Abstract

Introduction: Deer antlers are remarkable organs as they can regenerate seasonally and leave no scars. Antler-derived stem cell therapy applications are of increasing interest in food, beauty, and medicine.

Method: Antler stem cells (ASCs) were isolated from antlers, and their expression of markers CD73, CD90, CD105, Nanog, and Oct4 was detected by PCR. Their capacity to differentiate into osteoblasts, chondrocytes, and adipocytes was assessed through culture in selection media. In vitro evaluations included wound healing and angiogenic effects using scratch and tube formation assay, respectively. For in vivo assessment, a rat skin defect model was established surgically, and ASC mixtures were applied to the defective skin at a dose of 100μg/100μl. Treatment effects were also evaluated through histological analysis, immunohistochemical staining, and the expression of genes involved in wound healing (including TGF-B1, TGF-B3, TIMP1, Col1, Col3, MMP1, and MMP3) using qRT-PCR method.

Results: The results indicated that ASCs exhibited fibroblast-like cell shapes and positively expressed specific stem cell markers (CD73, CD90, CD105, Nanog, Oct 4). ASCs demonstrated the capability to differentiate into osteoblasts, chondrocytes, and adipocytes. The products derived from ASCs significantly enhanced NIH-3T3 cell proliferation and stimulated angiogenesis in HUVEC on Matrigel compared to the control group. In the rat skin defect model, wounds treated with the ASC mixture were quicker to heal, starting from day 8, compared to the sham group (day 16). The wound area treated with the ASC mixture was significantly smaller than the control group on day 4 (p < 0.05). Furthermore, the gene expression ratio of TGF-β3/TGF-β1, MMP1/TIMP1, and MMP3/TIMP1 was significantly increased in the ASC group compared to the sham group (p < 0.05).

Conclusion: This study highlights the robust wound-healing efficacy of ASC-derived products.

Introduction

Deer antler velvet is a prized repository of medicinal herbs, extensively used to produce food, nutraceuticals, and functional foods. In various Asian cultures, antler velvet holds a significant place in traditional medicine due to its perceived multitude of beneficial biological properties, including robust antioxidant capacity, anti-arthritis and anti-osteoporosis benefits, and its potential for treating reproductive disorders in women1, 2.

Exclusive to male deer, antlers are regarded as secondary sexual characteristics that undergo rapid growth during the summer. In the natural environment, antler velvet demonstrates a unique capacity to regenerate without scarring or inducing fibrosis. The growing tip, a critical region dictating antler growth, harbors a substantial concentration of stem cells1, 3.

Researchers have long been able to isolate stem cells from antler velvet and culture them in the laboratory for extended periods4. Consequently, the characteristics of antler stem cells (ASCs) have been comprehensively investigated. ASCs express mesenchymal stem cell markers (including CD73, CD90, and CD1055) as well as embryonic stem cells such as Oct4, Sox2, and Nanog6.

In recent years, components derived from antler velvet, notably stem cells and extracts, have garnered significant interest in research and application3. Injecting ASCs into the tail veins of mice with damaged skin demonstrated substantial healing effects without detectable scarring in the lesion area7. Kmiecik et al. (2021) treated 20 patients with foot venous ulcers using ASC extract, significantly reducing the area/circumference of the lesion8.

Yang et al. showcased the safety and efficacy of ASC extract in regenerating hair follicles: application to damaged mouse skin led to a significant increase in the number of hair follicles9. Notably, even using conditioned medium from ASCs in treating injured skin displayed positive regeneration outcomes in rats1, 10. These findings underscore the therapeutic efficacy of ASCs, demonstrating positive effects across diverse species. They suggest the potential for utilizing ASCs or ASC-derived secretions in xenotransplantation or other cosmetic applications.

In this study, ASCs were successfully isolated and characterized. The cells were verified to possess self-renewing capabilities and the capacity to differentiate into various cell lines. Products formulated with ASCs were assessed for their wound-healing properties both in vitro and in mouse models. The efficacy of these ASC-derived products was compared with a control group using DMEM/F12 media. The findings indicate that ASC secretions and extracts effectively treat lesions without scarring, showcasing significant potential in cell-free therapy.

Materials and methods Antler stem cell isolation

Fresh velvet from sika deer, Cervus nippon, was obtained from farmers in Dong Nai, Vietnam. It was collected by cutting and refrigerated during transportation to the laboratory. Biopsies were performed approximately 0.5–1.0 cm from the velvet tip to target the growth zone. The velvet tissue was dissected by making two perpendicular lines at a quarter of the velvet’s length, intersecting at the apical region. The collected tissue, comprising the velvet skin and dermis, was promptly placed into a sterile tube containing PBS supplemented with 5X penicillin/streptomycin. Tissue processing was carried out within 2 hours of the biopsy. The tissue samples were finely chopped into pieces measuring 0.5–1 mm3 using a sterile scalpel. Subsequently, each piece was incubated in an MSCCult Animal medium (Regenmedlab, Ho Chi Minh City, Viet Nam). The cells were cultured in T25 flasks at 37°C with 5% CO2.

Antler stem cell characterization

The expression of specific genes, including CD73, CD90, CD105, Oct4, and Nanog, was assessed using PCR after extracting total RNA with an Easy Blue Total RNA Extraction Kit. The PCR was performed using a Luna Universal One-Step RT-qPCR Kit and a real-time PCR device. Gel electrophoresis with ethidium bromide staining was utilized to visualize the target gene bands. The primers used for these evaluations are detailed in Table 1.

Table 1.

Primer sequences for ASCs evaluation

No Name Primer Sequence (5'-3') Size (bp) Accession Number 1 GAPDH F GGCGTGAACCACGAGAAGTATAA 119 XM_020902047.1 R CCCTCCACGATGCCAAACT 2 Nanog F CACCCTCGACACGGACACT 283 XM_043882000.1 R CTGCTTGTAGCTGAGGTTCAA 3 Oct4a F GTGGAGGAAGCTGACAACAA 352 XM_043907988.1 R AGCCTGGGGTACCAAAATG 4 CD73 F GGTCAAAGGTGCCTCCAATG 353 XM_043901016.1 R CAATCCCATTCTTCTCAACAGC 5 CD90 F TCAGCCTGACAGCCTGCCTG 334 XM_043901026.1 R CTTATGCCCCCACACCTGAC