) was used as the substrate for collagen hydrolysate production. The hydrolysis procedure was adapted from the method of Felician et al. (2019) with slight modifications. Briefly, 1 g of pepsin-solubilized collagen was dissolved in 200 mL of ultrapure water and incubated at 50 °C in a shaking water bath (Memmert, Schwabach, Germany). The collagen solution was then separately treated with 5% (w/w) alkaline protease and 5% (w/w) papain to produce SCHA and SCHP, respectively. Enzymatic hydrolysis was carried out at 50 °C for 2 h under continuous agitation. The reaction was subsequently terminated by heating the mixture at 95 °C for 15 min in a water bath. After cooling to 25 °C, the hydrolysates were centrifuged at 3000 rpm for 30 min, and the resulting supernatants were collected and freeze-dried using a lyophilizer (Labconco, Kansas City, USA). For the combined-enzyme treatment (SCHC), alkaline protease and papain were each added at 2.5% (w/w), and the subsequent hydrolysis steps were performed under the same conditions as described above. The freeze-dried collagen hydrolysates (SCHP, SCHA, and SCHC) were stored at freezing temperature until further analysis.

2.3. Fibroblast cell culture

BALB/3T3 clone A31 fibroblast cells were used in this study, and all experiments were conducted using cells at passages 5-8. The cells were cultured in T25 flasks (Jetbiofil, Guangzhou, China) containing 5 mL of complete medium (CM) composed of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (100 U/mL). Cell cultures were maintained at 37 °C in a humidified incubator with 5% CO2 (Memmert, Schwabach, Germany), and the culture medium was replaced every 2-3 days until the cells reached 70-80% confluency. At the desired confluency, the cells were detached by adding 1 mL of trypsin-EDTA solution and incubating for 3 min under the same conditions to facilitate trypsinization. The detached cells were then transferred into a 15 mL centrifuge tube, and complete medium was added to neutralize the trypsin-EDTA. The cell suspension was centrifuged at 1000 × g for 5 min, after which the supernatant was carefully removed and the cell pellet was resuspended in fresh complete medium. Cell viability and morphology were monitored daily using an Olympus BX53 inverted microscope (Tokyo, Japan) (Migone et al., 2022).

2.4. Cell viability assay

Before seeding, fibroblast BALB/3T3 cells were counted using a hemocytometer. The cells were then seeded into 96-well microplates (Jetbiofil, Guangzhou, China) at a density of 1 × 10⁴ cells/well and incubated for 24 h at 37 °C in a humidified atmosphere containing 5% CO₂ to allow cell attachment. After attachment, the cells were treated with different concentrations of lizardfish (Saurida tumbil) collagen hydrolysates (0, 6.25, 12.5, 25, 50, and 100 µg/mL) and further incubated for 24 h under the same conditions. Following treatment, 50 µL of MTT solution (0.5 mg/mL in 1× PBS) was added to each well, and the plates were incubated for an additional 6 h to allow the formation of formazan crystals. The supernatant was then carefully removed, and 100 µL of DMSO was added to each well to dissolve the formazan crystals. The plates were gently shaken and incubated for 15 min to ensure complete dissolution. Absorbance was subsequently measured at 570 nm using a Tecan Infinite M200 microplate reader (Tecan, Switzerland) (Migone et al., 2022).

2.5. Morphological change

BALB/3T3 fibroblast cells were seeded into 6-well plates (Jetbiofil, Guangzhou, China) at a density of 4 × 10⁵ cells/well in the presence of a cover glass and incubated for 24 h at 37 °C in a humidified atmosphere containing 5% CO₂. The attached cells were subsequently treated with SCHP, SCHA, and SCHC at concentrations of 0, 6.25, 12.5, 25, 50, and 100 µg/mL. After incubation, changes in cell morphology were observed and recorded using an Olympus BX53 inverted microscope (Tokyo, Japan) (Lin et al., 2019).

2.6. Scratch test

The scratch assay was performed according to the method of Migone et al. (2022) with minor modifications. BALB/3T3 fibroblast cells were seeded into sterile 24-well plates (Jetbiofil, Guangzhou, China) at a density of 1 × 10⁵ cells/well and cultured for 24 h at 37 °C in a humidified 5% CO₂ incubator until a confluent monolayer was formed. A linear scratch was then created across the cell monolayer using a sterile 100 µL pipette tip. After scratching, the wells were washed twice with PBS to remove detached cells and debris. The scratched monolayers were subsequently incubated with FBS-free medium as the negative control or with lizardfish (S. tumbil) collagen hydrolysates at concentrations ranging from 6.25 to 100 µg/mL. Cells cultured in medium supplemented with 10% FBS served as the positive control. Images of the wound area were captured at 0, 24, and 48 h using an Olympus BX53 inverted microscope (Tokyo, Japan) equipped with a camera at 4× magnification. The percentage of wound closure was quantified using ImageJ software (NIH, USA) by measuring the remaining scratch area at each time point. The closure percentage was calculated using the following Equation 1:

$Scratchclosurerate\left(\%\right)=\frac{S_0-S_t}{S_0}\times 100$ (1)

where S₀ is the scratch area at 0 h and S_t is the scratch area at the specified time point.

2.7. Biocompatibility study

All animal procedures were approved by the Animal Ethics Committee of Universiti Malaysia Sabah (JEHUMS), Institute for Tropical Biology and Conservation, Universiti Malaysia Sabah (ethical approval no. UMS/IBTP7.2/800-2/1/17; approved on 21 March 2023). BALB/c mice weighing 30-35 g were housed in a colony room under controlled conditions at 24 ± 1 °C with a 12 h light/dark cycle and were provided with standard rodent feed (Gold Coin Pte Ltd., Scotts Road, Singapore) and water ad libitum. A total of 28 mice were randomly allocated into four treatment groups (n = 6 per group): sterile saline water (SSW, group 1), lizardfish skin collagen hydrolysate produced using alkaline protease (SCHA, group 2), lizardfish skin collagen hydrolysate produced using a combination of papain and alkaline protease (SCHC, group 3), and commercial fish collagen hydrolysate (CFCH, group 4). All animals received oral administration at the same dose of 0.9 g/kg body weight (Felician et al., 2019). The mice were then anaesthetised by intraperitoneal injection of a 1% ketamine-xylazine cocktail at doses of 80 mg/kg and 10 mg/kg, respectively. After anaesthesia, the dorsal area was shaved using an electric clipper and disinfected with 70% alcohol. A full-thickness excision wound was created by surgically removing a circular section of skin approximately 2 cm in diameter. The animals were housed individually and orally administered the respective samples daily. Wound progression was monitored and photographed every three days. The mice were euthanised on days 4 and 14, and the wound-surrounding tissues were excised and fixed in 10% formaldehyde for subsequent histological analysis.

2.8. Full-thickness wound healing evaluation

The wound-healing effect of each treatment group was assessed by measuring wound size with a ruler and recording wound appearance using a Samsung digital camera, as described by Peng et al. (2020). Wound area was measured on days 0, 4, 8, 12, and 14 after wound induction. The percentage of wound closure was then calculated using the following Equation 2:

$Woundclosurerate\left(\%\right)=\frac{W_0-W_t}{W_0}\times 100$ (2)

where W₀ is the wound area at 0 day and W_t is the wound area at a particular day.

2.9. Histological examination

Samples collected on days 4 and 14 were fixed in 10% formaldehyde for 1 week, embedded in paraffin, and sectioned into 4 µm-thick slices using a microtome (Leica, Tokyo, Japan). The tissue sections were subsequently stained with haematoxylin-eosin (H&E) and Masson’s trichrome, followed by dehydration, washing, mounting, and microscopic observation, according to the method of Felician et al. (2019).

2.10. Statistical analysis

The collected data were processed using a SPSS version 29.0. (IBM Corp., Armonk, New York) statistical software. All experimental results are expressed as the mean ± standard deviation (SD) of at least three independent experiments. One-way analysis of variance (ANOVA) and Tukey’s multiple range test were applied to evaluate statistical significance. Values of p < 0.05 between two independent groups were considered to be statistically significant.

3. Results and Discussion

3.1. Cell viability study

Cell migration and proliferation are fundamental events in the wound-healing cascade and involve the coordinated activity of multiple cell populations. Among these, fibroblasts play a central role throughout the repair process, particularly from the late inflammatory phase to re-epithelialisation and extracellular matrix formation (Talbott et al., 2022). In the present study, BALB/3T3 mouse embryonic fibroblasts were used as the model cell line to evaluate the effects of lizardfish (S. tumbil) collagen hydrolysates on cell viability and proliferative activity. As shown in Figure 1, treatment of BALB/3T3 cells with lizardfish collagen hydrolysates (SCHP, SCHA, and SCHC) and commercial fish collagen hydrolysate (CFCH) at concentrations of 6.25, 12.5, 25, 50, and 100 µg/mL for 24-48 h did not induce cytotoxicity. Instead, all hydrolysate-treated groups exhibited cell viability values exceeding 100%, indicating a proliferative effect rather than growth inhibition. After 24 h of incubation, cell viability ranged from 110% to 213% across the tested concentrations (Figure 1a), suggesting that hydrolysed collagens promoted fibroblast growth in a concentration-dependent manner. All lizardfish-derived hydrolysates significantly increased BALB/3T3 cell viability relative to the negative control (serum-free medium) at all tested concentrations (p < 0.001), whereas CFCH showed a significant effect only at 50-100 µg/mL (p < 0.05). Notably, lizardfish collagen hydrolysates consistently produced higher viability values than the commercial hydrolysate. Among the tested samples, SCHA and SCHC exhibited the strongest proliferative responses across the entire concentration range, and at concentrations of 12.5 µg/mL and above, their effects were not significantly different from those of the positive control supplemented with 10% fetal bovine serum (p ≥ 0.05). With prolonged incubation to 48 h (Figure 1b), BALB/3T3 fibroblasts treated with collagen hydrolysates exhibited a further significant increase in viability compared with the negative control (p < 0.001). In particular, the SCHA and SCHC groups showed the most pronounced proliferative effect, with viability values ranging from 223% to 260%, exceeding twofold that of the untreated group. Notably, no significant difference was observed between these hydrolysate-treated groups and the positive control supplemented with 10% FBS (p ≥ 0.05), indicating that their proliferative activity was comparable to that of serum-supported growth. These results were consistent with the 24 h findings and suggest that the bioactivity of the hydrolysates was maintained or enhanced with longer exposure.

Figure 1
Effect of lizardfish skin collagen hydrolysates on BALB/3T3 fibroblast cell viability. a) At 24 h of treatment. b) At 48 h of treatment. NC: negative control (serum free medium). PC: positive control (serum supplemented medium). CFCH: commercial fish collagen hydrolysate. SCHP: PSC hydrolysed with papain. SCHA: PSC hydrolysed with alkaline protease. SCHC: PSC hydrolysed with papain and alkaline protease. Statistically significant difference vs. control according to One-way ANOVA: *p < 0.05; **p < 0.01; ***p < 0.001 compared with the control group (0.00 µg/mL).

The proliferative response observed in this study may be associated with the molecular characteristics of the hydrolysates, particularly peptide size, amino acid composition, and peptide sequence, all of which are known to influence cellular proliferation potential (Chang et al., 2024). Hydrolysed collagen peptides enriched in glycine, proline, and alanine have previously been shown to stimulate the growth of L929 and MRC5 fibroblasts as well as bone marrow-mesenchymal stem cells (Benjakul et al., 2018; Chotphruethipong et al., 2021). In addition, proline-hydroxyproline dipeptides have been reported to promote fibroblast and osteoblast proliferation (Kimura et al., 2017). Accordingly, the superior viability observed in the SCHA and SCHC groups may be attributed to their abundance of low-molecular-weight peptides and hydrophobic amino acids, which may serve as readily available substrates and signalling molecules for fibroblast growth. These findings are in agreement with previous studies demonstrating the excellent biocompatibility of fish-derived collagen materials. Lin et al. (2019) reported that collagen isolated from bigeye tuna (T. obesus) skin enhanced NIH-3T3 fibroblast growth without inducing significant cytotoxicity. Similarly, Manjushree et al. (2022) showed that hydrolysed collagen from pirapitinga (P. brachypomus) skin increased the viability of L292 mouse fibroblasts and remained non-toxic even at 3 mg/mL. Gharahgheshlagh et al. (2023) further demonstrated that collagen-based films derived from narrow-barred Spanish mackerel (S. commerson) skin promoted 3T3 cell growth and proliferation. Beyond fibroblasts, fish collagen hydrolysates from silver carp (H. molitrix) bone and giant croaker (N. japonica) swim bladder have also been reported to stimulate the proliferation of HaCaT keratinocytes and HUVECs, respectively (Chen et al., 2019; Iosageanu et al., 2021). Collectively, these results confirm that the collagen hydrolysates investigated in the present study were non-cytotoxic and exhibited good biocompatibility, supporting their potential use in dermal and wound-healing applications.

3.2. Cell migration analysis

Fibroblast proliferation and migration are pivotal events in the wound-healing cascade and are tightly regulated by multiple cell types and microenvironmental cues. Among the in vitro approaches available, the scratch assay is widely used to evaluate the wound-healing potential of bioactive compounds by mimicking an artificial wound on a confluent cell monolayer (Migone et al., 2022). In the present study, BALB/3T3 fibroblasts were treated with lizardfish (S. tumbil) collagen hydrolysates at concentrations ranging from 6.25 to 100 µg/mL for up to 48 h. Cell migration was monitored at 0, 24, and 48 h, and the wound closure area was quantified using ImageJ software. As shown in Figure 2, both the positive control supplemented with 10% FBS and the collagen hydrolysate-treated groups exhibited visibly enhanced cell migration compared with the untreated control. Quantitative analysis of fibroblast migration is presented in Figure 22c. After 24 h of incubation, the hydrolysates promoted wound closure to varying extents, with SCHA showing the most pronounced effect at concentrations of 25-100 µg/mL (p < 0.05), resulting in wound closure values of 54-57% relative to the negative control (FBS-free medium). Notably, the migratory response induced by SCHA was not significantly different from that of the positive control containing 10% FBS (p ≥ 0.05). Following 48 h of treatment, the migration rate of BALB/3T3 cells increased further, and all collagen hydrolysate groups, including SCHP, SCHA, SCHC, and CFCH, produced significantly greater wound closure than the untreated group in a concentration-dependent manner. Among them, SCHA again exhibited the strongest response, reaching a migration rate of 94% at 25 µg/mL, with no significant difference relative to the FBS-supplemented medium (p ≥ 0.05).

Figure 2
Effect of lizardfish skin collagen hydrolysates on BALB/3T3 fibroblast cell migration. a) Representative scratch test photographs of different hydrolysates treated at 24 h and 48 h. b) Cell migration rate at 24 h. c) Cell migration rate at 48 h. NC: negative control (serum free medium). PC: positive control (serum supplemented medium). CFCH: commercial fish collagen hydrolysate. SCHP: PSC hydrolysed with papain. SCHA: PSC hydrolysed with alkaline protease. SCHC: PSC hydrolysed with papain and alkaline protease. Statistically significant difference vs. control according to One-way ANOVA: *p < 0.05; **p < 0.01; ***p < 0.001 compared with the control group (0.00 µg/mL).

The superior activity of SCHA suggests that collagen hydrolysates generated using alkaline protease may possess molecular characteristics favourable for fibroblast chemotaxis and migration. Felician et al. (2019) reported that low-molecular-weight collagen peptides can enhance wound cell migration due to their chemotactic properties. In addition, hydroxyproline is considered an important marker of collagen deposition during tissue repair, and collagen materials rich in hydroxyproline have been shown to accelerate wound healing in vivo (Chen et al., 2019). Likewise, collagen hydrolysates enriched in glycine, proline, and alanine have been associated with improved wound closure activity (Chotphruethipong et al., 2021). Therefore, the enhanced migratory response observed in the present study may be attributed to the combined contribution of low-molecular-weight peptides and bioactive amino acid residues that support both cell movement and proliferation.

3.3. Morphological analysis

Morphological evaluation of BALB/3T3 fibroblasts treated with lizardfish (S. tumbil) skin collagen hydrolysates at concentrations of 6.25, 12.5, 25, 50, and 100 µg/mL revealed no observable morphological abnormalities compared with the reference groups cultured in serum-free medium or medium supplemented with 10% fetal bovine serum (Figure 3). Both treated and control cells maintained their typical fibroblastic morphology and displayed uniform growth patterns throughout the incubation period. These observations indicate that the tested hydrolysates did not induce adverse morphological alterations in normal fibroblast cells. As presented in Figure 3, all collagen hydrolysates supported BALB/3T3 cell growth in a concentration-dependent manner, in agreement with the MTT viability results. Among the tested samples, SCHA promoted the most pronounced cell growth, exceeding that observed for SCHC, SCHP, and the commercial fish collagen hydrolysate (CFCH). This finding suggests that the lizardfish-derived collagen hydrolysates, particularly SCHA, are highly compatible with normal skin fibroblasts and may contribute positively to cellular responses associated with wound repair. The present results are consistent with previous reports on the biocompatibility of fish-derived collagen materials. Lin et al. (2019) demonstrated that collagen isolated from bigeye tuna (T. obesus) skin did not induce morphological changes in 3T3 F442A fibroblasts and supported cell growth in a concentration-dependent manner. Similarly, Khong et al. (2018) and Tan and Chang (2018) reported that collagen derived from giant croaker (N. japonica) skin and jellyfish (A. hardenbergi) did not alter the morphology of NIH-3T3 and 3T3 F442A fibroblast cell lines, respectively, while still promoting cell proliferation. Collectively, these findings confirm that lizardfish collagen hydrolysates are non-toxic to fibroblasts and highlight their potential as bioactive ingredients for wound-healing applications.

Figure 3
Morphological changes of BALB/c 3T3 fibroblast cells treated with different concentrations of collagen hydrolysates isolated from lizardfish (S. tumbil) skin at 48 h. CFCH: commercial fish collagen hydrolysate. SCHP: PSC hydrolysed with papain. SCHA: PSC hydrolysed with alkaline protease. SCHC: PSC hydrolysed with papain and alkaline protease.

3.4. Mice’s body weight

Following the in vitro evaluation of cell viability and migration, all lizardfish collagen hydrolysates demonstrated good biocompatibility and no cytotoxic effects toward BALB/3T3 fibroblasts across the tested concentration range (6.25-100 µg/mL), thereby supporting their suitability for subsequent in vivo assessment (Lin et al., 2019). To further investigate their wound-healing potential under physiological conditions, BALB/c mice were used as the in vivo wound model. Changes in body weight were monitored throughout the experimental period as a general indicator of animal health and treatment tolerance, as shown in Figure 4. The results indicated that all mice remained in good overall condition during the course of the experiment. A slight reduction in body weight was observed on the day of wound induction, which was most likely attributable to acute stress and pain associated with the injury procedure. However, from the following day onward, body weight gradually recovered in parallel with progressive wound contraction. Importantly, no significant differences in body weight were observed between the collagen hydrolysate-treated groups and the untreated control group throughout the experimental period (p ≥ 0.05). This finding suggests that oral administration of the hydrolysates did not adversely affect appetite, metabolism, or general physiological status. These observations further support the biocompatibility and safety of the tested collagen hydrolysates in vivo. Comparable findings have been reported in previous studies. Felician et al. (2019) observed no significant changes in body weight in mice treated with jellyfish collagen hydrolysates, while Guo et al. (2019) similarly reported that treatment with a polypyrrole hydrogel dressing did not significantly influence body weight during wound healing. Taken together, the present results indicate that the lizardfish collagen hydrolysates were well tolerated and did not induce systemic adverse effects during the treatment period.

Figure 4
The effect of lizardfish (S. tumbil) collagen hydrolysates on the mice body weight. Control: saline water. SCHP: PSC hydrolysed with papain. SCHA: PSC hydrolysed with alkaline protease. SCHC: PSC hydrolysed with papain and alkaline protease.

3.5. Wound healing rate

To evaluate the in vivo wound-healing efficacy of the collagen hydrolysates, the experiment was conducted in four groups: the control group treated with sterile saline water, CFCH, SCHA, and SCHC groups. As shown in Figure 5, all groups exhibited progressive wound contraction over the 14-day observation period; however, the rate of reduction differed markedly among treatments. Quantitative analysis of relative wound area (Figure 5) demonstrated that SCHA consistently produced the most rapid wound closure. On day 4, the wound area in the SCHA group (77.50%) was significantly smaller than that in the SCHC (86.25%) and CFCH (87.50%) groups (p < 0.01), and markedly lower than that in the control group (91.25%) (p < 0.001). On day 8, wound reduction remained more pronounced in the SCHA group than in the CFCH and control groups, while SCHC did not differ significantly from SCHA (p < 0.05). By day 12, mice treated with SCHA showed a wound area of only 2.87%, which was lower than that of all other groups, although no significant difference was observed relative to SCHC. By day 14, wounds in the SCHA-treated mice were almost completely healed, with a residual wound area of 0.62%, compared with 3.59% in SCHC, 5.22% in CFCH, and 7.31% in the control group. These findings indicate that oral administration of SCHA was the most effective treatment for accelerating wound repair.

Figure 5
Effect of lizardfish skin collagen hydrolysates upon in vivo wound healing test. (a) Representative wound healing photographs of hydrolysates treated over 14 days. (b) Schematic images of wound contraction on day 0, 4, 8, 12, and 14 after wound treatment. (c) Relative wound area of each group at days 4, 8, 12, and 14. *p < 0.05, **p < 0.01, ***p < 0.001. CFCH: commercial fish collagen hydrolysate. SCHA: PSC hydrolysed with alkaline protease. SCHC: PSC hydrolysed with papain and alkaline protease.

The temporal wound-healing profiles further support this observation. Although all groups showed a gradual decline in wound area from day 4 to day 14, the untreated control exhibited the slowest healing response, indicating that spontaneous healing occurred but at a lower efficiency. The CFCH group also showed steady wound reduction, but its healing trajectory remained slower than those of SCHA and SCHC, particularly during the later healing stages. In contrast, the SCHA group displayed the fastest and most substantial wound contraction between days 4 and 12, culminating in near-complete closure by day 14. The SCHC group also showed progressive healing with marked wound reduction by day 12 and near-complete closure by day 14, although its response remained slightly inferior to that of SCHA. Notably, these in vivo findings were consistent with the in vitro results, in which SCHA also produced the strongest stimulatory effects on fibroblast viability and migration.

The superior wound-healing effect of lizardfish collagen hydrolysates, particularly SCHA, is in line with previous reports on orally administered marine collagen peptides. Felician et al. (2019) demonstrated that jellyfish (R. esculentum) collagen hydrolysates produced using collagenase II and a combination of alkaline protease and papain significantly reduced wound area in mice (p < 0.001) compared with the vehicle-treated group. Similarly, Hou and Chen (2023) reported that low-molecular-weight collagen hydrolysate nanoemulsions from sturgeon skin (Acipenser baerii × Huso huso) achieved superior wound closure in mice relative to higher-molecular-weight fractions. Yang et al. (2018) further showed that low-molecular-weight collagen hydrolysates from Alaska pollock (T. chalcogramma) accelerated wound repair in rats between days 4 and 12 post-injury. Comparable outcomes were also described by Mei et al. (2020), who found that oral administration of collagen hydrolysates from Atlantic salmon (S. salar) and tilapia (O. niloticus) improved wound healing in Sprague-Dawley rats, with complete closure observed by day 12. In diabetic wound models, tilapia collagen peptide mixture TY001 likewise enhanced wound repair more effectively than whey protein and untreated controls (Xiong et al., 2020).

The enhanced healing response observed in the present study may be attributed to the low molecular weight and bioactive composition of the hydrolysates, especially in SCHA. Low-molecular-weight collagen peptides generally exhibit greater gastrointestinal absorption and more efficient delivery to wound sites, thereby facilitating the rapid supply of amino acids required for tissue regeneration (Manjushree et al., 2022). In addition, bioactive residues such as proline and hydroxyproline are known to stimulate fibroblast proliferation and migration, thereby enhancing extracellular matrix deposition and collagen synthesis (Manjushree et al., 2022; Yang et al., 2019). These processes are essential for accelerating wound closure and restoring tissue strength (Sugihara et al., 2018). The antioxidant capacity of collagen hydrolysates may further contribute to the healing response by reducing oxidative stress at the wound site. Since excessive reactive oxygen species can damage lipids, proteins, and DNA and delay tissue repair, antioxidant peptides may protect cells from oxidative injury and help regulate the transition from inflammation to the proliferative phase of healing (León-López et al., 2019; Sugihara et al., 2018). Moreover, amino acids such as hydroxyproline, glutamine, and arginine support collagen integrity and provide essential substrates for rapid tissue repair (Yang et al., 2019; Lin et al., 2019).

Another factor that may explain the strong healing effect is the essential amino acid profile of the hydrolysates. Yang et al. (2018) reported that fish protein hydrolysates from Alaska pollock containing high levels of essential amino acids accelerated wound repair. Wang et al. (2022) likewise emphasized the nutritional value of collagen- and essential amino acid-enriched supplements in supporting wound healing. Fuentes-Orozco et al. (2004) further showed that hydrolysates rich in glutamine and arginine could enhance tissue repair because these amino acids become conditionally essential after trauma. In addition, the biological activity of collagen hydrolysates is closely linked to their peptide sequence characteristics. Specific peptide motifs, such as Gly-Leu and Gly-Pro, have been associated with radical-scavenging activity (Liping et al., 2018), while fragments such as Gln-Glu, Gly-Pro, and Gln-Gly-Pro have been identified in T. chalcogramma collagen peptides and implicated in wound-healing promotion. Taken together, these findings suggest that the remarkable wound-healing efficacy of SCHA is likely associated with its low molecular weight and favourable amino acid and peptide composition, which collectively enhance nutrient bioavailability and support tissue regeneration.

3.6. Histological analysis

Histological evaluation was performed using haematoxylin-eosin (H&E) and Masson’s trichrome (MT) staining to assess tissue regeneration and collagen deposition in the wound area on days 4 and 14. As shown in Figure 6a, H&E staining revealed that granulation tissue containing abundant fibroblasts, extracellular matrix components, and regenerative elements progressively occupied the wound bed in all groups, contributing to the gradual formation of neo-dermal tissue. By day 14, the SCHA-treated group exhibited the most advanced histological features of repair, characterized by reduced inflammatory cell infiltration, improved epithelial organization, enhanced neovascularization, and greater fibroblast accumulation compared with the SCHC, CFCH, and control groups. Quantitative analysis of epidermal thickness using ImageJ further demonstrated that all collagen hydrolysate-treated groups developed a thicker epidermal layer than the untreated control on day 14 (Figure 6c). Notably, the SCHA group showed significantly greater epidermal thickness than the SCHC (p < 0.05), CFCH (p < 0.01), and control (p < 0.001) groups. No obvious re-epithelialisation was detected on day 4, whereas clear epithelial reconstruction was evident by day 14, particularly in the SCHA-treated wounds, indicating faster wound contraction and neo-tissue formation.

Figure 6
Microscopic pictures and histological analysis of the haematoxylin-eosin (H&E) and Masson’s trichrome (MT) stained tissue sections of mice treated with collagen hydrolysates and saline water (control) at day 14. (a) H&E-stained tissue section; (b) MT-stained tissue section; (c) measurement of epidermal layer thickness; d) measurement of collagen volume fraction. *p < 0.05, **p < 0.01, ***p < 0.001. New blood vessels (red arrow), fibroblast cells (green arrow), keratinocyte cells (blue arrow), hair follicles (black arrow), and inflammatory cells (white arrow). CFCH: commercial fish collagen hydrolysate. SCHA: PSC hydrolysed with alkaline protease. SCHC: PSC hydrolysed with papain and alkaline protease.

The MT-stained sections (Figure 6b) provided further evidence of improved dermal regeneration in the collagen hydrolysate-treated groups, in agreement with the H&E observations. MT staining is particularly useful for visualizing collagen fibre distribution within granulation and neo-dermal tissues, thereby reflecting the structural quality of regenerated skin. On day 4, collagen fibres were already detectable in the treated groups, whereas the control group showed markedly less collagen deposition. By day 14, collagen accumulation became more prominent in all groups, with the SCHA-treated wounds exhibiting the highest collagen fraction (Figure 6d). Quantitative analysis confirmed that the collagen content in the SCHA group was significantly greater than that in the SCHC, CFCH, and control groups (all p < 0.001). In addition, capillary vessels and hair follicles were observed in the granulation and neo-dermal tissues of all groups on day 14, with the most pronounced development again found in the SCHA group. Interestingly, early signs of angiogenesis and hair follicle formation were already detectable on day 4 only in the SCHA-treated wounds, whereas these features were minimal or absent in the other groups.

Overall, the histological findings demonstrated that SCHA treatment promoted more mature granulation tissue, enhanced dermal reconstruction, and improved skin appendage formation relative to the other hydrolysate-treated groups and the untreated control. These results were fully consistent with the wound closure data, further supporting the superior wound-healing efficacy of SCHA. The greater epidermal thickness and collagen deposition observed in this group may be attributed to the rapid absorption and bioactivity of low-molecular-weight collagen peptides, which can provide critical substrates for collagen-synthesizing cells and support tissue remodeling. Similar findings have been reported in previous studies. Felician et al. (2019) showed that jellyfish (R. esculentum) collagen hydrolysates (Mw 25 kDa) enhanced re-epithelialisation, tissue regeneration, and collagen deposition in wounded mice compared with the vehicle group. Likewise, Xiong et al. (2020) reported increased collagen deposition following oral administration of tilapia collagen peptide mixture TY001. Zhang et al. (2011) and Wang et al. (2015) further demonstrated that orally administered hydrolysed collagen from chum salmon (O. keta) promoted fibroblast infiltration, vascularisation, epithelialisation, and collagen formation in rat wounds. In addition, Yang et al. (2018) found that low-molecular-weight collagen hydrolysates from Alaska pollock (T. chalcogramma) restored a nearly normal epidermal architecture, including complete re-epithelialisation and the appearance of hair follicles, whereas the vehicle-treated wounds showed poor epithelial recovery.

Collectively, these findings indicate that low-molecular-weight collagen hydrolysates derived from lizardfish (S. tumbil) skin by-products possess substantial wound-healing potential in both in vitro and in vivo systems, highlighting their promise as safe and effective bioactive ingredients for future wound management applications.

4. Conclusion

Collagen hydrolysates derived from lizardfish (Saurida tumbil) skin demonstrated promising wound-healing activity in both in vitro and in vivo models. All hydrolysates showed good biocompatibility toward BALB/3T3 fibroblast cells by enhancing cell viability and supporting normal cell morphology. Among the tested samples, collagen hydrolysed with alkaline protease (SCHA) exhibited the strongest bioactivity, significantly promoting fibroblast proliferation and migration, accelerating wound closure in BALB/c mice, and producing superior histological outcomes, including improved re-epithelialisation, greater epidermal thickness, and enhanced collagen deposition. These findings indicate that the wound-healing efficacy of lizardfish collagen hydrolysates is influenced by the enzymatic treatment used, with alkaline protease producing the most effective hydrolysate compared other treatments. Overall, lizardfish skin, an underutilized tropical marine by-product, represents a safe and valuable source of bioactive collagen hydrolysates with considerable potential for development as functional ingredients in wound management, nutraceutical, and biomedical applications. However, the molecular mechanisms and peptide characteristics responsible for these wound-healing effects remain to be fully elucidated. Therefore, further studies are needed to characterize the bioactive peptide profile of lizardfish collagen hydrolysates and clarify the cellular and molecular pathways underlying their wound-healing activity.

Acknowledgements

The authors gratefully acknowledge the financial support provided by the Ministry of Higher Education Malaysia through the Fundamental Research Grant Scheme (FRGS), grant number FRGS/1/2019/STG03/UMS/02/5. The authors also sincerely thank Universitas Brawijaya (UB) and the Indonesia Endowment Fund for Education Agency (LPDP) for supporting the completion of this research and manuscript under the 2025 funding scheme, contract number 02567.5/DST/UN10.A0501/B/PG/2025.

Data Availability Statement

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

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*

e-mail: [e-mail]

Editor:

Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  14 Aug 2026
• Date of issue
  2026

History

• Received
  14 Apr 2026
• Accepted
  11 June 2026