Introduction
Breast cancer is the most common cancer in women, and its prevalence is increasing every year [1]. Accordingly, since total mastectomy is the standard procedure for treating breast cancer, autologous tissue or implants are widely utilized for breast reconstruction. When opting for autologous tissue preservation, implant reconstruction offers the distinct advantage of minimal scarring as there is no need for additional incisions. As a result, the increasing use of implants in breast reconstruction has led to a growing focus on developing techniques to minimize complications such as capsular contracture, with various studies currently underway [2].
Delayed breast reconstruction using implants was first introduced in 1961, after which immediate reconstruction techniques were reported, inserting implants directly under the chest wall skin flaps after mastectomy [3,4]. However, this subcutaneous implant insertion technique led to numerous complications such as implant exposure, capsular contracture, and implant displacement. Later, the submuscular implant insertion technique was introduced, where the entire implant was placed under the pectoralis major muscle. This method also presented several issues, prompting the development of partial muscle coverage or dual plane approaches to overcome its drawbacks.
With the development of implant technology and the advent of acellular dermal matrix (ADM), an improved dual plane surgical method was devised. In various studies, ADM has been shown to reduce the rate of capsular contracture by downregulating the expression of myofibroblasts, fibroblasts, vascularity markers, macrophages, TGF-β1 (transforming growth factor beta-1), and PDGF-B (platelet derived growth factor subunit B) in capsular tissue, resulting in a more natural and aesthetically pleasing breast shape and providing a different layer of soft tissue coverage between the implant and the skin flap, thus increasing the success rate of breast reconstruction [3-5].
Notably, one study has investigated the therapeutic effects of adipose-derived stem cells (ADSCs) in improving immunological tolerance, where they mitigate host reactions to foreign bodies, which is one of the major causes of capsular contracture [6]. As ADSCs lack HLA-DR (human leukocyte antigen-DR), the main antigen of the major histocompatibility complex, they do not induce immune rejection even after allogeneic or heterogeneous cultivation and transplantation, effectively suppressing foreign body reactions that occur in capsular contracture [7]. In addition, ADSCs have been demonstrated to promote angiogenesis and help rapid attachment to the recipient tissue [8].
This study aimed to investigate whether the application of ADSCs to ADM could reduce the incidence of capsular contracture in a rat model of breast reconstruction. Specifically, we sought to evaluate the extent to which ADSCs can enhance the anti-inflammatory and angiogenic properties of ADM, thereby contributing to improved outcomes in implant-based breast reconstruction.
Methods
The animal experiment was conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) (Approval number: DGMIF-21081805-00).
Materials
Sprague-Dawley rats
A total of 26 Sprague-Dawley rats (female, 7 weeks old), each weighing over 200 g, were purchased from the Yeongnam branch of Orient Bio Inc. After a 1-week acclimation period, 12 rats were selected and divided into four groups of three rats each, ensuring equal mean weights.
Adipose-derived stem cells
Consent was obtained for the experimental use of ADSCs from a 44-year-old female donor scheduled for liposuction in the abdomen, with no underlying disease. Tissue containing adipocytes was harvested using a suction cannula connected to a 10 mL syringe, following the Coleman technique. The tissue obtained from the lower abdomen through liposuction was centrifuged at 3,000 rpm for three minutes to separate and remove oily fractions and the serum, and pure adipose tissue samples were extracted. The pure fat samples were digested with a collagenase solution at 37 °C for 1 hour, followed by neutralization with DMEM/F-12 medium. Subsequently, the solution was centrifuged at 300 ×g for 5 minutes to remove the supernatant, and the cell pellet was washed with phosphate buffered saline and centrifuged again. The washed cells were resuspended in DMEM/F-12 medium and seeded into flask, then incubated at 37 °C with 5% CO2. When the cells reached 80% to 90% confluence, they were treated with 0.25% trypsin-EDTA (ethylenediaminetetraacetic acid) to detach the cells for the next passage. A total of three passages were performed in this study. The cells were used for in-vivo animal experiments.
Methods
An animal model was established with 12 rats (7-week-old female Sprague-Dawley rats: range, 212.56–234.23 g; mean weight, 226.75 g). After 2 cm-long incisions were made at 4 cm from the base of the tail towards the head, along the vertebral center line of the rats, a silicone implant with a diameter of 2 cm and a height of 1 cm, specifically manufactured for animal experiments, was inserted. The experimental animals were divided into two groups as follows (Fig. 1): Group A, insertion of silicone implant and ADM; Group B, insertion of silicone implant and ADM with ADSC seeding.
Insertion of silicone implant
Following intraperitoneal anesthesia with Zoletil 50 (30 mg/kg; Virbac) and Rompun (10 mg/kg; Elanco), a silicone implant (2 cm in diameter, 1 cm in height; Hans Biomed Co.), specifically designed for animal experiments, was inserted.
Insertion of ADM
After insertion of the silicone implant, a square-shaped ADM (3 cm in width, 3 cm in length, and 2 mm thick; CGBio), manufactured for animal experiments, was inserted. The ADM was positioned to fully cover the anterior surface of the silicone implant and was subsequently secured in place using sutures with 4-0 nylon.
Evaluation
Visual evaluation
After transplantation, to examine capsule formation around the silicone implant and ADM, the experimental animals were euthanized, and the surgical site was incised again and photographed with a digital camera. The formation and thickness of the capsule were visually evaluated at weeks 4 and 8 into the experiment.
Capsular thickness
Capsular thickness was evaluated in hematoxylin and eosin (H&E)-stained slides, using a microscope at weeks 4 and 8 of the experiment. Capsule thickness was measured at five different locations in each specimen and mean values were calculated.
Histopathological evaluation
For histopathological evaluation, a random selection of three animals from each group at 4 and 8 weeks into the experiment were euthanized. Incisions were performed as previously described, and the silicone implant, ADM, and surrounding capsule were harvested. Subsequently, the capsule tissue harvested from center of the dorsal aspect was trimmed to obtain a tissue block through a series of processes: each block was stained with H&E stain and Masson’s trichrome stain, and fibrous tissue, granulomatous tissue, angiogenesis, and inflammatory reactions were observed using an optical microscope.
Reverse transcription-polymerase chain reaction
Using tissue blocks obtained at 4 and 8 weeks, the expression levels of vascular endothelial growth factor A (VEGFA), hypoxia-inducible factor 1-alpha (HIF-1α), tumor necrosis factor-alpha (TNF-α), matrix metallopeptidase-2 (MMP-2), and tissue inhibitor of metalloproteinase-2 (TIMP-2) were measured and confirmed by reverse transcription-polymerase chain reaction (RT-PCR). These genes were selected because they play crucial roles in angiogenesis, inflammation, and the formation of fibrotic tissue, all of which are key processes in capsular contracture development. VEGFA is involved in promoting angiogenesis, HIF-1α is associated with the response to hypoxia and fibrosis, TNF-α is a major pro-inflammatory cytokine, and MMP-2 and TIMP-2 are critical regulators of extracellular matrix remodeling.
Statistical analysis
Data analysis was performed using SPSS 22.0 (IBM Corp.), and an independent samples t-test was conducted. When performing the multiple t-tests, Bonferroni correction was applied to adjust the significance level, reducing the probability of errors from multiple comparisons. Capsule thicknesses were compared using the Mann-Whitney U test.
Results
To assess normal growth progression, the body weight of the Sprague-Dawley rats was measured weekly. During the experiment, the silicone implant of one rat in Group A was exposed; however, the rat survived until the end of the experiment. No obvious complications were observed in the other rats, and all of them showed normal growth. At the start of the experiment, the mean weight of Group A was 227.04±10.34 g, and 228.35±9.45 g in Group B. At 4 weeks, the mean weights of rats in Group A and Group B were 337.45±18.34 g and 323.83± 16.78 g, respectively. At 8 weeks, the mean weights of rats in Group A and Group B were 360.75±16.75 g and 349.41±15.77 g, respectively. No statistical difference was observed in the growth of the groups (Fig. 2).
Visual observation
In each group, three rats were euthanized at 4 and 8 weeks, and capsule samples were harvested. Visual observation confirmed that the capsule samples of rats in Group A were thicker than those of Group B (Fig. 3).
Capsular thickness
At 4 weeks, the mean capsular thickness was 177.16 μm in Group A and 170.76 μm in Group B. By 8 weeks, it was 196.69 μm in Group A and 176.10 μm in Group B (Fig. 4). The Mann-Whitney U test results showed no statistically significant differences in capsule thickness between the groups (P>0.05).
Histological findings
H&E staining
Capsule samples harvested from each group at 4 and 8 weeks were subjected to H&E staining and were observed under an optical microscope. More inflammatory cells were observed in Group A than in Group B both at 4 weeks and 8 weeks, and reduced angiogenesis was observed in Group A (Fig. 5).
Trichrome staining
Capsule samples harvested from each group at 4 and 8 weeks were stained with Masson’s trichrome stain and observed using an optical microscope. In comparison with Group B, Group A had a larger stained area of collagen fibers with greater density at weeks 4 and 8 (Fig. 6).
RT-PCR test
RT-PCR was performed using capsule samples harvested from each group at 4 and 8 weeks to measure gene expression levels. For VEGFA, the expression level in Group B was 14% higher at 4 weeks and 156% higher at 8 weeks compared to Group A (P=0.0321). For HIF-1α, the expression level in Group A was 2% higher at 4 weeks and 89% higher at 8 weeks compared to Group B (P=0.0266). For TNF-α, the expression level in Group A was 18% higher at 4 weeks and 348% higher at 8 weeks compared to Group B (P=0.0133). For MMP-2, the expression level in Group A was 23% higher at 4 weeks and 63% higher at 8 weeks compared to Group B (P=0.0193). For TIMP-2, the expression level in Group A was 53% higher at 4 weeks and 52% higher at 8 weeks compared to Group B (P=0.0433, P=0.0271) (Figs. 7, 8).
Discussion
During breast reconstruction, the placement of an implant typically triggers capsule formation, a critical component of the host’s response to foreign bodies. However, excessive capsule formation, commonly known as capsular contracture, can present with a variety of clinical symptoms that correlate with its severity. These symptoms include abnormal sensations, pain, breast shape deformation, and capsule hardening, causing significant discomfort to the patient [9]. Various theories exist on the causes, including the anatomical position of the implant, asymptomatic infection or inflammation, bacterial biofilm, and material properties of the implant surface [10]. Though many animal experiments and clinical studies have been conducted to identify the causes and mechanisms of capsular contracture, they remain unclear [11,12]. One widely supported hypothesis suggests that capsular contracture is related to a foreign body reaction, where an inflammatory response and collagen fiber deposition lead to excessive fibrosis in the capsule formed as a normal defense mechanism against the implanted foreign material [11,12].
ADSCs, which have recently attracted considerable attention, are relatively easy to harvest; patients report less discomfort during ADSC harvesting compared to the more invasive process of bone marrow stem cell extraction. Meanwhile, ADSCs are known to have different differentiation potencies depending on the method of harvesting; specifically, harvesting through direct excision or the Coleman technique with centrifugation can obtain the most ADSCs [13]. Harvested ADSCs exhibit immunomodulatory properties, but do not affect various immune cells including T cells, B cells, and antigen-presenting cells. Furthermore, they do not elicit immune rejection responses even after allogeneic or xenogeneic transplantation [14,15]. In addition, studies reported that ADSCs could reduce host reaction to foreign bodies, preventing capsular contracture [16,17]. For this reason, we hypothesized that when ADSCs are applied to ADM, they would suppress foreign body reactions and further enhance the capsular contracture inhibitory effect of ADM; we planned and conducted this study based on this hypothesis.
In this study, visual evaluation confirmed that capsule samples were thicker at 8 weeks in Group A, which was not treated with ADSCs. However, statistical analysis showed no significant difference in capsule thickness between Group A and Group B. Similarly, at 4 weeks, there was no statistically significant difference in capsule thickness between the two groups. H&E staining revealed that the degree of angiogenesis was lower in Group A than in Group B at 4 weeks and 8 weeks, and the number of inflammatory cells was higher in Group A than in Group B. This result may be attributed to the ability of ADSCs to suppress the immune process and preventing foreign body reactions. In addition, Masson’s trichrome staining demonstrated a higher density of collagen fibers in Group A than in Group B at 4 weeks and 8 weeks. Capsular contracture is characterized by the presence of dense, thick, cable-like bundles of collagen fibers. The study results suggest that ADSCs effectively inhibit the formation of these collagen fibers, which are the primary structural component of capsules [18]. This outcome may be associated with the inhibition of the inflammatory response.
RT-PCR was performed to quantitatively evaluate inflammatory reactions and angiogenesis. In comparison with Group A, Group B had a higher expression level of VEGFA, known as a prominent factor in angiogenesis. The higher expression level of VEGFA in Group B is consistent with the result of H&E staining showing more new blood vessels in Group B compared to Group A [19]. Meanwhile, the expression level of HIF-1α was lower in Group B than in Group A. HIF-1α is a protein expressed in the absence of oxygen, preventing cell death and contributing to the development and progression of fibrosis; several studies have found high levels of HIF-1α in the capsule samples of patients whose capsular contracture has progressed [20]. The lower expression level of HIF-1α observed in the ADSC group compared with the control group is consistent with the effectiveness of ADSCs in inhibiting capsule formation.
Next, the expression level of TNF-α was lower in Group B than in Group A. TNF-α is one of the main factors that induce an inflammatory response, and it is known that it promotes the formation of capsular contracture when its expression level increases [21]. It was confirmed that the expression level of TNF-α was significantly lower in Group B compared to Group A. This finding suggests that ADSCs may be effective in preventing capsular contracture and deterioration. Both MMP-2 and TIMP-2 showed lower expression levels in Group B than in Group A. While the precise mechanisms of MMP-2 and TIMP-2 in capsular contracture are not fully elucidated, these molecules are recognized as significant contributors to the development of this condition [22]. The significantly lower expression levels of both MMP-2 and TIMP-2 in the ADSC group suggest that ADSCs may contribute to the prevention of capsular contracture.
In this study, compared with the group that received only silicone implants and ADM, the group that received ADSCs as well showed a reduced inflammatory response, increased angiogenesis, and suppressed collagen fiber production. These results are consistent with the effects of ADSCs observed in previous studies. It is presumed that the reduced inflammatory response and suppression of collagen fiber production induce a decrease in the host reaction to foreign bodies, and increased angiogenesis leads to rapid attachment to the recipient tissue, effectively suppressing foreign body reactions, which are widely supported as a major cause of capsular contracture.
This study has several limitations. Primarily, the sample size of the experimental groups was limited. Although statistically significant results were obtained and the possibility of clinical applications was demonstrated, further experiments are warranted with a larger sample size to increase the significance of the results. Second, further research is needed regarding the number of ADSCs to be applied. Though 1.0×105 ADSCs were applied to each animal in this study, based on existing literature [11], it is necessary to increase the efficiency of treatment by optimizing the number of cells applied. In addition, the experiment should be conducted by harvesting samples according to the stage of capsular contracture following the Baker classification. Third, previous studies conducted on rat models have shown that capsular contracture can be observed and assessed within an 8-week period [23]. However, in clinical human studies, the observation periods typically range from 6 months to 1 year to fully evaluate the progression and severity of capsular contracture. Fourth, most markers observed in this study were involved in the acute inflammatory phase. There was a lack of evaluation of cells or markers related to the formation of fibrosis. Although the study’s initial background mentioned the direct suppression of immune cells, this evaluation was not performed.
According to Muehlberg et al. [24], the use of stem cells in animal experiments can induce or promote the progression of breast cancer. However, in another study, when stem cells were used clinically, there was no significant difference in the growth or recurrence of breast cancer [25]. Therefore, when using stem cells in breast reconstruction, studies on the progression and recurrence of breast cancer should also be conducted.
This animal study aimed to evaluate the efficacy of ADSCs in augmenting the preventive effects of ADM against capsular contracture during breast reconstruction with silicone implants. We were able to assess the histological changes and conducted a quantitative evaluation of factors influenced by ADSCs on capsular formation. The results demonstrated that ADSCs effectively suppressed inflammatory responses and inhibited the formation of collagen fibers, the main components of the capsule, while also promoting angiogenesis. These findings suggest that ADSCs could be sufficiently effective in preventing capsular contracture when applied clinically.
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