Breast adipose tissue-derived extracellular vesicles from obese women alter tumor cell metabolism.

Breast adipose tissue is an important contributor to the obesity-breast cancer link. Extracellular vesicles (EVs) are nanosized particles containing selective cargo, such as miRNAs, that act locally or circulate to distant sites to modulate target cell functions. Here, we find that long-term education of breast cancer cells with EVs obtained from breast adipose tissue of women who are overweight or obese (O-EVs) results in increased proliferation. RNA-seq analysis of O-EV-educated cells demonstrates increased expression of genes involved in oxidative phosphorylation, such as ATP synthase and NADH: ubiquinone oxidoreductase. O-EVs increase respiratory complex protein expression, mitochondrial density, and mitochondrial respiration in tumor cells. The mitochondrial complex I inhibitor metformin reverses O-EV-induced cell proliferation. Several miRNAs-miR-155-5p, miR-10a-3p, and miR-30a-3p-which promote mitochondrial respiration and proliferation, are enriched in O-EVs relative to EVs from lean women. O-EV-induced proliferation and mitochondrial activity are associated with stimulation of the Akt/mTOR/P70S6K pathway, and are reversed upon silencing of P70S6K. This study reveals a new facet of the obesity-breast cancer link with human breast adipose tissue-derived EVs causing metabolic reprogramming of breast cancer cells.

Use of Banked Fibula for Improved Nasal Reconstruction Following Free Fibula Midface Reconstruction.

OBJECTIVE: Anterior maxillary deficiency caused by trauma or oncologic resection presents a complex reconstructive challenge. The authors present a technique for 2-stage midface reconstruction utilizing a vascularized free fibula flap for maxillary reconstruction, followed by nasal reconstruction at a second stage utilizing a banked fibula graft. METHODS: This case series utilizes a 2-stage technique for midface reconstruction. In the first stage, a fibula-free flap was used to reconstruct the maxilla with the excess banked in the abdomen. In the second stage, this bone graft was used to restore the nasal dorsum. RESULTS: Two patients were included in this series. Patient 1 was a 28-year-old man who presented after a remote gunshot wound to his face, resulting in complete loss of his anterior maxilla and nasal support with midface collapse. Patient 2 was a 65-year-old man who presented with squamous cell carcinoma of the hard palate with extension into the maxilla and nasal septum. In both cases, the flaps healed without complication, providing midface restoration. Placement of the banked fibula graft in a second stage resulted in restoration of dorsal nasal projection. CONCLUSION: The authors describe the use of "spare" fibula parts for nasal reconstruction after loss of the maxilla and cartilaginous septum. The use of the fibula bone as a graft to restore the nasal dorsum in a delayed manner allows for a better assessment of the esthetic needs after the massive swelling from the initial surgery has abated. Further, this approach eliminates the need for a second donor site for nasal reconstruction.

Improving Fat Transplantation Survival and Vascularization with Adenovirus E4+ Endothelial Cell-Assisted Lipotransfer.

Autologous fat transplantation is plagued by an unpredictable and often significant degree of graft loss. AdE4+ endothelial cells (ECs) are human endothelial cells that have been transduced with the E4ORF1 region of human adenovirus type 5, resulting in long-term preservation of EC proliferation and angiogenic capability without immortalization. We hypothesized that AdE4+ EC-enriched fat grafts would demonstrate improved volume retention secondary to enhanced angiogenesis. Three experimental groups were prepared by admixing 400 µL of patient lipoaspirate with 100 µL of AdE4+ EC suspensions (high AdE4+ EC concentration-enriched [5 × 106/mL], low AdE4+ EC concentration-enriched [1.25 × 106/mL], or PBS) and injected subcutaneously into the bilateral dorsa of nude mice. Fat transplants were explanted at 90 and 180 days for volumetric and histologic analyses. After both 90 and 180 days, AdE4+ EC-enriched fat grafts showed greater mean volume preservation compared to control grafts (p < 0.05). Regions of focal necrosis were only noticed in low AdE4+ EC concentration-enriched and control groups after 180 days. Histologic analysis demonstrated the presence of healthy adipocytes in all AdE4+ EC-enriched fat grafts in which both human and host ECs were evident after 90 and 180 days. AdE4+ EC enrichment improved fat graft volume preservation and vascularization in this murine xenograft model. Though further study is warranted, AdE4+ ECs demonstrated to be promising as a potential off-the-shelf adjunct for improving the volume, quality, and consistency of fat engraftment.

Reducing Peri-implant Capsule Thickness in Submuscular Rodent Model of Breast Reconstruction With Delayed Radiotherapy.

INTRODUCTION: Capsular contracture remains the most common complication following device-based breast reconstruction, occurring in up to 50% of women who also undergo adjuvant radiotherapy either before or after device-based reconstruction. While certain risk factors for capsular contracture have been identified, there remains no clinically effective method of prevention. The purpose of the present study is to determine the effect of coating the implant with the novel small molecule Met-Z2-Y12, with and without delayed, targeted radiotherapy, on capsule thickness and morphologic change around smooth silicone implants placed under the latissimus dorsi in a rodent model. METHODS: Twenty-four female Sprague Dawley rats each had 2 mL smooth round silicone breast implants implanted bilaterally under the latissimus dorsi muscle. Twelve received uncoated implants and twelve received implants coated with Met-Z2-Y12. Half of the animals from each group received targeted radiotherapy (20 Gray) on postoperative day ten. At three and 6 months after implantation, the tissue surrounding the implants was harvested for analysis of capsular histology including capsule thickness. Additionally, microCT scans were qualitatively analyzed for morphologic change. RESULTS: Capsules surrounding Met-Z2-Y12-coated implants were significantly thinner (P = 0.006). The greatest difference in capsule thickness was seen in the irradiated 6-month groups, where mean capsule thickness was 79.1 ± 27.3 µm for uncoated versus 50.9 ± 9.6 µm for Met-Z2-Y12-coated implants (P = 0.038). At the time of explant, there were no capsular morphologic differences between the groups either grossly or per microCT. CONCLUSIONS: Met-Z2-Y12 coating of smooth silicone breast implants significantly reduces capsule thickness in a rodent model of submuscular breast reconstruction with delayed radiotherapy.

Successful reconstruction of full-thickness skin defects in a swine model using simultaneous split-thickness skin grafting and composite collagen microstructured dermal scaffolds.

Reconstitution of normal skin anatomy after full-thickness skin loss may be accomplished using a combination of a dermal regeneration template (DRT) and a split thickness skin graft (STSG). However, because of the relatively low rate of cell infiltration and vascularisation of currently available DRTs, reconstruction is almost always performed in a two-step procedure over the course of several weeks, resulting in multiple dressing changes, prolonged immobilisation and increased chance of infection. To mitigate the potential complications of this prolonged process, the collagen-based dermal template DermiSphere™ was developed and tested in a single-step procedure wherein DermiSphere and STSG were implanted simultaneously. When evaluated in a porcine, full thickness, excisional wound model, DermiSphere successfully supported simultaneous split thickness skin graft take and induced functional neodermal tissue deposition. When compared to a market leading product Integra Bilayer Wound Matrix, which was used in a multistep procedure (STSG placed 14 days after product implantation according to the product IFU), DermiSphere induced a similar moderate and transient inflammatory response that produced similar neodermal tissue maturity, thickness and vascularity, despite being implanted in a single surgical procedure leading to wound closure 2 weeks earlier. These data suggest that DermiSphere may be implanted in a single-step procedure with an STSG, which would significantly shorten the time course required for the reconstruction of both dermal and epidermal components of skin after full thickness loss.

Topical α-Gal Nanoparticles Enhance Wound Healing in Radiated Skin.

PURPOSE: Surgery within radiated tissue is associated with increased complication rates. It is hypothesized that impaired wound healing may result from aberrant inflammatory responses that occur in previously radiated tissues. Previous work has demonstrated that the topical application of naturally occurring antigen α-gal (Galα1-3Galß1-(3)4GlcNAc-R) nanoparticles (AGNs) within wounds accelerates macrophage recruitment and subsequent healing in both normal and diabetic wounds. Herein, we hypothesize that application of this antigen would similarly enhance wound healing in irradiated tissues. METHODS: To simulate human physiology, α-1,3-galactosyltransferase knockout (KO) mice were exposed to the antigen to produce anti-α-gal antibodies (anti-Gal). Ten days prior to wounding, the dorsal skin was irradiated with 1 session of 40 Gy. Bilateral dorsal 6-mm splinted full-thickness wounds were created within the radiated skin and treated with 50 µL of AGNs (50 mg/mL) immediately after wounding and again on postoperative day 1. A control KO group underwent similar irradiation and wounding protocols but was treated with phosphate-buffered saline (PBS) vehicle. Wild-type (WT) mice, which do not produce anti-Gal, went through the same irradiation and wounding. RESULTS: Histologic analysis demonstrated enhanced epithelial migration in the radiated/AGN-treated KO wounds, which was significantly elevated in comparison to radiated/PBS-treated KO wounds beginning by day 15 and continuing until the end of the study (p < 0.01). In WT mice, treatment with AGNs showed no effect on epithelial migration. CONCLUSIONS: Topical application of AGNs onto irradiated wounds significantly ameliorates the delayed wound healing classically seen in radiated skin and results in faster wound closure with only transient application.

Production of a Low-Cost, Off-the-Shelf, Decellularized Cartilage Xenograft for Tissue Regeneration.

BACKGROUND: Reconstruction of cartilaginous deformities is a well-established surgical challenge with high levels of unpredictability and complication. Because of the morbidity associated with autologous cartilage grafting, combined with its limited supply and the significant expense of commercially decellularized allografts, increasing efforts have sought to produce an acellular, nonimmunogenic cartilage xenograft. We have developed and validated a novel protocol for high throughput decellularization of ovine costal cartilage with immediate translational potential for preclinical investigation of novel strategies for cartilaginous reconstruction. METHODS: Floating ribs were isolated from freshly slaughtered rack of lamb and after cleaning, the ribs were either minced into 2-mm cubes or zested into 1-mm flakes. Tissue was then decellularized via a protocol consisting of 4 freeze/thaw cycles, digestion with trypsin, incubation in hyperosmolar and hypoosmolar salt solutions, with incubation in 1% Tween following both the hyperosmolar and hypoosmolar steps, a 48-hour incubation in nucleases, DNA elution via EDTA, and 2 terminal sterilization steps. Protocol success was evaluated via histologic analysis with hematoxylin and eosin, DAPI, and safranin-O staining, as well as DNA quantification. RESULTS: Histologic analysis of the decellularized tissue revealed a significant reduction in nuclei as evidenced by hematoxylin and eosin and DAPI staining (P < 0.01). Safranin-O staining demonstrated a depletion of glycosaminoglycan content in the decellularized cartilage but with preservation of tissue architecture. Unprocessed lamb cartilage contained 421 ± 60 ng DNA/mg of lyophilized tissue, whereas decellularized zested and minced costal cartilage contained 27 ± 2 ng DNA/mg lyophilized tissue (P < 0.0001) and 24 ± 2.3 ng DNA/mg lyophilized tissue (p < 0.0001), respectively, well below the threshold of 50 ng accepted as evidence of suitable decellularization. In comparison, commercial allograft cartilage contained 17 ± 5 ng DNA/mg of lyophilized tissue. CONCLUSIONS: We have developed a novel protocol for the decellularization of xenogeneic cartilage graft. This structurally stable, low immunogenicity decellularized cartilage can be produced at low cost in large quantities for use in preclinical investigation.

Off-the-Shelf Nipple Engineering: Neonipple Formation via Implantation of Scaffolded Decellularized Ovine Xenograft.

BACKGROUND: Nipple reconstruction is widely regarded as the final step in postmastectomy breast reconstruction. While grafts, local flaps, or combination approaches have been used in nipple reconstruction, none has been able to achieve reliable long-term projection preservation. In response, we have sought to bioengineer neonipples in situ via the implantation of processed, decellularized cartilage xenografts placed within 3-dimensional-printed polylactic acid (PLA) scaffolds. MATERIALS AND METHODS: External nipple scaffolds were designed in-house and 3-dimensional-printed with PLA filament. Decellularized ovine xenograft infill was prepared and processed by mincing or zesting. All nipple scaffolds were placed subcutaneously on the dorsa of Sprague-Dawley rats and explanted after 1, 3, and 6 months for analysis. RESULTS: Explanted nipple scaffolds demonstrated gross maintenance of scaffold shape, diameter, and projection with accompanying increases in tissue volume. Histologic analyses revealed preservation of native cartilage architecture after 6 months without evidence of degradation. Analysis of formed tissue within the scaffolds revealed a progressive invasion of fibrovascular tissue with identifiable vascular channels and adipose tissue after 6 months in vivo. Confined compression testing revealed equilibrium moduli of both minced and zested samples that were within the expected range of previously reported human nipple tissue, while these data revealed no differences in the mechanical properties of the neotissue between time points or processing techniques. CONCLUSIONS: These preliminary data support potential use of decellularized allograft to foster healthy tissue ingrowth within a PLA scaffold, thereby offering a novel solution to current limitations in nipple reconstruction.

Microsurgical Head and Neck Reconstruction in Patients With Fanconi Anemia.

ABSTRACT: Patients with Fanconi anemia (FA) are at increased risk for head and neck cancers that often necessitate extensive reconstructions. Such patients have multiple comorbidities including anemia and thrombocytopenia frequently requiring bone marrow transplant, and they are at an increased risk of cancer recurrence and need for further extirpation. in the present study, charts from 3 patients with FA who underwent microvascular free tissue transfer by the senior author were retrospectively reviewed for pertinent pre- and peri-operative details in addition to functional and cosmetic outcomes. Two of these patients ultimately required metachronous free flap reconstructions for recurrence. All patients had acceptable functional and cosmetic outcomes following each instance of free flap reconstruction, thereby demonstrating the utility of microvas- cular free tissue transfer in patients with FA. The authors herein present each patient's clinical history in addition to a discussion of the current literature and an outline of our approach to these challenging cases.

Poly-4-hydroxybutyrate (Phasix™) mesh onlay in complex abdominal wall repair.

BACKGROUND: Poly-4-hydroxybutyric acid (P4HB, Phasix™) is a biosynthetic polymer that degrades by hydrolysis that can be woven into a mesh for use in soft tissue reinforcement. Herein, we describe our initial experience performing complex abdominal wall repair (CAWR) utilizing component separation and P4HB mesh as onlay reinforcement. METHODS: All patients undergoing CAWR between June 2014 and May 2017 were followed prospectively for postoperative outcomes. Only those patients who underwent components separation with primary repair of the fascial edges followed by onlay of P4HB mesh were included in this study. RESULTS: 105 patients (52 male, 53 female; mean age 59.2 years, range 22-84) met inclusion criteria. Mean BMI was 29.1 (range 16-48); 52% patients had prior attempted hernia repair, most with multiple medical comorbidities (71% of patients with ASA 3 or greater). 30% of cases were not clean at the time of repair (CDC class 2 or greater). Median follow-up was 36 months (range 9-63). Eighteen patients (17%) developed a hernia recurrence ranging from 2 to 36 months postoperatively. Five (5%) patients developed a localized superficial infection treated with antibiotics, three (2.8%) required re-operation for non-healing wounds, and six (6%) patients developed seroma. CONCLUSIONS: These data demonstrate a relatively low rate of hernia recurrence, seroma, and other common complications of CAWR in a highly morbid patient population. Importantly, the rate of mesh infection was low and no patients required complete mesh removal, even when placed into a contaminated or infected surgical field.

Obesity-associated Adipose Stromal Cells Promote Breast Cancer Invasion Through Direct Cell Contact and ECM Remodeling.

Obesity increases the risk and worsens the prognosis for breast cancer due, in part, to altered adipose stromal cell (ASC) behavior. Whether ASCs from obese individuals increase migration of breast cancer cells relative to their lean counterparts, however, remains unclear. To test this connection, multicellular spheroids composed of MCF10A-derived tumor cell lines of varying malignant potential and lean or obese ASCs were embedded into collagen scaffolds mimicking the elastic moduli of interstitial breast adipose tissue. Confocal image analysis suggests that tumor cells alone migrate insignificantly under these conditions. However, direct cell-cell contact with either lean or obese ASCs enables them to migrate collectively, whereby obese ASCs activate tumor cell migration more effectively than their lean counterparts. Time-resolved optical coherence tomography (OCT) imaging suggests that obese ASCs facilitate tumor cell migration by mediating contraction of local collagen fibers. Matrix metalloproteinase (MMP)-dependent proteolytic activity significantly contributes to ASC-mediated tumor cell invasion and collagen deformation. However, ASC contractility is also important, as co-inhibition of both MMPs and contractility is necessary to completely abrogate ASC-mediated tumor cell migration. These findings imply that obesity-mediated changes of ASC phenotype may impact tumor cell migration and invasion with potential implications for breast cancer malignancy in obese patients.

Topical α-gal nanoparticles accelerate diabetic wound healing.

An inadequate response from macrophages, key orchestrators of the wound healing process, has been implicated in the pathophysiology of impaired healing in diabetes. This study explored the utility of nanoparticles presenting the α-gal (Galα1-3Galß1-4GlcNAc-R) epitope to induce anti-Gal antibody-mediated local transient recruitment of macrophages to accelerate wound closure and healing in a diabetic murine model. α1,3galactosyltrasferase knockout mice were stimulated to produce anti-Gal antibodies and subsequently diabetes was induced by streptozotocin-induced ß-cell destruction. Six mm full-thickness skin wounds were made and α-gal nanoparticles (AGN) were topically applied on postwounding days 0 and 1. Wounds were analysed histologically for macrophage invasion and markers of wound healing, including epithelialization, vascularization and granulation tissue deposition through postoperative day 12. We found that application of AGN transiently but significantly increased macrophage recruitment into the wounds of diabetic mice. Treated wounds demonstrated more rapid closure and enhanced wound healing as demonstrated by significantly accelerated rates of epithelialization, vascularization and granulation tissue deposition. Thus, topical treatment of full-thickness wounds in diabetic mice with α-gal nanoparticles induced a transient but significant increase in macrophage recruitment resulting in an accelerated rate of wound healing. Using α-gal nanoparticles as a topical wound healing adjunct is a simple, safe and effective means of augmenting dysregulated macrophage recruitment present in the diabetic state.

Reconstruction of Full Thickness Scalp Defects in Extremely Elderly Patients Using Dermal Regeneration Templates.

The reconstruction of full thickness scalp defects following oncologic resection poses a unique challenge that is further magnified in "extremely elderly" patients, defined as those in at least their ninth decade of life, who are often unsuitable candidates for complex reconstruction. A "simpler" option is two-stage reconstruction: placement of Integra dermal regenerative template (Integra Life Science, Plainsboro, NJ) followed by a split thickness skin graft (STSG). This case series illustrates the success of this technique in the extremely elderly. A retrospective analysis of patients > 80 years at the time of surgery who underwent full thickness scalp reconstruction following tumor extirpation in a two-stage approach under the care of single surgeon from January 2010 to June 2019 was conducted. Variables reviewed were medical history, surgical treatment response, time to split thickness skin graft, follow up, and success of wound coverage. Fourteen patients, with a mean age of 87 years (range: 80 to 101, median: 87), met inclusion criteria. Split thickness skin grafts were placed after an average of 18 days. Twelve patients had successful two-stage reconstruction with 100% take. One patient developed a hematoma under a portion of the template that neither required reoperation nor delayed split thickness skin graft placement. A second suffered from insufficient vascularization of the template with delay to split thickness skin graft and incomplete wound closure. This two-stage approach is a successful primary reconstructive option for definitive management of full thickness scalp defects following oncologic resection in extremely elderly patients.

Periprosthetic Capsule Formation and Contracture in a Rodent Model of Implant-Based Breast Reconstruction With Delayed Radiotherapy.

INTRODUCTION: Capsular contracture (CC) is the most common complication of breast implantation, with an incidence of nearly 50% in patients undergoing breast reconstruction with subsequent radiotherapy. Although the move toward submuscular (SM) device placement led to a decreased incidence of CC, subcutaneous (SQ) implantation has seen a resurgence. The purpose of this study was to use a rodent model of breast reconstruction with smooth silicone implants and delayed radiotherapy to assess the occurrence of CC in SQ versus SM implantation. METHODS: Custom 2 mL smooth round silicone implants were placed bilaterally into 12 female Sprague Dawley rats that were randomized into 4 groups of 3, with each group differing by implantation plane (SQ vs SM) and irradiation status (irradiated vs nonirradiated). Rats from the SQ group received implants bilaterally underlying the skin on the flank. Rats in the SM groups received implants bilaterally under the latissimus dorsi muscle. Irradiated rats received 20 Gy localized to each implant on postoperative day 10. One rat from each group was imaged with a micro-computed tomography scanner at baseline and at explant 3 months later, whereupon capsules from all rats were examined histologically. RESULTS: Rats in the SQ group showed evidence of contracture on gross examination and greater evidence of morphologic disruption per micro-computed tomography scan. There was no evidence of contracture or morphologic disruption in either SM group. Mean ± SD capsule thickness was 39.0 ± 9.0 µm in the SQ versus 37.6 ± 9.8 µm in the SM nonirradiated groups and 43.9 ± 14.9 µm in the SQ versus 34.3 ± 8.3 µm in the SM irradiated groups (all P > 0.05). CONCLUSIONS: In a rodent model of smooth silicone breast implantation and delayed radiotherapy, although there did not appear to be differences in capsule thickness regardless of device placement plane, SQ implants demonstrated gross evidence of CC. These data indicate that capsule thickness is only part of a larger pathogenetic picture, which should take into consideration the contribution from all peri-implant tissue.

Functional and Aesthetic Outcomes in Free Flap Reconstruction of Intraoral Defects With Lip-Split Versus Non-Lip-Split Incisions.

BACKGROUND: Traditional free flap reconstruction of complex intraoral defects often uses large lip-splitting incisions. To reduce morbidity and preserve aesthetics, we have adopted a more technically demanding visor technique obviating an incision through the lower lip through which the resection and reconstruction are performed. METHODS: A retrospective review was performed of patients who underwent free flap reconstruction of intraoral defects over 7 years by a single plastic surgeon (C.H.R.) at a single institution. Patients were included if they underwent a resection from the mandible, tongue, or floor of mouth followed by free tissue transfer as a reconstructive approach. Patients were excluded if they underwent reconstruction of an area that does not traditionally require a lip incision, such as a maxillectomy or laryngeal defect. An ablative approach was taken via a lip-split technique or visor technique. Wound complications, margins of resection, and functional outcomes were assessed. Two standardized questionnaires (Derriford Appearance Scale Short Form and Quality of Life Questionnaire for Head and Neck Cancer) were used to assess psychological distress and dysfunction from disfigurement, speech quality, and oral function. Preoperative and postoperative patient photos were evaluated. RESULTS: Of 27 patients (mean ± SD age, 58.33 ± 13.02 years), 52% (14) had visor reconstructions whereas 48% (13) had lip-splitting reconstructions. About 78.6% of visor patients had widely-free margins compared with 46.2% of the lip-split patients. No differences in surgical-site complications between the lip-split and visor group (38.5% vs 28.6%) or in operative times were observed. Ninety-three percent of visor patients versus 54% of lip-split patients tolerated oral feeds at 1 year. Lip-split patients rated their quality of eating and speech worse than the visor patients (Quality of Life Questionnaire for Head and Neck Cancer mean score, 2.2 vs 1.56). Patients and clinical staff deemed visor reconstructions resulted in less visible sequelae. CONCLUSIONS: A visor technique with no lip-split incision for intraoral free flap reconstruction is an oncologically safe technique to consider that may improve cosmetic and functional outcomes for head and neck reconstruction patients.

Tissue Engineering Auricular Cartilage Using Late Passage Human Auricular Chondrocytes.

PURPOSE: The significant shortcomings associated with current autologous reconstructive options for auricular deformities have inspired great interest in a tissue engineering solution. A major obstacle in the engineering of human auricular cartilage is the availability of sufficient autologous human chondrocytes. A clinically obtainable amount of auricular cartilage tissue (ie, 1 g) only yields approximately 10 million cells, where 25 times this amount is needed for the fabrication of a full-scale pediatric ear. It is thought that repeated passaging of chondrocytes leads to dedifferentiation and loss of the chondrogenic potential. However, little to no data exist regarding the ideal number of times that human auricular chondrocytes (HAuCs) can be passaged in a manner that maximizes the cellular expansion while minimizing dedifferentiation. METHODS: Human auricular chondrocytes were isolated from discarded otoplasty specimens. The HAuCs were then expanded, and cells from passages 3, 4, and 5 were encapsulated into discs 8 mm in diameter made from type I collagen hydrogels with a cell density of 25 million cells/mL. The constructs were implanted subcutaneously in the dorsa of nude mice and harvested after 1 and 3 months for analysis. RESULTS: Constructs containing passages 3, 4, and 5 chondrocytes all maintained their original cylindrical geometry. After 3 months in vivo, the diameters of the P3, P4, and P5 discs were 69 ± 9%, 67 ± 10%, and 73 ± 15% of their initial diameter, respectively. Regardless of the passage number, all constructs developed a glossy white cartilaginous appearance, similar to native auricular cartilage. Histologic analysis demonstrated development of an organized perichondrium composed of collagen, a rich proteoglycan matrix, cellular lacunae, and a dense elastin fibrin network by Safranin-O and Verhoeff stain. Biochemical analysis confirmed similar amounts of proteoglycan and hydroxyproline content in late passage constructs when compared with native auricular cartilage. CONCLUSIONS: These data indicate that late passage HAuCs (up to passage 5) form elastic cartilage that is histologically, biochemically, and biomechanically similar to native human elastic cartilage and have the potential to be used for auricular cartilage engineering.

Antigen-Mediated, Macrophage-Stimulated, Accelerated Wound Healing Using α-Gal Nanoparticles.

BACKGROUND: Macrophages are known to be crucial to timely and efficacious wound healing. They have been shown to modulate inflammation and the migration and proliferation of regenerative cells, promoting tissue deposition and wound closure. This study explored the use of the natural antigen Galα1-3Galß1-4GlcNAc-R (α-gal), present in lower mammals yet absent in Old World primates and humans, to induce a transiently enhanced macrophage response and thereby direct accelerated wound closure and healing in a standard murine model. METHODS: α1,3galactosyltransferase knockout mice were stimulated to produce anti-Gal antibodies at levels comparable with humans. α-Gal-containing micelle nanoparticles were generated and applied to full-thickness splinted wounds on the mice. At 1, 2, 3, 6, and 9 days postoperatively, mice were killed, and wounds were analyzed histologically for macrophage invasion, epithelialization, vascularization, and granulation tissue deposition. Flow cytometry of wound tissue was performed to quantify relative levels of proinflammatory M1 to anti-inflammatory M2 macrophage subtypes. RESULTS: Treatment of splinted full-thickness murine wounds with α-gal-containing nanoparticles led to accelerated wound healing and closure as demonstrated by accelerated rates of keratinization, vascular growth, and wound tissue deposition. Furthermore, treated wounds demonstrated early and enhanced macrophage invasion, as well as a lower M1-M2 ratio. CONCLUSION: Application of α-gal-containing nanoparticles to wounds stimulated a transiently increased inflammatory response, accelerating the rate of wound healing. Use of α-gal may be a simple and effective way to stimulate the wound healing response in both normal and pathologic wound beds.

A 3-Dimensional Biomimetic Platform to Interrogate the Safety of Autologous Fat Transfer in the Setting of Breast Cancer.

INTRODUCTION: Obesity is a known risk factor for the development and prognosis of breast cancer. Adipocytes have been identified as a source of exogenous lipids in other cancer types and may similarly provide energy to fuel malignant survival and growth in breast cancer. This relationship is of particular relevance to plastic surgery, because many reconstructions after oncologic mastectomy achieve optimal aesthetics and durability using adjunctive autologous fat transfer (AFT). Despite the increasing ubiquity and promise of AFT, many unanswered questions remain, including safety in the setting of breast cancer. Clinical studies to examine this question are underway, but an in vitro system is critical to elucidate the complex interplay between the cells that normally reside at the surgical recipient site. To study these interactions and characterize possible lipid transfer between adipocytes to breast cancer cells, we designed a 3-dimensional in vitro model using primary patient-derived tissues. METHODS: Breast adipose tissue was acquired from patients undergoing breast reduction surgery. The tissue was enzymatically digested and sorted to retrieve adipocytes and adipose stromal cells. Polydimethylsiloxane wells were filled with type I collagen-encapsulated adipocytes labeled with the fluorescent lipid dye boron dipyrromethene, as well as unlabeled adipose stromal cells. A monolayer of red fluorescently labeled MDA-MB-231 and MDA-MB-468 breast cancer cells was seeded on the surface of the construct. Lipid transfer at the interface between adipocytes and breast cancer cells was analyzed. RESULTS: Confocal microscopy revealed a dense culture of native adipocytes containing fluorescent lipid droplets in the 3-dimensional collagen culture platform. RFP-positive breast cancer cells were found in close proximity to lipid-laden adipocytes. Lipid transfer from adipocytes to breast cancer cells was observed by the presence of boron dipyrromethene-positive lipid droplets within RFP-labeled breast cancer cells. CONCLUSION: We have established a 3-dimensional model to study complex breast cancer-adipose tissue interactions. Direct transfer of fluorescently labeled lipids from adipocytes to breast cancer cells may indicate aberrant metabolism to fuel malignant growth and adaptive survival. Our novel platform can untangle the complex interplay within the breast cancer tumor microenvironment for high-throughput analysis and better elucidate the safety of AFT in postoncologic mastectomy.

In Search of a Murine Model of Radiation-Induced Periprosthetic Capsular Fibrosis.

INTRODUCTION: Capsular contracture after breast reconstruction is a morbid complication, occurring in 30.0% to 47.5% of patients undergoing postoperative radiotherapy. Although it is well known that radiation increases rate of capsular contracture, there are few well-established animal models that faithfully replicate standard-of-care clinical practice, that is, prosthesis placement at the time of mastectomy followed by delayed radiotherapy. To better recapitulate current clinical practice, we developed a murine model in which the implant sites were irradiated 10 days postoperatively, rather than at time of surgery. METHODS: Hemispherical implants were created from polydimethylsiloxane and implanted bilaterally in the subcutaneous dorsa of 20 C57Bl/6 mice. Mice were randomized to 5 treatment groups, differing in irradiation dose: 0 to 40 Gy. Ten days postoperatively, irradiation was performed using 250-kVp x-rays (XRAD225Cx, Precision X-ray, North Branford, Conn). In 1 mouse per group, dosimeters were placed subcutaneously to measure the delivered absorbed dose. Thirty-one days postoperatively, the mice were sacrificed and examined grossly, and periprosthetic tissues were removed for histologic analysis of periprosthetic capsule thickness and cellular deposition. RESULTS: Total radiation dose was calculated by the treatment planning software and confirmed by the in vivo dosimeters. Physical examination of the irradiated region demonstrated evidence of local radiation delivery, including circular patterns of hair blanching and thinning directly over the implants. Furthermore, histologic analysis of the irradiated epidermis demonstrated dose-dependent radiation changes including keratin whorls and patches of uneven epidermal thickness. There was no statistically significant difference in capsule thickness among the groups. Mice in the 30 and 40 Gy groups endured complications including shortness of breath, coagulopathy, and death, signs of systemic radiation poisoning. CONCLUSIONS: There was no evidence of increased periprosthetic capsule thickness with localized irradiation, irrespective of dose up to 20 Gy. These results differ from those previously published, which demonstrated increased capsule thickness with 10 Gy irradiation. Given the evidence of local radiation delivery, we believe that the lack of increase in capsule thickness observed in our experiment is a real phenomenon and demonstrate the difficulty in creating an easily reproducible rodent model that mimics the effects of postmastectomy implant-based reconstruction and irradiation.

Perioperative Antibiotics in the Setting of Oropharyngeal Reconstruction: Less Is More.

BACKGROUND: Recipient-site infection after oropharyngeal reconstruction is a potentially disastrous complication. Although studies suggest that perioperative antibiotics reduces infection rates in these patients from 87% to 20%, there is no consensus regarding what constitutes the most appropriate antibiotic regimen and duration of treatment. METHODS: A retrospective review of perioperative antibiotic administration was performed of all patients who underwent local, pedicled, or free flap oropharyngeal reconstruction after oncologic resection by a single surgeon at a single institution between 2007 and 2013 to assess for recipient-site complications. RESULTS: Ninety-seven patients underwent 100 reconstructions (61 free flap reconstructions, 39 pedicled/local flap reconstructions) and all received a combination of intravenous (IV) antibiotic agents designed to cover oral flora. There were 23 (23%) recipient-site complications, which included cellulitis (9%), mucocutaneous fistula (5%), abscess (5%), and wound dehiscence (4%). Duration of antibiotic prophylaxis, defined as less than 48 hours (short-course) or greater than 48 hours (long-course), was not a significant predictor of recipient-site complication. Significant risk factors for recipient-site complications were clindamycin prophylaxis (P < 0.008), increased duration of surgery (P < 0.047), and advanced age (P < 0.034). Recipient-site complication was found to be a significant predictor of both increased length of hospital stay (P < 0.001) and increased time to the resumption of enteral feeds (P < 0.035). CONCLUSIONS: These data suggest that extended courses of perioperative antibiotics do not confer additional benefits in patients undergoing oropharyngeal reconstruction. We recommend a limited 48-hour course of prophylactic antibiotics with sufficient aerobic and anaerobic coverage to help minimize the incidence of antibiotic-related morbidities.