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. 2024 Jan 31;10(2):e1359. doi: 10.1002/vms3.1359

Effect of dietary supplementation with chitooligosaccharides on wound healing in dogs

Guoqiang Cheng 1, Yu Zeng 2, Yongjin Wang 1, Jieying Xia 1, Xindong Lei 1, Dong Chen 3, Huaqiao Tang 2, Li Zhang 1,✉, Yanglu Liu 1,✉
PMCID: PMC10828722

Abstract

Background

Wound healing has long been a subject of great interest for pathologists. Previous research has established the effectiveness of topical application of chitooligosaccharides (COSs) on wound healing. However, studies on their potential usefulness as food additives to improve wound healing are lacking.

Objective

Here, we investigated whether dietary supplementation with COSs could promote wound healing using Beagle dogs as an animal model.

Methods

Nine dogs were evenly assigned randomly into control, 0.5% COSs and 1% COSs groups. Control dogs were fed with a non‐supplemented dietary formula (vehicle). Dogs in 0.5% COSs and 1% COSs groups were fed with a dietary formula supplemented with 0.5% and 1% COSs. After 8 weeks of feeding, dogs in each group underwent surgery with three matching full‐thickness 2 × 2‐cm skin defects were created with a scalpel on the left side of the dorsal midline, 2 cm apart from each other. Wounds were photographed and the healing percentage was determined on day 0, 2, 4, 7, 9, 11, 14, 16, 18 and 21. The healing process was assessed by daily gross examination, histologic examination and Masson's trichrome staining on days 7, 14 and 21. Considering the important role of the transforming growth factor‐β (TGF‐β1) signalling pathway in wound healing, immunostaining was used to evaluate TGF‐β1 expression.

Results

The results showed that wound healing was accelerated; collagen synthesis and TGF‐β1 expression were enhanced in response to dietary supplementation with COSs.

Conclusions

This study makes an original contribution to the understanding of wound healing in dogs and other animals and supports the use of COSs as a supplement to be added to animal diets.

Keywords: chitooligosaccharides, dog food, TGF‐β, wound healing

We investigated whether dietary supplementation with COSs could promote wound healing using Beagle dogs as an animal model. Results showed that wound healing was accelerated; collagen synthesis and TGF‐β1 expression were enhanced in response to dietary supplementation with COSs. This was the first study to describe the effects of dietary supplementation with COSs on wound healing. The results of this study demonstrate that, in addition to its topical application, oral administration of COSs is also effective in promoting wound healing.

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1. INTRODUCTION

As the largest and most exposed organ in the body, the skin plays a pivotal role in protecting internal structures from the external environment (Su et al., 2014). In daily life, the skin can be easily injured in various ways, including trauma, burns and ulcers (Groeber et al., 2012). After the skin is injured, the wound will gradually heal, preventing dehydration, blood loss and invasion by potentially pernicious microorganisms (Burger et al., 2019). The process of wound healing is highly complex, and it can be divided into several phases: blood clot formation, inflammation, proliferation and maturation (Raghow, 1994). Multiple factors have a considerable impact on wound healing, including ischemic tissue, foreign bodies, infection and contamination, which potentially cause serious complications, and even death (Broughton et al., 2006). A growing body of research has recognized the importance of promoting wound healing, not only through the development of novel drugs but also via dietary supplements.

Chitooligosaccharides (COSs), oligomers of glucosamine (GlcN) or N‐acetylglucosamine subunits linked by a β‐1,4 O‐glycosidic bond, are the products of the degradation of chitin, one of the most abundant elements of the biomass commonly found in crustacean shells, insect cuticles and fungal cell walls (Mahata et al., 2014). They are biodegradable polymers that have previously been observed to have therapeutic and regenerative effects in the process of wound healing (Naveed et al., 2019). COSs have low molecular weight and water solubility, are non‐toxic, and can be naturally absorbed. The market demand for COSs is considerably higher than that of precursor molecules (Liaqat & Eltem, 2018). They exhibit immunoregulatory, antibacterial, antifungal, antiviral, antitumor and antioxidant activity and can regulate fat and cholesterol metabolism as well as blood pressure (Kim & Rajapakse, 2005).

Recent evidence suggests that COSs may also affect wound healing by stimulating the secretion of cytokines essential for this process (Okamoto et al., 2003). COSs have been shown to stimulate fibroblast proliferation by regulating the production of transforming growth factor β (TGF‐β1), with the resulting increase in collagen levels promoting the development of connective tissue (Jun et al., 2014; Zi‐Wei et al., 2017). TGF‐β1 reportedly plays a critical role in wound healing by enhancing extracellular matrix deposition, regulating fibroblast proliferation and promoting angiogenesis (Jun et al., 2014). Smad2 and Smad3 are phosphorylated upon binding TGF‐β1, which then combine with Smad4 (Hong et al., 2008). The receptor complex then enters the nucleus to regulate gene transcription (Schiller et al., 2004).

Although previous studies on the effect of the external application of COSs on rat wound healing have been carried out, few studies have investigated the effect of oral administration of COSs on larger animals. Therefore, this study assessed the effect of dietary supplementation with COSs on wound healing in dogs. Considering the pivotal role of TGF‐β1 in this process, we determined its levels of expression via immunofluorescence staining. We assessed the degree of wound healing by measuring collagen formation via hematoxylin–eosin and Masson's trichrome staining (Rabau et al., 1995).

2. MATERIALS AND METHODS

2.1. Reagents and diet composition

The COSs used in the study were prepared via enzyme reaction‐membrane separation coupling technology at the Dalian Institute of Physical Chemistry (Chinese Academy of Sciences, Dalian, China). The polymerization degree was 2–7, and the final purity was above 90% (molecular weight <1000 Da). The experimental subjects were fed with a basic, nutritionally complete dietary formula based on extruded dry puppy food (22.0% crude protein, 11.6% crude fat, 5.0% crude fibre, 10.0% crude ash, 0.77% lysine, 1.2% calcium, 1.0% phosphorus, 10.0% moisture and 0.45% water‐soluble chloride). COSs were added to the dietary formula as a powder using a mixer.

2.2. Animals, experimental groups and feeding schedule

Male Beagle dogs (6 months old, 7.4 ± 0.4 kg) were obtained from the Beagles Breeding Centre. All dogs were considered healthy following examination. The dogs were given 2 weeks to adapt to the experimental conditions.

After the 2‐week acclimation period, the dogs were assigned randomly into three groups. Each dog was individually housed in a 1.5 × 1.0‐m kennel at a constant temperature (24°C ± 2) under a 12‐h light/12‐h dark cycle. Each kennel was equipped with a feeder and a water bucket. Fresh water was provided ad libitum. The dogs were weighed every week. Control (n = 3) was fed with a non‐supplemented dietary formula (vehicle) and served as a control group. The 0.5% COSs group (n = 3) was fed with a dietary formula supplemented with 0.5% COSs, and the 1 % COSs group (n = 3) was fed with a dietary formula supplemented with 1% COSs. The dogs were fed twice a day (at 9:00 AM and 6:00 PM) in sufficient amounts to meet their metabolic energy requirements (Dobenecker et al., 2013). They were maintained under this regimen during the 8‐week study period.

2.3. Establishment of a skin excision wound model

After the 8 weeks of feeding, the dogs underwent surgery. In preparation for surgery, the dogs were intravenously injected with butorphanol (0.1 mg/kg) and acepromazine (0.05 mg/kg) for sedation and analgesia 20 min before the surgery. Propofol (5 mg/kg) was used at the beginning of the surgery for anaesthetic induction. Anaesthesia was maintained with isoflurane mixed with oxygen, and the dogs received Ringer's lactate solution (10 mL/kg/h IV) during this period. The dorsolateral area of the trunk was clipped and aseptically prepared for surgery.

The dogs were positioned in right lateral recumbency, and three matching full‐thickness 2 × 2‐cm skin defects were created with a scalpel on the left side of the dorsal midline, 2 cm apart from each other (Figure 1). Sterile non‐adherent bandage pads were used to cover each wound, and a cotton roll gauze was wrapped around the trunk, crisscrossed in front of the forelimbs. Elastic bandage was used as a tertiary bandage layer. Elizabethan collars were fitted on the dogs after surgery to prevent them from biting and licking the wounds. The bandages were replaced every 2 days and monitored at least three times per day for integrity until the end of the experiment using sterile gloves, instruments and bandage material. Butorphanol (0.2 mg/kg IV) was provided for analgesia during the initial 24 h after surgery at intervals of 4–6 h as needed. Dietary formula after surgery was the same as before.

FIGURE 1.

FIGURE 1

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Diagram of wound creation and sample collection. Three matching full‐thickness 2 cm × 2‐cm skin defects were created on the left side of the dorsal midline, 2 cm apart from each other. The wounds were excised in a cranial‐to‐caudal sequence each week (shown in blue).

Wounds were photographed with a digital single‐lens reflex camera during each bandage change. To determine the healing percentage, we estimated the average diameter of the remaining open wound area using wound tracing software (ImageJ; National Institutes of Health, Bethesda, MD).

2.4. Histopathological examination

The first (cranial), second (middle) and third (caudal) wounds were used for histological examination on days 7, 14 and 21, respectively. The wounds, along with a 2–3‐mm diameter area encompassing the surrounding skin, were excised for histological examination. The surgical procedure was performed under the same conditions used to create the wounds. The wound samples were fixed using 4% paraformaldehyde for 12 h, rinsed with water for 30 min, and then dehydrated using the following sequence of solutions: 75% ethanol for 6 h, 85% ethanol for 10 h, 95% ethanol for 4 h, anhydrous ethanol I and III for 2 h, anhydrous ethanol II for 2 h, xylene I for 20 min and xylene II for 15 min. Thereafter, they were immersed in paraffin for 3 h and embedded in paraffin. The tissues were cut into 5‐μm thick slices with a Leica RM2235 microtome (Leica Microsystems) and were subsequently mounted on slides. The slides were then baked at 60°C for at least 2 h. Finally, the sections were stained with hematoxylin–eosin and Masson's trichrome and analysed using an optical microscope (Olympus BX51; Olympus) at a magnification of ×100.

2.5. Immunohistochemistry staining

Tissue sections with a thickness of 4 μm were baked at 60°C for at least 2 h. Paraffin sections were soaked in fresh xylene, anhydrous ethanol, 95% ethanol and 75% ethanol. After a blocking step, the primary antibody against TGF‐β1 (AF1027, Affinity Biosciences) was added at a 1:100 dilution and incubated overnight at 4°C. After washing, the tissue was incubated at room temperature for 10–15 min in the presence of biotin‐labelled secondary antibody (histochemical secondary antibody kit 19149A01, Beijing Zhongshan Jinqiao Biotechnology Co., LTD.), washed again, and incubated under the same conditions in the presence of horseradish enzyme‐labelled streptomycin working solution. DAB colour rendering solution was finally added, and the tissue was further incubated for 5–8 min. The slices were also stained with hematoxylin staining solution for 20 s at the end of the staining protocol. Imaging of the stained slides was performed using a confocal laser scanning microscope (Leica Microsystems (Shanghai) Co., LTD.) at a magnification of ×800.

2.6. Statistical analysis

Statistical analysis was performed using SPSS (version 22.0; IBM). Differences between the two groups were analysed using one‐way analysis of variance. Statistical significance was established at p < 0.05.

3. RESULTS

3.1. COS dietary supplementation accelerated wound healing after skin trauma in dogs

The wound changes were observed on day 0, 2, 4, 7, 9, 11, 14, 16, 18 and 21. Figure 2A shows representative images of the full‐thickness excisional wounds 0, 4, 7, 11, 16 and 21 days after surgery. Wound shrinkage in the 0.5% COSs group was significantly faster than that in the control group (p < 0.01). On day 16, the wounds in the 0.5% COSs group were almost healed. Wound healing in 1% COSs group was at an intermediate level compared to that in the other two groups. Figure 2B shows that there was a steep decrease in the wound area in all three groups, and this was particularly evident in the case of 0.5% COSs group: The total wound area relative to the initial size in this group was 74% on day 4 and 5% on day 14. The wound had disappeared by day 18.

FIGURE 2.

FIGURE 2

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(a) Representative images of full‐thickness excisional wounds at 0, 4, 7, 11, 16 and 21 days after surgery. (b) Results of wound area quantification using image analysis software. The size recorded on each measurement is compared with the size of the wound area on day 0. Values are expressed as mean ± standard deviation. *p < 0.05 and **p < 0.01, compared with control.

3.2. Cytological examination confirmed more efficient wound healing in 0.5% COSs and 1% COSs groups

Hematoxylin–eosin staining was used to histologically assess wound healing. Figure 3 shows representative histological images from tissue slices obtained on days 7, 14 and 21 after the induced trauma. At the incipient stage of the healing process (days 0–7), there was no proliferation of epidermal cells in most of the skin samples, and bleeding and inflammatory cell infiltration were evident. Fibrous connective tissue collagen fibre hyperplasia under the wound was obvious, and multiple new capillaries were observed perpendicular to the wound surface, mainly forming granulation tissue. In the control group, the epidermis was exfoliated, the wound was infiltrated by numerous inflammatory cells, and it showed no obvious signs of healing. However, the 0.5% COSs and 1% COSs groups showed obvious hyperplasia of skin epithelioid cells covering the wound surface. At the middle stage of the healing process (days 8–14), the epidermal layer was still missing in the control group and many inflammatory cells remained in the area. Wound repair was minimal. In contrast, epithelioid cells had obviously proliferated in the 0.5% COSs and 1% COSs groups, generating epidermis that covered the surface of the wound; dermal fibre tissue grew perpendicular or parallel to the wound after reconstruction, and few inflammatory cells, abundant fibrous tissue, and new capillaries growing perpendicularly were observed in the deep layers. At the last stage of the healing process (days 15–21), the epidermis was still not completely repaired in the control group, but it had completely covered the wound surface in the 0.5% and 1% COSs groups, the subcutaneous collagen fibres had been reconstructed, and a small number of inflammatory cells could still be observed in the wound area.

FIGURE 3.

FIGURE 3

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Histological analysis of the wound healing process on days 7, 14 and 21 after surgery. Hematoxylin–eosin staining shows collagen fibres in pale pink, cytoplasm in purple, nuclei in blue and erythrocytes in cherry red. The stratum basale (sb), stratum corneous (sc), stratum granulosum (sg), stratum spinosum (ss), sebaceous gland (seb), hair follicles (hf) and blood vessels (bv) are indicated by black arrows (scale bar = 250 μm).

3.3. Collagen synthesis and TGF‐β1 expression were enhanced in 0.5% COSs and 1% COSs groups

Collagen synthesis plays a major role in wound healing. Hence, we stained the skin tissue with Masson's trichrome (Figure 4A) to evaluate the effect of dietary supplementation with COSs on collagen synthesis. The intensity of the staining of the deposited collagen fibres (in blue) is positively associated with wound healing. The results showed that there was a steady increase in collagen deposition in all experimental groups, but it was significantly higher in the 0.5% COSs (p < 0.05) and 1% COSs (p < 0.01, p < 0.05) groups than in the control group on days 7 and 21. On day 14, collagen deposition in the 0.5% COSs group (p < 0.05) was significantly higher than in the control group (Figure 4B).

FIGURE 4.

FIGURE 4

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(a) Representative images of biopsy tissue from each group on days 7, 14 and 21 after surgery, stained with Masson's trichrome (scale bar = 50 μm). Collagen fibre appear as blue–green, whereas cell cytoplasm, nuclei and erythrocytes are stained red. (b) Mean optical density of collagen fibres discriminated by group on days 7, 14 and 21. Values are expressed as mean ± standard deviation. *p < 0.05 and **p < 0.01 vs. control.

As TGF‐β1 is crucial for collagen synthesis, we evaluated the expression of this cytokine using immunostaining (Figure 5A). The results showed that during the incipient stage of the wound healing process, the expression of TGF‐β1, estimated through the optical density of the immunostaining, rose to a high level and then gradually decreased (Figure 5B). TGF‐β1 expression in the 0.5% COSs group (p < 0.01, p < 0.05) was significantly higher than that in the control group at all timepoints, and this was also the case for the 1% COSs group (p < 0.05) on days 7 and 14.

FIGURE 5.

FIGURE 5

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(a) Representative images of transforming growth factor‐β (TGF‐β1) immunostaining of wound tissue on days 7, 14 and 21 after surgery (scale bar = 100 μm). TGF‐β1 staining appears brown. (b) Mean optical density of TGF‐β staining discriminated by experimental group on days 7, 14 and 21. Values are expressed as mean ± standard deviation. *p < 0.05 and **p < 0.01 vs. control.

4. DISCUSSION

Skin plays a pivotal role in the maintenance of physiological homeostasis and in the protection against mechanical forces, infections, fluid imbalance and thermal dysregulation (Sorg et al., 2017). Wound healing is particularly important for the maintenance of skin integrity after trauma. Many factors may lead to inadequate wound healing, resulting in infections, thermal dysregulation and fluid loss that may require medical intervention (Landén et al., 2016). Therefore, there has been sustained interest in the development of a biomedical material capable of accelerating the healing process without inconveniencing the patient. Previous studies have underlined the suitability of COSs for this purpose as they reportedly have antibacterial, antifungal, antiviral, antitumor, anti‐oxidation and immune‐regulating properties (Wang et al., 2015). The present study was designed to determine the effect of dietary supplementation with COSs on wound healing, and the results provide morphological, cytological and molecular evidence indicating that COS supplementation can accelerate skin repair after trauma.

We found that dietary supplementation with COSs had a strong effect on wound closure and remodelling. A visual analysis indicated that, in comparison with a control group, 0.5% COSs achieved complete wound closure in 16 days. These results are in agreement with those of recent studies indicating that COSs, as natural polymers with attractive biological activities, can promote wound healing (Chandika et al., 2021; Li et al., 2016; Zhang et al., 2019).

Histopathological examination revealed that, at incipient stages of the healing process (days 0–7), epithelioid repair began to occur in subjects that had received COSs as a dietary supplement, but not in the control group. At the middle stage of the healing process (days 8–14), obvious reduction in wound size had taken place in subjects that had been fed with a COS‐containing diet and the wound was completely covered by epidermal cells, whereas in most subjects in the control group, an open wound was still present at this stage. Finally, at the last stage of the healing process (days 15–21), the epidermis had covered the wounds in the two COSs treatment groups, but not in the control group. In summary, dietary supplementation with 0.5% and 1% COSs effectively accelerated skin repair after wound infliction. As collagen is the main protein involved in wound healing, its synthesis is increased in response to a skin wound to help repair it and return it to its normal structure and function (Corsetti et al., 2017). The results of Masson's trichrome staining further support the notion that supplementation with COSs could be useful for accelerating skin repair after a wound.

The TGF‐β superfamily, especially TGF‐β1, is essential for wound healing (Isenberg et al., 2005). As a main regulator of extracellular matrix synthesis and remodelling, TGF‐β1 induces fibroblast differentiation into myofibroblasts, thus initiating the processes of inflammation, granulation tissue formation and wound contraction (Gabbiani, 2003). Furthermore, TGF‐β1 promotes collagen synthesis and keratinocyte migration during wound closure (Abarca‐Buis et al., 2021; Ling & Robinson, 2002). The relevance of TGF‐β1 signalling for wound healing has been demonstrated using two experimental models: TGF‐β1‐deficient mice (Brown et al., 1995) and mice treated with a neutralizing anti‐TGF‐β1 antibody (Shah et al., 1999). To determine the relationship between COSs and the TGF‐β1/Smad signalling pathway in the process of wound healing, Chen et al. conducted in vivo experiments using male Sprague–Dawley rats with COS‐containing nanofibres. The results showed that COS‐containing nanofibres promote wound healing by upregulating cytokines associated with the TGF‐β1/Smad signalling pathway (Li et al., 2016; Zi‐Wei et al., 2017). The increase in the expression of TGF‐β1 in response to COS administration that we reported in this study corroborates these earlier findings. Thus, dietary COS supplementation promotes TGF‐β1 expression and regulation during the entire wound healing process, reducing the time needed for healing and improving the quality of wound repair.

5. CONCLUSIONS

This was the first study to describe the effects of dietary supplementation with COSs on wound healing. The results of this study demonstrate that, in addition to its topical application, oral administration of COSs is also effective in promoting wound healing. The use of synthetic polymers has gradually been replaced by biodegradable polymer alternatives (Mahata et al., 2014). Our findings shed new light on wound healing and have many important implications for future practice. We hope that this study will contribute to a deeper understanding of the process of wound healing and to the further development of effective drugs or food additives for animal and human use.

Many studies have been done on the effects of COSs on the wound healing process. And It was demonstrated that the effect of COSs, which was administered intragastrically, on the wound healing process can be accelerated via higher proliferation of fibroblasts in mice (Lv et al., 2019). In addition, COSs have been used to enhance cartilage repair during treatments involving angiogenesis in vivo (Jafari et al., 2020; Wei et al., 2013). These studies suggest that the role of COSs is related to angiogenesis and fibroblast migration. In this study, the increase in the expression of collagen and TGF‐β1 in response to COS administration was demonstrated. These results further support the notion that supplementation with COSs could be useful for accelerating skin repair. But here you can see limitations in this study. The first one is small sample size. As mentioned above, we only used nine animals, three for each group. The second one is limited COSs groups (dietary supplementation with only 0.5% and 1% COSs) and no crossover between groups. So, in a follow‐up study, the above questions will be refined. Further, to claim and prove the effects of this compound orally, as well as the direct relationship between TGFβ, angiogenesis and fibroblast migration, the underlying mechanism will be elucidated.

AUTHOR CONTRIBUTIONS

Conceptualization; formal analysis and methodology: Guoqiang Cheng, Yu Zeng and Yongjin Wang. Data curation; validation and formal analysis: Jieying Xia and Xindong Lei. Writing – original draft: Dong Chen and Huaqiao Tang. Conceptualization; project administration; methodology; writing – review and editing: Yanglu Liu and Li Zhang. All authors have read and agreed to the published version of the manuscript.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

FUNDING INFORMATION

Science &Technology Department of Sichuan Province, Grant Number: 2022JDKY0013; Sichuan Provincial Administration of Traditional Chinese Medicine, Grant Number: Z‐2023N‐6

ETHICS STATEMENT

All procedures used in this study were approved by the Animal Care Advisory Committee of the Sichuan Academy of Chinese Medicine Sciences (SCACMS‐20180406).

PEER REVIEW

The peer review history for this article is available at https://publons.com/publon/10.1002/vms3.1359.

Cheng, G. , Zeng, Y. , Wang, Y. , Xia, J. , Lei, X. , Chen, D. , Tang, H. , Zhang, L. , & Liu, Y. (2024). Effect of dietary supplementation with chitooligosaccharides on wound healing in dogs. Veterinary Medicine and Science, 10, e1359. 10.1002/vms3.1359

Contributor Information

Li Zhang, Email: zhangli556@126.com.

Yanglu Liu, Email: nczxlyl@126.com.

DATA AVAILABILITY STATEMENT

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

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

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

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