Epithelial Defect Repair in the Auricle and Auditory Meatus by Grafting with Cultured Adipose-Derived Mesenchymal Stem Cell Aggregate-Extracellular Matrix
Introduction
Surgical removal is the primary treatment for various ear conditions, including congenital and acquired concha atresia deformity, external auditory canal malignant tumors, and chronic suppurative attic-antral disease in the middle ear cleft. However, these procedures often result in concha skin defects and exposure of the temporal bone mastoid cavity to the external bone surface. The complete healing of these wounds depends on the epidermal level of the wound. Delayed epithelialization can lead to long-term otorrhea, superinfection, and granulation formation, which further impede epithelial healing and negatively impact the patient’s quality of life. Clinical studies have shown that in nearly one-third of patients, epithelialization is either absent or occurs too slowly before the onset of infection.
Various techniques have been employed to address these issues, including the use of thick skin grafts, temporal muscle fascia-periosteum flaps, bone pate, and beta-tricalcium phosphate. However, none of these methods have achieved satisfactory results. Skin tissue engineering techniques, which involve the application of various cells, scaffolds, and growth factors, have been explored to accelerate skin regeneration. These techniques have shown promise in controlling otorrhea in several centers, but they also present significant disadvantages, such as long waiting periods, difficulty in handling brittle and thin cell sheets, low success rates after transplantation, and low vascularization.
To overcome these challenges, this study investigates the use of adipose-derived mesenchymal stem cell (ADMSC) sheets combined with extracellular matrix (ECM) scaffolds (referred to as ADMSC aggregate-ECM) to treat full-thickness defects in a rabbit skin injury model. ADMSCs have been shown to secrete various tissue-healing promoters and growth factors, exhibit minimal immune response in allogeneic hosts, and expand easily in vitro, making them a promising candidate for therapeutic tissue engineering. ECM scaffolds, which have been successfully used for wound repair in preclinical animal studies and human clinical applications, provide a supportive medium for cell growth and promote vascularization in vivo.
Methods
Ethical Approval
The study was conducted in accordance with the Declaration of Helsinki and was approved by the Animal Use and Care Committee of the Fourth Military Medical University (License number: SCXK 2007-007).
Isolation and Culture of ADMSCs
Adipose tissue was obtained from the inguinal region of two-week-old New Zealand white rabbits. The tissue was washed extensively with phosphate-buffered saline (PBS) and digested with collagenase type I. The digested tissue was then filtered, centrifuged, and resuspended in a-minimum essential medium (MEM) supplemented with fetal bovine serum (FBS), glutamine, penicillin, and streptomycin. The cell suspension was placed in T75 flasks and incubated at 37°C in a humidified chamber containing 5% CO2. After reaching 80% to 90% confluency, the cells were harvested with trypsin and sub-cultured.
Characterization of ADMSCs
The phenotype of cultured ADMSCs was evaluated using flow cytometry. Cells at passage five were incubated with fluorescein isothiocyanate-conjugated antibodies against CD14, CD90, CD29, and CD45. The proliferation of ADMSCs was quantified using the MTT assay. Adipogenic and osteogenic differentiation assays were performed to confirm the multipotency of the ADMSCs.
Preparation of ADMSC Aggregate-ECM
ECM scaffolds were cut into 15mm pieces and placed in six-well plates. ADMSCs were seeded onto the scaffolds and incubated under standard culture conditions. The ADMSC aggregate-ECM was examined using scanning electron microscopy (SEM) and confocal laser scanning microscopy.
Effect of Scaffold on Growth Factor Secretion by ADMSCs
Real-time polymerase chain reaction (q-PCR) was used to analyze the mRNA expression of transforming growth factor beta (TGF-b), hepatocyte growth factor (HGF), and epidermal growth factor (EGF) by ADMSCs cultured on ECM. Western blotting was performed to evaluate the protein expression levels of these growth factors.
Full-Thickness Cutaneous Wound Models
Full-thickness cutaneous wounds were created in the ear auricle and auditory meatus of New Zealand white rabbits. The rabbits were randomized into three treatment groups: ADMSC aggregate-ECM, ECM, and control. Wound healing was assessed by general observation, hematoxylin and eosin (H&E) staining, and measurement of the scar elevation index.
Results
Characterization of ADMSCs
Rabbit ADMSCs exhibited a long spindle shape and a radial arrangement. Flow cytometry analysis confirmed the expression of mesenchymal stem cell markers CD90 and CD29, while the monocyte marker CD14 and hematopoietic cell marker CD45 were negative. The cells showed exponential growth, reaching a peak at 5 to 6 days. ADMSCs differentiated into osteoblasts and adipocytes under appropriate differentiation conditions.
Characteristics of Scaffolds and Effects of Scaffolds on Proliferation of ADMSCs
SEM micrographs showed that the ECM scaffold had an imperforate bottom with widely interconnected collagen bundles. ADMSCs adhered tightly to the ECM and formed cell sheets rapidly. The percentage of ADMSCs in the S phase was higher in the ECM group compared to the control group. q-PCR results indicated that the expression levels of TGF-b, EGF, and HGF were increased in ADMSCs cultured on the ECM.
Repairing Rabbit Skin Defects of Auditory Meatus
At 4 weeks post-surgery, the ADMSC aggregate-ECM group showed nearly complete skin formation consistent with the surrounding tissue, while the ECM and control groups showed partial skin formation. H&E staining revealed a larger range of normal skin-like structure in the ADMSC aggregate-ECM group compared to the ECM group. Western blot results showed higher levels of bFGF, EGF, HGF, VEGF, and TGF-b1 in the ADMSC aggregate-ECM and ECM groups compared to the control group.
Repairing Rabbit Skin Defects of Ear Auricle
At 2 weeks post-surgery, the wound healing rates in the ADMSC aggregate-ECM (69.02±6.36%) and ECM (59.32±4.10%) groups were higher than that in the control group (43.74±12.15%). At 7 weeks, the scar elevation index was significantly lower in the ADMSC aggregate-ECM (2.08±0.87) and ECM (2.31±0.33) groups compared to the control group (4.06±0.45). The ADMSC aggregate-ECM group reached the lowest scar elevation index 4 weeks in advance.
Discussion
The findings of this study demonstrate that ADMSC aggregate-ECM and ECM are effective materials for promoting rapid skin regeneration in the ear auricle and external auditory canal of rabbits. ADMSC aggregate-ECM exhibited superior performance, particularly in reducing scar formation and promoting normal skin-like structure. The ECM scaffold provided a supportive medium for ADMSC survival and proliferation, while ADMSCs secreted growth factors that contributed to tissue repair and remodeling.
Previous studies have explored the use of autologous-cultured keratinocyte layers, autologous epithelial layers from the buccal epithelium, and mucosal cell sheets for promoting the regeneration of external auditory canal and middle ear skin or mucosa. However, these methods present significant challenges, such as the difficulty in obtaining sufficient normal tissue for culture and ethical concerns related to the decomposition of normal nasal and buccal mucosal tissue. The use of ADMSC aggregate-ECM offers a promising alternative that addresses these issues while providing a supportive environment for cell growth and tissue repair.
The ECM scaffolds used in this study were derived from porcine peritoneum and processed to remove resident cells while retaining the integrity and ratios of the collagen structure. These scaffolds supported the adherence, proliferation, and secretion of growth factors by ADMSCs, creating a favorable environment for tissue regeneration. The results of q-PCR and Western blotting indicated that the ECM scaffold enhanced the expression of growth factors such as TGF-b, EGF, and HGF, which are essential for wound healing and tissue remodeling.
In conclusion, this study demonstrates the potential of ADMSC aggregate-ECM as a graft material for wound healing in the auricle and auditory meatus. The combination of ADMSCs and ECM scaffolds provides a supportive environment for cell growth and tissue repair, promoting rapid skin regeneration and reducing scar formation. These findings offer a meaningful experimental basis for the development of new therapeutic approaches for mastoid epithelium repair in clinical trials.
doi.org/10.1097/CM9.0000000000000125
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