Pathological characteristics of breast nodules after large‐volume fat grafting for breast augmentation

The complications of large‐volume fat grafting (LVFG) for breast augmentation remain unpredictable and include palpable breast nodules, oil cysts, and calcifications.


| INTRODUC TI ON
In recent years, autologous fat grafting has been widely used in cosmetic and reconstructive plastic surgery. 1 In addition, as the harvesting and processing of aspirated adipose tissues become easier and safer, 2 an increasing number of people are willing to apply large-volume fat grafting (LVFG) to the breast.However, complications remain unpredictable, including palpable breast nodules, oil cysts, and calcifications. 3east nodules caused by fat necrosis have been frequently reported. 4,5It has been reported that new nodules unexpectedly occur with an incidence of 11.0%, 4 which has led to dissatisfaction for both patients and plastic surgeons.Fat necrosis manifests as nonviable adipose cells from injured or ischemic breast tissue that presents as a palpable nodule. 5Nevertheless, it remains unclear how breast nodules form and how surgeons can remove them in a minimally invasive manner.
Vacuum-assisted breast biopsy (VABB) system allows for a larger tissue sample to be obtained from a single minimally invasive procedure, 6 and it has been used for diagnostic biopsy and the treatment of gynecomastia. 7In recent years, an increasing number of surgeons have applied the VABB system to resection of breast nodules after augmentation by LVFG. 8Compared to the traditional methods that expose the skin incision to treat breast nodules, the use of the VABB system requires a very small skin incision of approximately 5 mm and ensures complete resection with no exposure to air.
In this study, we effectively used the VABB system for patients with breast nodules after LVFG and characterized the histological changes in collagen in the breast nodules.This study may have important clinical implications for treating unexpected complications in a minimally invasive manner and relieving pain in patients.Furthermore, we innovatively revealed the collagen characteristics of breast nodules, which may provide a potential target for regulating the development of fibrosis.

| Study sample
Twenty-nine female patients (Table 1) with palpable subcutaneous nodules or who were informed by the doctor of the existence of nodules during physical examination from December 2019 to May 2022 were recruited retrospectively at our department for this study.All of them had undergone breast augmentation by LVFG in unknown cosmetic surgery clinics.Ultrasonography was performed preoperatively.And then the breast nodules were categorized by the Breast Imaging Reporting and Data System (BI-RADS).

| Procedure of treatment using the vacuum-assisted breast biopsy system
Under the guidance of an ultrasound device, we performed the VABB system (ENCOR ENSPIRE; SenoRX Inc) for the patients.And then the locations of the breast nodules were marked preoperatively.When local anesthesia was injected into the excision area, a 5-mm skin incision was then made, positioned inferior or lateral to the identified nodules.Under ultrasound monitoring, the nodule was sequentially removed with little adhesions of the surrounding tissues.After the ultrasound scan confirmed the absence of residual nodules, the procedure was completed.

| Histological assessment and immunohisto chemical staining
The excised breast nodules were fixed in 4% paraformaldehyde for histological evaluation and immunohistochemical staining.Briefly, the samples were embedded in paraffin after fixing at room temperature for 48 h and then cut into 4μm sections serially.For histological evaluation, tissue sections were then dehydrated in alcohol (70% for 1 min, 96% for 2 min, and 100% for 2 min) and in (Thermo Fisher Scientific).Expression levels were calculated by the 2 −ΔΔC t method using GAPDH as normalization.

| Statistical analysis
Statistical analyses were conducted using GraphPad Prism 9 (Version 9.2.0).All data are expressed as mean ± standard deviation (SD).
Student's t-test was used to compare the two groups.Differences were considered statistically significant at p < 0.05.

| Complete resection of breast nodules via a minimal skin incision using the VABB system under ultrasound guidance
All 29 patients with multiple nodules underwent ultrasound-guided needle biopsy in our department.Briefly, the subcutaneous nodules were removed surgically using the VABB System under the guidance of ultrasound (Figure 1A, Figure S1 and Video 1).All of the nodules were located in the posterior mammary gland.These excised nodules appeared stripe-shaped and the sizes of the nodules ranged from 1.7 to 3.8 cm (Figure 1C).Some of them also have muscle tissue, with the nodules tightly adherent to the pectoralis major muscle.After ultrasound confirmed that the nodules were completely removed, they were sent for pathology testing and histologic examination.Nearly all patients recovered well and only one patient experienced postoperative hematoma.The overall treatment effect was satisfactory (Table 1).

| Deposition of Type I, III, IV and VI collagens in breast nodules
The

| Immunofluorescence staining of breast nodules
To explore the reason for collagen production in some fibrotic areas, we performed immunofluorescence staining to study the relationship between collagen and inflammatory cells (Figure 4).
The result shows mac2+ macrophages surround around large oil droplets and form crown structure.Furthermore, we found that the type VI collagen+ area appeared around mac2+ macrophages (white arrows) and α-SMA+ myofibroblasts (white asterisks).This strong colocalization may indicate a possible direct relationship between macrophages, myofibroblasts, and type VI collagen formation.

| DISCUSS ION
The first report of autologous breast fat grafting dates to 1987 at the time when Bircoll used fat injection for breast augmentation. 9 Nevertheless, the American Society of Plastic and Reconstructive Surgeons (ASPS) banned fat grafting for breast augmentation because it could interfere with breast cancer detection.Subsequently,

Spear et al. and Coleman reported surprising effects of autologous
fat injection to correct contour deformities. 10,11Since then, a variety of fat grafting techniques have been developed and applied clinically with good results, including the 3M3L technique, 12 cellassisted lipotransfer, 13 nanofat grafting 14 and SVF gel injection. 15wever, breast augmentation by LVFG is sometimes accompanied by postoperative complications, including oil cysts, calcification and palpable breast nodules.These problems directly influence surgical efficacy and may result in unsatisfied outcomes. 16minimally invasive biopsy system is an effective tool for thoroughly excising benign breast lesions with no open surgical incision, a low infection rate, and high patient comfort.The advantage of using a negative pressure suction system is that the closed system can accurately locate nodules, and the negative pressure can suck out the liquid in oil cysts.In 2019, breast surgeons first reported the application of the VABB system to resect breast nodules after autologous fat grafting and achieved a satisfactory therapeutic effect. 8However, plastic surgeons need to focus more on the fat injection skills, adequate injection volume and injected anatomic plane. 17We should be alert to the potential complications and accept more training to be skillful in advanced treatment methods such as breast ultrasound detention and the VABB system.In our study, we found all of the breast nodules were located in the posterior mammary gland, which indicates that the posterior mammary gland may not be a wonderful place for adipocytes' survival and regeneration.Relatively speaking, the posterior part of the pectoralis major muscle may be the preferred plane for fat injection which provides volume and projection. 17We are preparing to publish experimental studies in this regard (unpublished data).When performing autologous breast fat injection, experienced and well-trained plastic surgeons are required to evaluate the reasonable injection volume, take control of the injection plane and master appropriate methods to prevent complications to the most extent after LVFG.
After autologous fat injection into the breast, necrosis and nodules sometimes may occur unexpectedly, which was histologically defined as fibrosis and calcification. 18Fibrosis is characterized by the thickening and scarring of tissues during pathological repair. 19This may be because that after LVFG, the adipose tissues need to be adapt to the hypoxic and ischemic environment, 20 and then the inflammatory cells such as neutrophils 21 ， macrophages 22 and fibroblasts infiltrate in the inner area of the grafted adipose tissue, which may be responsible for the formation of oil cysts and nodules.Eventually, fibrosis develops, with inflammatory infiltration and collagen deposition.It is conceivable that if the volume of fat injected in a single plane is too large, adipose tissues may not easily survive, resulting in oil cysts and fat necrosis; if the fat is injected in a multi-plane, multitunnel, multi-point manner into the breast, adipose tissues are more likely to survive and regenerate, which may result in less tissue fibrosis. 12In the future, large-mass fat injection in a single plane must be avoided to prevent postoperative nodules and fibrosis.
The balance between collagen synthesis and degradation plays a significant role in the progression of fibrosis.Generally, the dense extracellular structure in fibrotic tissues is often referred to as an extracellular matrix, or simply as collagen. 23However, the precise process of collagen synthesis and degradation after autologous fat grafting remains to be explored.Fibroblasts are ECM-producing mesenchymal cells that consists of subsets with distinct capacities.Furthermore, fibroblasts can be activated or transformed into different phenotypes with specific behaviors and functions, including proto-myofibroblasts, myofibroblasts, and fibrocytes. 24ofibroblasts can also originate from various other precursor cells, such as perivascular adventitial cells, pericytes, epithelial cells and etc. 25 Recently, it is reported that the crosstalk between macrophage and fibroblasts exerts a significant role in the formation of

TA B L E 1 2 . 4 |
xylene (100% for 2 min) and stained with hematoxylin-eosin (HE) and Masson's trichrome working solution.Immunohistochemical staining was performed using anti-collagen I (1:250, ab21286; Abcam), anti-collagen III (1: 250, SAB#33341; Progen), anticollagen IV (1:150, ab19808; Abcam), and anti-collagen VI (1: 1500, SAB#49207; Progen), followed by appropriate secondary antibodies.Immunofluorescent staining for collagen VI and MAC2 (1: 300, Cedarlane Corp) and α-SMA (1: 400, SAB#40482; Progen) was performed, following by corresponding secondary antibodies.Nuclei were then stained with DAPI.•Category 4: 5(17%) Location of the nodules •Posterior mammary gland: 29(100%) •Posterior pectoralis major muscle: 0 Complications •Hematoma:1(3%) Satisfaction with postoperative result 28(97%) Patient characteristics after large-volume fat grafting for breast augmentation.Quantitative reverse transcription polymerase chain reaction (qRT-PCR) After being excised, breast nodules were snap-frozen in liquid nitrogen and stored at −80°C overnight.Total RNA was extracted using Trizol according to the manufacturer's instructions.Then, reverse transcription was performed by Thermo Scientific RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific).mRNA analysis were performed by SYBR Green quantitative PCR SuperMix (Thermo Fisher Scientific) on QuanStudio 5 real-time PCR system HE and Masson staining of the excised nodules demonstrated that fibrosis exists in the center of the nodule accompanied by degenerated adipocytes (Figure 2A,B).Then, to investigate the expression and precise role of collagen in subcutaneous nodules, we used immunohistochemistry to study the expression of type I, III, IV and VI collagens in serial tissue sections.Interestingly, the serial immunohistochemistry sections showed that type I and VI collagens were strongly expressed in the fibrotic area and type III collagen was moderately expressed, while type IV collagen were barely expressed in this region (Figure 2C-F).However, type IV collagen were positively expressed around the blood vessel.Then, we further evaluated type I, III, IV, VI collagens and transforming growth factor (TGF)β expression in the nodules with control subcutaneous fat that was excised together by VABB.The expression of type I, III, VI collagens and TGFβ was markedly upregulated in the nodules compared to the fat (p < 0.05), whereas type IV collagen expression was not significantly different between the two groups (Figure 3).

F I G U R E 1 F I G U R E 3
Intraoperative pictures showing nodule removal, real-time ultrasound images and macroscopic appearance of the excised nodules.(A) Surgical operation under ultrasound guidance.(B) Ultrasound images demonstrating a large oil and nodule in the right breast with internal soft tissue components preoperatively.The ultrasound image showed a 3.8 × 2.2 cm solid hyperechoic mass (red arrows) between the gland tissue and pectoralis major muscle at 6 o'clock in the right breast.The nodules were excised by the VABB system (black arrows).(C) The nodules obtained by the vacuum-assisted breast biopsy system were stripe-shaped with areas of adipogenesis and fibrosis.F I G U R E 2 Serial HE, Masson's trichrome staining, and immunohistochemistry of collagen I, III, IV and VI.(A, B) The HE stainging and Masson's trichrome staining of the serial sections.(C-F) The immunohistochemistry of collagen I(C), III(D), IV(E) and VI(F) of the serial sections.The results showed that type I and VI collagens were strongly expressed in the fibrotic area and type III collagen was moderately expressed, while type IV collagen were barely expressed in this region.However, type IV collagen were positively expressed around the blood vessel(black arrows).Scale bar = 100 μm.Assessment of collagen formation and fibrosis level of the breast nodules.(A-E) Quantitative real-time polymerase chain reaction of the mRNA expression of COL1(A), COL3(B), COL4(C), COL6(D), and TGFβ(E) in the excised nodules and fat.The expression of COL1, COL3, COL6 and TGFβ were markedly up-regulated in nodules compared to fat, whereas the expression of COL4 between the two gruops was not significantly different.Results are presented as the mean ± SD. *p < 0.05; **p < 0.01.