Optimizing the radiation dose in a murine model of breast implant capsular fibrosis

Abstract

Breast implant capsular contracture (CC) is the leading complication following reconstructive breast surgery, especially in breast cancer patients who undergo radiotherapy after reconstruction surgery. The exact pathophysiology of this complication is unclear with limited treatment options and high-quality research models are greatly needed. Here, we aimed to determine the optimal protocol to create an easily reproducible murine model that recapitulates the effects of radiotherapy following breast implantation. We implanted smooth mini silicone breast implants into a dorsal subcutaneous pocket of 8-week-old, female C57Bl/6 mice. Then, we immediately applied a directed dose of 10-Gy (N = 11), and 15-Gy (N = 11) slit-beam radiation to the implanted area along with non-irradiated mice (N = 11). The follow-up period was 42 -days post-radiation after which histological and tissue gene expression studies were done. Circulating TGF-b concentrations were also measured. Radiation therapy led to a significant decrease in total body weight and was proportional to the radiation dose. The 10-Gy dose of targeted radiotherapy significantly increased mean capsular thickness measured at 42 days following the procedure. However, mice who received a 15-Gy dose did not present with increased mean capsular thickness compared with implant-only mice. The 10-Gy dose upregulated pro-fibrotic and pro-inflammatory genes such as Collagen-3 and TIMP1 while the 15-Gy dose did not. Targeted radiotherapy with a 10-Gy and not 15-Gy dose in implanted mice induced significant CC along with an elevated pro-fibrotic response. Our findings demonstrate the successful development of a robust mouse model of breast implant CC using a 10-Gy unfractionated dose of radiotherapy immediately after implant insertion.

Introduction

Breast implant capsular contracture (CC) is the most common complication following implant-based breast reconstruction surgery^1,2. This complication often leads to deformed and firm breasts accompanied by extreme pain. CC manifests as an exaggerated fibrotic foreign body response to the implant, accompanied by excessive inflammation and collagen deposition³. Women with breast cancer who undergo postmastectomy radiation therapy (PMRT) present with the highest risk for CC ranging from 70 to 100%^4,5. PMRT induces tissue damage and leads to increased inflammation, fibrosis, and collagen deposition ultimately contributing to the formation of a contracted thicker periprosthetic capsule^3,6.

Despite its high prevalence, the underlying etiology and treatment of CC remain poorly understood, and the only currently effective treatment options are limited to revision surgery which carries a high risk of recurrent CC and secondary complications^1,2.

Thus, experimental models to study breast implant CC induced by radiation are highly sought for better understanding the underlying cellular mechanisms and for discovering new therapeutic targets. While several animal models have been previously described⁷, the optimal study protocol to create a reproducible murine model that recapitulates the effects of PMRT is not clear.

Past attempts to create such a model have focused on different doses of radiation delivered after implant insertion including 10, 20, 30 and 40 Gray (Gy) unfractionated doses^7–9. Yet it seems that there is no strict linear correlation between rising radiation doses and phenotypical features associated with CC such as capsular thickness.

In this study we aimed to compare the effect of a 15-Gy vs 10- Gy unfractionated dose of radiation administered immediately after silicone implant insertion into the dorsum of mice. We measured histological features and tissue gene expression profiles, along with a clinical assessment of the overall well-being of the animals. Our goal was to determine the optimal protocol for establishing a murine model for breast implant CC induced by radiotherapy.

Methods

Murine model for capsular contracture following radiation:

To recapitulate CC following radiation, we used 8-week-old, female C57Bl/6 mice. The study was approved by the Tel Aviv Sourasky Medical Center—Institutional Animal Care and Use Committee, Israel. All of the experiments were carried out in accordance with relevant guidelines and regulations. Eight-week-old female C57Bl/6 mice were purchased from Envigo (Israel) and housed in the Animal Research Laboratory at Tel Aviv Sourasky Medical Center. All mice were acclimatized one week upon arrival and were weighed and marked. All methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).

On day 0, the mice underwent anesthesia via intraperitoneal injection of ketamine (2 mg/0.47 mg) and xylazine (200 µl/mouse). Once anesthetized, the surgical site was shaved and treated with an iodine solution. A 1.5-cm transverse incision was made on the dorsal side of the mouse at the sacral spine level. A subcutaneous cranial pocket was then dissected to accommodate the implant. Smooth silicone 1 cc mini-implants (Polytech Health and Aesthetics, Germany) were inserted into the pocket after irrigation with betadine. The incision was closed with interrupted sutures using 4–0 nylon (Ethicon, Inc., Somerville, N.J.) (Fig. 1).

Fig. 1.

Intra-and-post-operative images of silicone mini-implant insertion. (A) A 1.5-cm transverse incision was made on the dorsal aspect of the mouse at the level of the sacral spine, followed by dissection of a cranial pocket, and placement of the mini-implant subcutaneously. (B) Mice received a directed 10-Gy or 15-Gy dose of radiation from a slit-beam cesium-137 source. (C) Representative Post procedural image during follow-up.

The mice were divided into 3 groups: A control group with no radiation (N = 11) and 2 radiated groups that received a directed 10-Gy (N = 11) or 15-Gy (N = 11) dose of radiation from a slit-beam cesium-137 source (Fig. 1). This radiation was targeted to induce periprosthetic capsular fibrosis while avoiding the systemic effects of radiation.

Follow-up visits were conducted on day 3 post-surgery and then once weekly throughout the experiment. The visits included: (i) evaluation of the general well-being of the mouse and signs of distress; (ii) physical examination, and (iii) measurement of the mouse’s body weight.

On postoperative day 42, mice were euthanized using CO2 inhalation in accordance with approved animal welfare protocols. Following euthanasia, a total capsulectomy was performed and the capsule was sent for histology and gene expression analysis using Real-Time Polymerase Chain Reaction (PCR). Intracardiac blood was taken for the Enzyme-linked immunosorbent assay (ELISA) for serum TGF-β.

Histopathological examination

Harvested capsules were fixed in 10% neutral buffered formalin. Fixed tissues were embedded in paraffin blocks. Serial longitudinal sections were prepared and stained with Hematoxylin and Eosin for histological analysis of capsular thickness. All slides were scanned and capsular thickness was measured blindly by a pathologist. We performed 6 measurements of the three thickest and three thinnest areas of the capsules. The mean values of these measurements were defined as the capsular thickness of the implant.

Quantitative real-time PCR

RNA from capsules was isolated with a Tri-Reagent (Sigma, USA) and reverse transcribed with a High-Capacity cDNA Reversed transcription kit (Applied Biosystems, USA) according to the manufacturer’s instructions. PCRs were performed with the SYBER green PCR Master Mix (Applied Biosystems, USA). Quantification was done with Step One software (V2.2). The genes of interest: Transforming Growth Factor beta (TGF-β), Collagen type 1 (Coll1), Alpha Smooth Muscle Actin (αSMA), Tissue Inhibitor of Metalloproteinase 1 (TIMP1), Interleukin-1 beta (IL-1β) and Tumor Necrosis Factor-alpha (TNFα) were compared with Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene. Primer sequences (forward and reverse, respectively) are shown in Supplementary Table 1.

Enzyme-linked immunosorbent assay (ELISA)

On day 42, intracardial blood was extracted for measurement of serum TGF-β using TGF beta-1/LAP Mouse Uncoated ELISA Kit (INVITROGEN, USA) according to the manufacturer’s protocol.

Statistical analysis

Variables were expressed as mean ± standard error of the mean (SEM). Specific statistical tests are detailed in the Figure legends. In brief, differences between values were tested by a One-Way ANOVA followed by Bonferroni correction for multiple comparisons. If values were not normally distributed (tested by the D’Agostino Pearson omnibus normality test), we used the non-parametric Kruskal–Wallis test. P values of < 0.05 were considered statistically significant. All statistical analyses were performed with GraphPad Prism version 9.00 (GraphPad Software, La Jolla, CA, USA).

Results

High-dose radiation reduced total body weight over time

We first sought to assess the effect of radiation on the general well-being of the animal, assessed primarily by total body weight. Radiation therapy with both a 10-Gy and 15-Gy dose led to a significant decrease in body weight immediately after the procedure compared to the implant-only non-radiated group (Fig. 2). The body weight loss was proportionate to the radiation dose, as seen in Fig. 2.

Fig. 2.

Total body weight across time. Total body weight was measured at day 3 post-surgery and then once weekly throughout the experiment. Radiation therapy led to a significant decrease in body weight, and the higher dose of 15-Gy was associated with the lowest total body weight over the entire follow-up period. P values were calculated using a mixed model followed by Bonferroni correction for multiple comparisons.

Radiation effect on capsular contracture formation

A 10-Gy dose of targeted radiotherapy significantly increased the mean capsular thickness, as measured by histopathological examination at 42 days following the procedure. However, mice who received a 15-Gy dose did not demonstrate a significant increase in the mean capsular thickness compared with implant-only mice (Fig. 3).

Fig. 3.

Capsular thickness assessed by histopathology. At the end of the experiment (day 42), implant capsules were stained with H&E staining and analyzed using planimetry software. A targeted radiation dose of 10-Gy increased mean capsular thickness while the 15-Gy dose did not. Left side: Representative images of stained capsules. Black lines mark capsular thickness. P values were calculated by a One-Way ANOVA followed by Bonferroni correction for multiple comparisons.

Thereafter, we examined the expression of pro-fibrotic genes in the capsular tissue. A 10-Gy dose of radiotherapy significantly upregulated Collagen3, and TIMP1 expression while the 15-Gy dose did not (Fig. 4). Similarly, the fibrotic genes TGF-β and αSMA were also increased in the 10-Gy group albeit not statistically significant (Fig. 4). We also found a trend for increased levels of the pro-inflammatory genes IL-1β and TNFα in capsular tissues of all irradiated mice (10-Gy and 15-Gy) suggesting an aggravated local inflammatory response induced by radiation (Fig. 4).

Fig. 4.

Gene Expression Analysis in capsular tissues 42 days post-radiation. Radiotherapy at a 10 Gy -dose significantly upregulated the expression of the pro-fibrotic genes COLL3A and TIMP1 in the capsular tissue while the 15 Gy dose did not. A similar trend was observed for the pro-fibrotic genes TGF-β and αSMA, and pro-inflammatory genes IL-1β and TNFα, albeit not statistically significant. Quantitative RT-PCR 2 − ΔΔCT values were calculated using GAPDH as a housekeeping gene. Data are shown as mean ± standard error of the mean (SEM) with individual measurements. P values were calculated by a One-Way ANOVA followed by Bonferroni correction for multiple comparisons. TGF-β-Transforming Growth Factor beta; Coll1-Collagen type 1; αSMA: Alpha Smooth Muscle Actin; TIMP1: Tissue Inhibitor of Metalloproteinase 1; IL-1β: Interleukin-1 beta; TNFα: Tumor Necrosis Factor-alpha.

Finally, serum TGF-β concentrations in 10-Gy irradiated mice were slightly higher compared to implant-only and 15-Gy irradiated mice albeit not statistically significant (Fig. 5).

Fig. 5.

Circulating serum TGF-β concentrations. Blood samples were collected 42 days after surgery and serum transforming growth factor beta (TGF-β) concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Data are shown as the mean ± SEM. p values were calculated by a one-way analysis of variance, followed by Bonferroni correction for multiple comparisons.

Taken together, targeted radiotherapy with a 10-Gy and not 15-Gy dose in implanted mice induced significant CC along with an elevated pro-fibrotic response.

Discussion

Breast implant CC is the leading complication in implant-based surgery and the main reason for reconstructive failure, revision, and re-operations^1,3. Patients receiving radiation therapy to implant-based breast reconstruction or implants after previous augmentation, have a very high chance to develop CC^2,4,5. The exact pathophysiology is still unclear, with limited treatment options. In-vivo radiation-induced periprosthetic capsular fibrosis models are sought to better understand the pathophysiology of the inflammatory process and to seek new treatment modalities for this complication^10–13. As breast implant CC has clinical features for diagnosis, In-vivo models demonstrate capsular thickness and levels of inflammatory chemokines to compare the contracture grade^14–16.

In this study we have demonstrated that the insertion of smooth silicone mini-implant devices into the dorsum of mice subcutaneously, followed by immediate targeted radiotherapy of a 10-Gy dose increases capsular thickness and the expression of pro-fibrotic genes. Specifically, we displayed that a higher radiation dose of 15-Gy did not induce significant phenotypical changes in the capsular tissue, while reducing the total body weight and comprising the overall well-being of mice.

Our findings are in line with the published murine model of breast implant CC described by Katzel et al.⁸. Notably, our current study provides further details on capsular tissue gene expression along with circulating TGFb concentrations following radiotherapy.

A higher radiation dose of 15-Gy did not lead to increased capsular thickness nor to significant up-regulation of pro-fibrotic and pro-inflammatory factors. Past reports have also shown that even higher doses of up to 40-Gy delivered immediately after implant insertion do not lead to increased capsular thickness^7,9. It has also been shown that while higher doses of radiation can exacerbate inflammation and fibrosis, the relationship is not strictly linear. Other factors such as dose thresholds, mitigating interventions, and unique tissue characteristics play significant roles in the tissue response to radiation^17–19. Accordingly, we hypothesize that the higher 15-Gy dose of radiation might have impaired inflammatory cells and downstream processes crucial for the formation of capsular fibrosis and thus is not an optimal regimen for the creation of an experimental model. The lack of increased fibrotic response at 15-Gy compared to 10-Gy is particularly intriguing also in the context of current clinical trends toward hypofractionation. Clinical trials by Poppee et al. and Punglia et al. have demonstrated that higher dose per fraction may actually be more favorable for CC outcomes compared to conventional 2-Gy fractionation^20,21. Our results suggest that the relationship between radiation dose and fibrotic response is complex and non-linear. Future investigations using our model should include detailed analysis of inflammatory cell infiltration, apoptosis markers, and vascular changes to better understand the mechanisms underlying this dose-dependent response. Such studies could provide valuable insights for optimizing clinical hypofractionation protocols.

Our findings have important clinical implications as CC in the clinical setting depends on multiple factors, including radiation dose (boost PMRT, bolus), radiation type (proton vs. photon), reconstruction types (prepectoral, single stage, expander, etc.), and other patient-specific factors. Large clinical series by Naoum et al. have demonstrated that proton therapy may actually increase CC rates compared to photon therapy²². Future studies on radiobiology modeling, such as ours, should further investigate the use of proton therapy in CC modeling and therapy. Additionally, the multicenter MROC studies by Jagsi et al. and Santosa et al. have provided valuable insights into the complex interplay between radiation parameters and CC formation in clinical practice^23,24. Our 10-Gy murine model provides a robust platform to further investigate these clinical variables and test potential therapeutic interventions that could translate to improved patient outcomes.

Our study is limited by the lack of histological and gene expression analysis at different time points besides the end of follow-up at 42 days post-operation. And yet, the significant changes in capsular thickness and gene expression profile observed at day-42, along with the original report by Katzel et al.⁸ which included additional time points at days 21, 28, and 35 support our main conclusion that the 10-Gy regimen is the optimal dose to recapitulate CC in mice over time. Nonetheless, longer follow-up periods may reveal different dose–response relationships as the fibrotic process evolves over time. Future studies utilizing our model should incorporate multiple time points to characterize the temporal progression of CC formation and determine whether the optimal radiation dose for modeling may change with extended follow-up.

An important limitation of our study is the use of single unfractionated radiation doses rather than the fractionated approach used clinically. In clinical practice, PMRT is typically delivered in smaller fractionated doses over several weeks, which allows for normal tissue repair and may modulate TGF-β signaling pathways, potentially reducing profibrotic effects. While our unfractionated protocol was chosen to create a reproducible and standardized experimental model, future studies should investigate fractionated radiation protocols to better replicate clinical scenarios and determine how fractionation affects CC formation in our murine model. Another limitation is our use of immediate post-surgical radiation rather than the delayed timing (4–8 weeks post-surgery) used clinically. As noted by Lin et al., delayed radiotherapy protocols do not induce significant CC features in murine models, which presents a challenge for creating clinically relevant experimental models⁷. While our immediate radiation protocol successfully induced measurable CC, future iterations of this model should continue to explore approaches that could bridge this temporal gap between experimental necessity and clinical practice. The timing factor remains an important consideration for the optimal use of this animal model in translational research.

Conclusions

In summary, our findings support the future use of an unfractionated 10-Gy dose delivered immediately after surgery to achieve optimal results in creating a robust mouse model of breast implant CC (Fig. 6). We show that a higher radiation dose of 15-Gy delivered immediately after silicone implant insertion does not induce significant features associated with breast implant CC.

Fig. 6.

Summary figure.

Abbreviations

αSMA

Alpha smooth muscle actin

ANOVA

Analysis of variance

CC

Capsular contracture

Coll1

Collagen type 1

ELISA

Enzyme-linked immunosorbent assay

GAPDH

Glyceraldehyde 3-phosphate dehydrogenase

Gy

Gray

IL-1β

Interleukin-1 beta

PCR

Polymerase chain reaction

PMRT

Postmastectomy radiation therapy

SEM

Standard error of the mean

TGF-β

Transforming growth factor beta

TIMP1

Tissue inhibitor of metalloproteinase 1

TNFα

Tumor necrosis factor alpha

Author contributions

OG, RYB, IS, EG, and YB participated in study conception and design. OG, IS, NS, GW, and YG conducted all of the experiments in mice. YZ conducted the histopathological staining and aided in interpreting the results. NS aided in radiotherapy procedures. RYB, OG, and IS participated in analysis and interpretation of the data.RYB, OG, and IS drafted the manuscript and EG, YB, ZO and NS helped in critical review of the manuscript. All of the authors have read and approved the final manuscript.

Funding

We did not receive financial support for this study. Polytech Health and Aesthetics, Germany provided the mini silicone implants gratis.

Data availability

The data generated during this study are available from the corresponding author upon reasonable request.

Declarations

Competing interests

Yoav Barnea participates in educational lectures for Johnson (Mentor) Corporation. All the remaining authors declare no conflict of interest.

Ethics approval and consent to participate

The study was approved by the Tel Aviv Sourasky Medical Center—Institutional Animal Care and Use Committee, Israel (TLVMC-IL-2203-111-4).

Consent for publication

Not applicable.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-16504-5.