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DOI 1011771090820X14533184

2014 34 769Aesthetic Surgery JournalIvan Maluf Jr

Guilherme Berto Roccedila Ruth Graf Renato da Silva Freitas Guataccedilara Salles Jr Julio Cesar Francisco Lucia Noronha andAutologous Fat Grafting for Treatment of Breast Implant Capsular Contracture A Study in Pigs

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Aesthetic Surgery Journal2014 Vol 34(5) 769 ndash775copy 2014 The American Society for Aesthetic Plastic Surgery IncReprints and permission httpwww sagepubcomjournalsPermissionsnavDOI 1011771090820X14533184wwwaestheticsurgeryjournalcom

Research

Since the introduction of silicone gel implants in 1963 the demand for breast augmentation and reconstruction has increased substantially In Brazil breast augmentation is a very common cosmetic surgery procedure12 Despite improve-ments in surgical techniques and the quality of implants patients continue to experience complications from this pro-cedure particularly capsular contracture (CC)3

Clinically CC may present as breast rigidity or harden-ing breast deformity asymmetry implant displacement

533184AESXXX1011771090820X14533184Aesthetic Surgery JournalRoccedila et alresearch-article2014

Dr Roccedila is a plastic surgeon in private practice in Curitiba Paranaacute Brazil Dr Graf is Professor Dr Freitas is Professor and Head of Unit and Mr Maluf is a resident in the Plastic Surgery Unit Federal University of Paranaacute Brazil Drs Salles Francisco and Noronha are Professors at Pontifical Catholic University of Paranaacute Brazil

Corresponding AuthorDr Guilherme Berto Roccedila Rua Joatildeo Geara 127 Apt 1802A Portatildeo Curitiba Paranaacute 80610-330 Brazil E-mail contatoguilhermerocacombr

Autologous Fat Grafting for Treatment of Breast Implant Capsular Contracture A Study in Pigs

Guilherme Berto Roccedila MD Ruth Graf MD PhD Renato da Silva Freitas MD PhD Guataccedilara Salles Jr MD PhD Julio Cesar Francisco MD PhD Lucia Noronha MD PhD and Ivan Maluf Jr

AbstractBackground Capsular contracture (CC) is a common complication after breast augmentation Autologous fat grafting may be effective for restoring tissue vascularization and functionObjective The authors evaluated the efficacy of autologous fat grafting in a porcine model as a treatment for CC after breast augmentationMethods This prospective study was performed in 20 female 30-day-old pigs Each animal was implanted with three 30-cc textured silicone implants (stage 1 of the experiment) Group A served as the untreated control group To induce CC 2 mL of autologous fibrin glue was applied to the pericapsular space in group B and C animals at implantation Three months after implantation (stage 2) the CCs of all groups were assessed by Baker classification and applanation tonometry (AT) Liposuction was performed in group B to harvest fat for these animals Three months after group B underwent fat grafting all 3 groups were reevaluated Reassessments included Baker classification AT histologic analysis and tensiometry (stage 3)Results The deposition of mature and immature collagen was similar for the 3 groups The amount of fat remaining around the implanted capsules did not differ significantly between the groups At stage 3 group B exhibited significantly larger tonometry areas than did group C The CCs in groups B and C were significantly thicker than those of group A but the difference between groups B and C was not significant Capsule rupture forces did not differ significantly between groups A and B but were significantly higher in group C compared with the other groupsConclusions Results in this animal model indicate that pericapsular lipoinjection may be a promising treatment for CC in humans

Keywordsautologous fat grafting breast augmentation capsular contracture silicone breast implants applanation tonometry

Accepted for publication November 1 2013

INTE

RNAT

IONAL CONTRIBUTION

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770 Aesthetic Surgery Journal 34(5)

prosthetic rupture andor discomfort Capsular contracture may develop slowly or rapidly and usually occurs within the first year of implantation However some patients experience CC after the first year Despite numerous theories and anec-dotal suggestions the etiology of CC remains unclear and is likely multifactorial Proposed contributors include filler mate-rial implant placement technique surface texture presence of foreign bodies (such as glove talc) and subclinical infec-tions near the area of implantation4 Capsular contracture is usually evaluated by Baker classification A less subjective method is applanation tonometry (AT) initially described by Moore5 (in 1979) and further developed by Gylbert6

Current treatments for CC are either surgical or pharma-cologic Surgical options include capsulotomy or capsulec-tomy and replacement of the implant Pharmacologic treatment usually involves intracapsular injection of ste-roids and antibiotics Another option to prevent or treat CC is zafirlukast a leukotriene receptor antagonist Moreover a growing body of clinical evidence strongly supports the therapeutic potential of mesenchymal stem cells to revas-cularize ischemic tissue and restore function78 Studies of fat grafting also indicate its effectiveness as a long-term filler and a treatment for CC among other conditions78

Grafted fat exhibits many qualities of an ideal filler it is autologous completely biocompatible available in suffi-cient quantities in most patients naturally integrated into host tissue removable if necessary and a potentially per-manent treatment Thus fat grafting has become very pop-ular in aesthetic and reconstructive surgery as a primary procedure and an adjunct to other procedures Its success is highly dependent on the technique of administration

Fat grafting the breast is currently utilized in the follow-ing situations to improve contour irregularities in breast reconstruction to correct defects after lumpectomy or par-tial injuries to cosmetically enhance or enlarge breasts to camouflage implants after augmentation and to recon-struct breasts after mastectomy (via fat infiltration only) To our knowledge there have been no studies of fat graft-ing as a treatment for CC We used a porcine model to study autologous fat grafting as a treatment for CC after breast augmentation with textured silicone gel implants

MethodsThis 6-month 3-stage prospective experiment was con-ducted in 30-day-old Duroc Gilt pigs (N = 20) The study design and methods were approved by the Committee for the Ethical Use of Animals at Pontifical Catholic University of Paranaacute Brazil Each pig received 3 implants A B and C In stage 1 each pig was anesthetized and then implanted with three 30-cc Silimed textured silicone implants (Rio de Janeiro Brazil) (Figure 1) the implants were placed in the ventral subglandular space (Figure 2) Group A served as the study control To induce CC in groups B and C autologous fibrin

glue (2 mL) was applied to the implant surface and into the pericapsular space during implantation (Figures 3 and 4) The glue was prepared with 4 mL of pig plasma 500 IU of 10 CaCl (Sigma-Tau Pharmaceuticals Gaithersburg Maryland) and 1000 IU of thrombin (Monarch Pharmaceuti-cals Bristol Tennessee) Three months after implantation (stage 2) the degree of CC was assessed in each animal according to Baker classification and AT (Figure 5) After these assessments group B animals underwent autologous fat harvesting via liposuction and the autologous fat was refined and injected (Figures 6 and 7)

TonometryTonometry was performed according to the method described by Moore5 and further developed by Gylbert6 who applied a known force to the breast then measured

Figure 1 Textured silicone implant (30 cc 5-cm diameter times 2-cm projection) (Silimed Rio de Janeiro Brazil)

Figure 2 Ventral suglandular implant pocket just above the aponeurosis of the rectusabdominis muscle

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Roccedila et al 771

the flattened area and calculated the intramammary pres-sure After trichotomy the animalrsquos skin was colored with

green gouache paint at each implant site A glass disk (8-cm diameter 10-mm thickness) was applied over the painted skin To calculate the area flattened by the disk a sheet of paper (same size as the glass disk) was placed between the glass and the painted skin surface After com-pression the image marked on the paper which was either round or elliptical was digitized with a scanner and measured with ImageJ software (Oracle Redwood City

Figure 3 Appearance of the fibrin glue applied to the pericapsular space during implantation in study groups B and C

Figure 4 Fibrin glue was applied to the breast implantrsquos surface immediately before implantation

Figure 5 Procedure for applanation tonometry at each implant location

Figure 6 After harvest the fat was centrifuged at 3000 rpm for 3 minutes The liquid and oil layers were then decanted

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772 Aesthetic Surgery Journal 34(5)

California) Larger areas indicated a lower degree of CC surrounding the implant

Fat Harvesting (Group B)Fat was harvested from the groin of each animal in group B Only autologous fat was injected The technique applied in our study has been described in detail elsewhere78 Briefly a blunt-tipped 2-hole Coleman harvesting cannula (Mentor Minneapolis Minnesota) was attached to a 10-mL Luer-Lok syringe (Becton Dickinson Franklin Lakes New Jersey) Three-millimeter incisions were made and the cannula was advanced through the groin of each animal while the surgeon applied digital manipulation to retract the syringersquos plunger thus creating a gentle negative pres-sure This slight negative pressure combined with the can-nularsquos curetting action through tissues allowed fat parcels to move through the cannula and Luer-Lok aperture into the syringe barrel When full the syringe was disconnected from the cannula and replaced with a plug that sealed the lock The plunger was removed from the syringe before its contents were centrifuged for fat refinement

Fat Refinement and Transfer (Group B)The fat was centrifuged to separate its components into dis-tinct layers To ensure sterile conditions we opted for a smaller centrifuge with a central rotor and sleeves which can be sterilized The harvested fat was processed at 3000 rpm for 3 minutes The upper level (the least dense) consisted primar-ily of oil The middle portion was primarily fatty tissue and the bottom layer comprised blood water and any aqueous elements After the oil layer was decanted the plug of the syringe was removed to release the liquid layer (the layer of

greatest density) Any remaining oil was wicked away with absorbent material The refined fat was then transferred into a 3-mL Luer-Lok syringe for injection

Lipoinjection Technique (Group B)The 2-mm cannulae used for lipoinjection are much smaller than those used for harvesting and each distal end contains a single hole Similar to the harvesting cannulae the proximal ends of these cannulae had a hub that fit into the Luer-Lok syringe The blunt cannulae allowed for immediate pericapsular placement of the autograft parcels within the subdermal plane and were more stable and less traumatic than other injection techniques Through retro-injections 5 mL of autologous fat was inserted (After withdrawal of the cannula the fatty-tissue parcels ldquofellrdquo into the natural tissue planes as the host tissue collapsed around them)

ReassessmentsThree months after lipoinjection of group B animals (stage 3) the following assessments were performed in every ani-mal (all groups) Baker classification AT histologic analysis of the capsule (hematoxylin-eosin [HampE] staining and Picrosirius Red staining [Polysciences Warrington Pennsylvania]) immunohistochemical analysis with adipo-nectin (rabbit polyclonal antibody adiponectin ABBiotec San Diego California) and tensiometry (Figures 8-10) The same surgeon (GBR) performed all explantations and no capsules or implants ruptured during this procedure

Histologic Analysis of CapsulesAfter AT the animals were euthanized A sample of sur-rounding skin and pericapsular tissue from each implanta-tion area was embedded in paraffin Sections of the samples were stained with HampE and Picrosirius Red The polarized Picrosirius demarcated areas of the capsules rich in collagen from areas that lacked collagen The total thick-ness of the pericapsular tissue and the thickness of the collagen and noncollagen layers were determined from an average of 10 measurements obtained from slides of each specimen

Rupture forces of all capsules were assessed via tensi-ometry with the EMIC DL 500 elastic testing machine (Emic Satildeo Paulo Brazil) The binomial test was utilized to compare the groups regarding the likelihood of normal contracture Repeated-measures analysis of variance was chosen to compare the stages (1 2 and 3) and groups A B and C with regard to quantitative variables The least significant difference test was used to compare stages and groups two by two P values less than 05 indicated statisti-cal significance

Figure 7 The lipoinjection instruments were 2-mm gauge cannulae Retro-injections of 5 mL of autologous fat were placed around the capsules

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Roccedila et al 773

ResultsThe deposition of collagen both mature (type I) and immature (type III) was similar for the 3 groups When the amount of fat remaining around the implant capsule was analyzed via immunohistochemistry no significant difference was found between the 3 groups At stage 3 the tonometry area of group B was significantly larger than that of group C (P lt 001) likely because group B capsules softened after lipoinjection The capsules of groups B and C (in which CC was induced with fibrin glue) were signifi-cantly thicker than those of group A (P lt 001) However there was no significant difference in capsule thickness between groups B and C (P = 342) With respect to rup-ture forces there was no significant difference between

groups A and B (P = 671) However group C had signifi-cantly higher rupture force than group A (P = 0017) or group B (P = 006) A difference in capsule softness favor-ing the fat-grafted animals (group B) was observed via AT

discussionCapsular contracture is the most common complication after aesthetic and reconstructive breast surgery the inci-dence ranges from 06 to 509 It is attributed to the gradual and progressive retraction of fibrous scar tissue around the prosthesis The periprosthetic fibrous capsule is similar to the dense fibrotic collagenous capsules of other fibrotic conditions it is composed of dense connec-tive tissue containing tightly packed collagen fibers reticu-lar fibers and an inner surface of fibrocytes and histiocytes in a single layer that forms an epithelium-like structure The most common feature of these capsules is dense fibro-collagenous or fibrovascular connective tissue containing foreign-body giant cells or granulomas which is consistent with an inflammatory or local immune response

Most studies of CC have focused on the effects of a par-ticular therapy on normal capsule formation Several ani-mal models primarily involving rats and rabbits have been developed to investigate CC510 Adams et al11 placed saline implants in rabbits to examine the relationship between capsule thickness and the degree of CC Ravin et al10 placed silicone implants in rats and induced CC with radiation Only a few studies of CC have been conducted in pigs Minami et al12 utilized AT and histologic examination to analyze the composition and behavior of capsules around smooth and textured breast implants in 33 pigs but CC was not induced in their study

We studied CC in Duroc Gilt pigs to determine whether our findings would parallel those of clinical studies of CC According to Clugston et al13 the epidermal structure of pigs resembles that of humans in certain respects the

Figure 8 Left The surface of an implant deformed by capsular contracture (after explantation) Right Appearance of the device before implantation Implants are 30-cc textured silicone devices (Silimed Rio de Janeiro Brazil)

Figure 9 Image shows the thickness of the capsule (077 mm) and its adherence to the surface of the implant Implants are 30-cc textured silicone devices (Silimed Rio de Janeiro Brazil)

Figure 10 Tensiometry was performed on all capsules to measure rupture force

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774 Aesthetic Surgery Journal 34(5)

dermis is thick and rich in elastic fibers Pigs proved to be an appropriate model in our study of CC

We induced CC with autologous fibrin glue which increased capsular thickness around the textured implant Fibrin glue appears to be more physiologically appropriate than methods used in previous studies Although Shah et al4 induced pathologic capsules with bacteria the repro-ducibility and control of their model have not been vali-dated Our model is the first to induce CC in pigs via fibrin glue This is important clinically because fibrin glue facili-tates conditions already known to produce capsule forma-tion in humans such as hematoma and infection

The therapeutic potential of mesenchymal stem cells for revascularization of ischemic tissue and restoration of func-tion appears to be based on the release of angiogenic and antiapoptotic growth factors in turn facilitating the recruit-ment of endothelial progenitor cells into newly sprouting vessels Recent studies have shown that the stromal-vascular cell portion of adipose tissue contains a rich reservoir of regenerative precursor cells with proangiogenic capabilities comparable to bone marrowndashderived stem cells1415 Given the capacity of stem cells for angiogenesis Coleman et al78 used liposuction and adipose fat injections in an effort to regenerate and repair damaged subcutaneous tissue Cardiologists who have researched preadipocytes for treat-ment of myocardial infarction have shown a positive corre-lation between stem cells and revascularization1617

Several studies have demonstrated the value of fat grafting in patients who undergo breast reconstruction procedures that include expanders and prostheses18-20 Fat grafting improves aesthetic results and tissue quality adds volume and may prevent CC However to our knowledge there have been no studies of fat grafting as the sole treatment for CC in aesthetic breast augmentation We analyzed the degree of CC via assessments described previously including Baker classi-fication AT histologic analyses of the capsule (HampE and Picrosirius Red staining) immunohistochemical analysis and tensiometry However it can be difficult to determine Baker classification in small animals such as rats and rabbits Moreover because the criteria for Baker classification are examiner dependent this method is less than idealmdasheven for pigs Despite this we found no significant differences in Baker classification between group B (fat grafted) and group C (not fat grafted)

Several researchers have evaluated CC with AT a more objective method18-21 To reduce the number of factors that could influence results all implants used in our study were textured Tonometry showed a reduction in mean applana-tion area after CC was induced with fibrin glue in groups B and C At the end of the 6-month study significant enlarge-ment of the mean applanation area was observed in fat-grafted animals (group B) this did not occur in untreated animals (group C) Therefore fat grafting may reduce the hardness of the capsule and surrounding tissue

Results of histologic staining showed no significant dif-ference in collagen deposition among the study groups As in other studies immature collagen deposition increased initially by the end of our study more mature collagen was observed It is noteworthy that despite the use of autologous fibrin glue to induce CC collagen deposition did not differ between the study groups The only signifi-cant difference was in capsule thickness The capsules that received fibrin glue (groups B and C) were significantly thicker than control capsules (group A) There was no dif-ference in capsule thickness between group B (fat-graft treated) and group C Therefore it is plausible that fat grafting does not alter collagen deposition or capsule thick-ness However the capsules did soften as the grafted fat created new vascularization in adjacent tissue

As observed in other studies12 all capsules in our exper-iment had the same basic 3-layer histologic structure The internal area abutting the silicone surface appeared to be either a single layer or multilayered containing macro-phages and fibroblasts In some cases a pseudoepithelial cellular layer was observed at the implant-capsule inter-face (synovia-like metaplasia) The middle layer consisted of loosely arranged connective tissue including internal vasculature The outer layer consisted of dense connective tissue with an external vascular supply22

In addition to histologic analysis we examined the quantity of fat that remained in surrounding tissue to ascertain whether the graft persisted around the capsule throughout the study Immunohistochemical analysis with adiponectin was employed Although a literature search yielded no other studies of adiponectin for this purpose our pathologist (LN) has had considerable experience with adiponectin and had already tested it in pigs with excellent results As expected more adipocytes were pres-ent in the surrounding tissue of fat-grafted animals (group B) but statistical analysis revealed no significant differ-ence in fat deposition among the 3 groups It is likely that many of the extremely sensitive adipocytes did not survive the histologic study

As noted by Coleman et al78 and Rigotti et al23 the pres-ence of stem cells in fat grafts may explain the apparent heal-ing and the improvement in tissue quality However the mechanisms of these effects have not been clarified Some investigators have suggested an interaction between the grafted fat and neighboring tissue which may promote repair of surrounding tissues directly or through angiogenesis or vasculogenesis24 Others have cited the plasticity between preadipocytes and macrophages such that some or all of the healing effect may be secondary to an enhanced immune response leading to permanent tissue remodeling25

Although our study was in animals fat-grafting applica-tions have been well established in humans It is notewor-thy that despite the lack of difference in Baker classification after lipoinjection the capsules of treated animals were

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Roccedila et al 775

softer than those of untreated animalsmdashaccording to AT and subjective evaluation by palpation

conclusionsPericapsular lipoinjection may be a promising treatment for CC We plan to continue this research by applying the technique in humans who experience mild to moderate CC after breast augmentation If patients perceive a greater degree of capsule softness the need for implant replace-ment may be eliminated or delayed

disclosuresThe authors declared no potential conflicts of interest with respect to the research authorship and publication of this article

FundingThe authors received no financial support for the research authorship and publication of this article

ReFeRences 1 Brazilian Society of Plastic Surgery Current situation of

plastic surgery in Brazil httpwwwcirurgiaplasticaorg br Accessed October 30 2013

2 The number of breast plastic surgery exceeds liposuction in Brazil Folha de Satildeo Paulo Newspaper httpwww folhauolcombr Accessed October 30 2013

3 Caffee HH Textured silicone and capsule contracture Ann Plast Surg 199024197-199

4 Shah Z Lehman JA Tan J Does infection play a role in breast capsular contracture Plast Reconstr Surg 19816834-43

5 Moore JR Applanation tonometry of breasts Plast Reconstr Surg 1979639-12

6 Gylbert LO Applanation tonometry for the evaluation of breast compressibility Scand J Plast Surg Hand Surg 198923(3)223-229

7 Coleman SR Saboeiro AP Fat grafting to the breast revisited safety and efficacy Plast Reconstr Surg 2007119(3)775-787

8 Coleman SR Structural fat grafting more than a perma-nent filler Plast Reconstr Surg 2006118(3)(suppl)108S-120S

9 Olbrich KC Meade R Bruno W Heller L Klitzman B Levin LS Halofuginone inhibits collagen deposition in fibrous capsules around implants Ann Plast Surg 200554293-296

10 Ravin AG Olbrich KC Levin LS Usala AL Klitzman B Long- and short-term effects of biological hydrogels on capsule microvascular density around implants in rats J Biomed Mater Res 200158313-318

11 Adams WP Jr Haydon MS Raniere J Jr et al A rabbit model for capsular contracture development and clinical implications Plast Reconstr Surg 20061171214-1221

12 Minami E Koh IHJ Ferreira JCR et al The composition and behavior of capsules around smooth and textured breast implants in pigs Plast Reconstr Surg 2006118(4) 874-884

13 Clugston PA Perry LC Hammond DC et al A rat model for capsular contracture the effects of surface texturing Ann Plast Surg 199433595-599

14 De Ugarte DA Morizono K Elbarbary A et al Comparison of multi-lineage cells from human adipose tissue and bone marrow Cells Tissues Organs 2003174101-106

15 Ryden M Dicker A Gotherstrom C et al Functional char-acterization of human mesenchymal stem cells derived adipocytes Biochem Biophys Res Commun 2003311391-397

16 Rehman J Traktuev D Li J Secretion of angiogenic and antiapoptotic factors by human adipose stromal cells Circulation 20041091292-1298

17 Planat-Bernard V Silvestre JS Cousin B et al Plasticity of human adipose lineage cells toward endothelial cells physiological and therapeutic perspectives Circulation 2004109656-663

18 Hakelius L Ohlsen L A clinical comparison of the ten-dency to capsular contracture between smooth and tex-tured gel-filled silicone mammary implants Plast Reconstr Surg 199290247-254

19 Hakelius L Ohlsen L Tendency to capsular contracture around smooth and textured gel-filled silicone mam-mary implants a five-year follow-up Plast Reconstr Surg 19971001566-1569

20 Wickman M Comparison between rapid and slow tissue expansion for breast reconstruction Plast Reconstr Surg 199391663-670

21 Wickman M Jurell G Low capsular contraction rate after primary and secondary breast reconstruction with a textured expander prosthesis Plast Reconstr Surg 199799692-697

22 Prantl L Schreml S Fichtner-Feigl S et al Clinical and morphological conditions in capsular contracture formed around silicone breast implants Plast Reconstr Surg 2007120(1)275-284

23 Rigotti G Marchi A Galiegrave M et al Clinical treatment of radiotherapy tissue damage by lipoaspirate transplant a healing process mediated by adipose-derived adult stem cells Plast Reconstr Surg 2007119(5)1409-1422

24 Nakagami H Maeda K Morishita R et al Novel autolo-gous cell therapy in ischemic limb disease through growth factor secretion by cultured adipose tissuendashderived stro-mal cells Arterioscler Thromb Vasc Biol 2005252542

25 Cousin B Andre M Casteilla L et al Altered macro-phage-like functions of preadipocytes in inflammation and genetic obesity J Cell Physiol 2001186380-386

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