A Model to Study Wound Healing Over Exposed Avascular Structures in Rodents With a 3D-Printed Wound Frame.

BACKGROUND: Soft tissue defects with exposed avascular structures require reconstruction with well-vascularized tissues. Extensive research is ongoing to explore tissue engineered products that provide durable coverage. However, there is a lack of controlled and affordable testbeds in the preclinical setting to reflect this challenging clinical scenario. We aimed to address this gap in the literature and develop a feasible and easily reproducible model in rodents that reflects an avascular structure in the wound bed. METHODS: We created 20 × 20 mm full thickness wounds on the dorsal skin of Lewis rats and secured 0.5-mm-thick silicone sheets of varying sizes to the wound bed. A 3D-printed wound frame was designed to isolate the wound environment. Skin graft and free flap survival along with exposure of the underlying silicone was assessed. Rats were followed for 4 weeks with weekly dressing changes and photography. Samples were retrieved at the endpoint for tissue viability and histologic analysis. RESULTS: The total wound surface area was constant throughout the duration of the experiment in all groups and the wound frames were well tolerated. The portion of the skin graft without underlying silicone demonstrated integration with the underlying fascia and a histologically intact epidermis. Gradual necrosis of the portion of the skin graft overlying the silicone sheet was observed with varying sizes of the silicone sheet. When the size of the silicone sheet was reduced from 50% of the wound surface area, the portion surviving over the silicone sheet increased at the 4-week timepoint. The free flap provided complete coverage over the silicone sheet. CONCLUSION: We developed a novel model of rodent wound healing to maintain the same wound size and isolate the wound environment for up to 4 weeks. This model is clinically relevant to a complex wound with an avascular structure in the wound bed. Skin grafts failed to completely cover increasing sizes of the avascular structure, whereas the free flap was able to provide viable coverage. This cost-effective model will establish an easily reproducible platform to evaluate more complex bioengineered wound coverage solutions.

Intramuscular Nanofat Injection Promotes Inflammation-Induced Gastrocnemius Regeneration in a Syngeneic Rat Sciatic Nerve Injury Model.

BACKGROUND: Mechanical emulsification of adipose tissue to concentrate protein and stromal cell components (ie, nanofat) has gained considerable interest in clinical practice. Although the regenerative potential of nanofat has largely been used in aesthetic applications, these effects have considerable potential in reconstruction as well. Here, the authors investigated the therapeutic properties of nanofat injected directly into the denervated gastrocnemius after a sciatic nerve injury in Lewis rats. METHODS: Muscle denervation was induced by transecting and immediately repairing the sciatic nerve. Inguinal and subcutaneous adipose was harvested from donor rodents, processed into nanofat, and then injected intramuscularly into the gastrocnemius. Gait analysis was performed weekly. Rodents were euthanized at 9 and 12 weeks, after which tetanic contraction force was measured, and gene expression, histology, and cytokine multiplexing were performed. RESULTS: Intramuscular injection of nanofat significantly increased maximum tetanic force generation at 9 and 12 weeks. The forces of the nanofat-injected gastrocnemii were better correlated to their contralateral gastrocnemii relative to controls. Muscle repair-associated inflammatory gene expressions were significantly up-regulated in nanofat-injected gastrocnemii. Cytokines interleukin (IL)-1ß, IL-18, vascular endothelial growth factor, granulocyte-macrophage colony-stimulating factor, and tissue inhibitor of metalloproteinase-1 were significantly higher in nanofat-injected gastrocnemii relative to control gastrocnemii, and the tetanic force was linearly and significantly correlated to IL-1ß and IL-18 and their interacting effects. CONCLUSIONS: Intramuscular injection of emulsified adipose tissue (nanofat) significantly increased gastrocnemii contraction force after sciatic nerve injury, with prolonged reconstructive inflammation by means of CD68, inducible nitric oxide synthase, IL-1ß, and IL-18 all being potential mechanisms for this recovery. This application could potentially increase the therapeutic breadth of nanofat to include muscular recovery after nerve injury. CLINICAL RELEVANCE STATEMENT: The authors' study investigates a clinically translatable therapy to mitigate muscle atrophy after nerve injury.

Automated Decellularization of the Rodent Epigastric Free Flap: A Comparison of Sodium Dodecyl Sulfate-Based Protocols.

BACKGROUND: Free tissue transfer to cover complex wounds with exposed critical structures results in donor-site morbidity. Perfusion decellularization and recellularization of vascularized composite tissues is an active area of research to fabricate complex constructs without a donor site. Sodium dodecyl sulfate (SDS)-based protocols remain the predominant choice for decellularization despite the deleterious effects on tissue ultrastructure and capillary networks. We aimed to develop an automated decellularization process and compare different SDS perfusion times to optimize the protocol. METHODS: A three-dimensional-printed closed-system bioreactor capable of continuously perfusing fluid through the vasculature was used for decellularization. The artery and vein of rat epigastric fasciocutaneous free flaps were cannulated and connected to the bioreactor. Protocols had varying durations of 1% SDS solution (3, 5, and 10 days) followed by 1 day of 1% Triton X-100 and 1 day of 1x phosphate-buffered saline. The residual DNA was quantified. Microarchitecture of the constructs was assessed with histology, and the vascular network was visualized for qualitative assessment. RESULTS: The structural integrity and the microarchitecture of the extracellular matrix was preserved in the 3- and 5-day SDS perfusion groups; however, the subcutaneous tissue of the 10-day protocol lost its structure. Collagen and elastin structures of the pedicle vessels were not compromised by the decellularization process. Five-day SDS exposure group had the least residual DNA content (p < 0.001). Across all protocols, skin consistently had twice as much residual DNA over the subcutaneous tissues. CONCLUSION: A compact and integrated bioreactor can automate decellularization of free flaps to bioengineer regenerative constructs for future use in reconstruction of complex defects. A decellularization protocol with 5 days of 1% SDS exposure was the most successful to keep the residual DNA content at a minimum while preserving the structural integrity of the tissues.

Establishment of a Robust and Reproducible Model of Radiation-Induced Skin and Muscle Fibrosis.

Radiation-induced skin fibrosis (RISF) can result from a plethora of scenarios including cancer therapy, accidental exposure, or acts of terrorism. Radioactive beams can penetrate through the skin and affect the structures in their path including skin, muscles, and internal organs. Skin is the first structure to get exposed to radiation and is susceptible to develop chronic fibrosis, which is challenging to treat. Currently, limited treatment options show moderate efficacy in mitigating radiation-related skin fibrosis. A key factor hindering the development of effective countermeasures is the absence of a convenient and robust model that could allow for translation of the experimental findings to humans. Here, a robust and reproducible murine hind limb skin fibrosis model has been established for prophylactic and therapeutic evaluation of possible agents for functional and molecular recovery. The right hind limb was irradiated using a single dose of 40 (Gray) Gy to induce skin fibrosis. Subjects developed edema and dermatitis in the early stages proceeded by visible skin constriction. Irradiated limbs showed a significantly reduced limb range of motion in the following weeks. In late stages, acute side effects subsided, yet chronic fibrosis persisted. A gait index was performed as an additional functional assay, which demonstrated the development of functional impairment. These non-invasive methods demonstrated reliable measurements for tracing fibrosis progression, which is supported by histological analyses. The radiation dose, application, and post-irradiation analyses employed in this model offer a vigorous and reproducible method for studying radiation-induced skin fibrosis and testing the efficacy of therapeutical agents.

Intramuscular injection of skeletal muscle derived extracellular matrix mitigates denervation atrophy after sciatic nerve transection.

Peripheral nerve injury and the associated muscle atrophy has an estimated annual healthcare burden of $150 billion dollars in the United States. When considering the total annual health-related spending of $3.5 trillion, these pathologies alone occupy about 4.3%. The prevalence of these ailments is rooted, at least in part, in the lack of specific preventative therapies that can be administered to muscle while it remains in the denervated state. To address this, skeletal muscle-derived ECM (skECM) was injected directly in denervated muscle with postoperative analysis performed at 20 weeks, including gait analysis, force production, cytokine quantification, and histological analysis. skECM was shown to be superior against non-injected muscle controls showing no difference in contraction force to uninjured muscle at 20 weeks. Cytokines IL-1ß, IL-18, and IFNγ appeared to mediate regeneration with statistical regression implicating these cytokines as strong predictors of muscle contraction, showing significant linear correlation.

A Report of Gender Bias and Sexual Harassment in Current Plastic Surgery Training: A National Survey.

BACKGROUND: Gender bias and sexual misconduct continue to pervade medicine. The authors hypothesize that gender bias and sexual misconduct disproportionately and negatively affect female plastic surgery trainees. METHODS: A national survey of plastic surgery trainees (2018 to 2019) was conducted using previously validated sexual harassment surveys adapted for relevance to plastic surgery. Respondents were queried about experiences with workplace gender bias and harassment; personal and professional impact; and reporting practices. Analyses included chi-square, logistic regression, and analysis of variance. Significance was accepted for values of p < 0.05. RESULTS: There were 236 responses (115 female respondents; 20.1 percent response rate). Most respondents were Caucasian (Asian/Pacific Islander, n = 34) residents (n = 123). The feeling of hindrance to career advancement was greater for women, by 10-fold (p < 0.001), and increased with age (p = 0.046). Women felt uncomfortable challenging attitudes regarding gender inequality (p < 0.001), regardless of training levels (p = 0.670) or race (p = 0.300). Gender bias diminished female trainees' career goals/ambition (p < 0.001). Women were more likely to experience sexual harassment, in the form of jokes (p = 0.003) and comments about their body or sexuality (p = 0.014). Respondents reported the majority of perpetrators of harassment to be attending physicians (30 percent) and other trainees (37 percent). Most common reasons to not report incidents included "futility" (29 percent) and "fear" (20 percent). Women experienced at least three symptoms of depression/anxiety, significantly higher than men (p = 0.001). CONCLUSIONS: Gender bias and sexual misconduct negatively affect female trainees' attitudes toward their career. Two-thirds of cases of sexual harassment originate from other physicians. Minority trainees are less prepared to address transgressions and more likely to experience sexual coercion. Trainees perceive a culture nonconducive to reporting. These findings can guide changes and discussions surrounding workplace culture in plastic surgery training.

Improved Testing and Design of Intubation Boxes During the COVID-19 Pandemic.

STUDY OBJECTIVE: Throughout the coronavirus disease 2019 pandemic, many emergency departments have been using passive protective enclosures ("intubation boxes") during intubation. The effectiveness of these enclosures remains uncertain. We sought to quantify their ability to contain aerosols using industry standard test protocols. METHODS: We tested a commercially available passive protective enclosure representing the most common design and compared this with a modified enclosure that incorporated a vacuum system for active air filtration during simulated intubations and negative-pressure isolation. We evaluated the enclosures by using the same 3 tests air filtration experts use to certify class I biosafety cabinets: visual smoke pattern analysis using neutrally buoyant smoke, aerosol leak testing using a test aerosol that mimics the size of virus-containing particulates, and air velocity measurements. RESULTS: Qualitative evaluation revealed smoke escaping from all passive enclosure openings. Aerosol leak testing demonstrated elevated particle concentrations outside the enclosure during simulated intubations. In contrast, vacuum-filter-equipped enclosures fully contained the visible smoke and test aerosol to standards consistent with class I biosafety cabinet certification. CONCLUSION: Passive enclosures for intubation failed to contain aerosols, but the addition of a vacuum and active air filtration reduced aerosol spread during simulated intubation and patient isolation.

Adipose Stem Cells Enhance Nerve Regeneration and Muscle Function in a Peroneal Nerve Ablation Model.

Severe peripheral nerve injuries have devastating consequences on the quality of life in affected patients, and they represent a significant unmet medical need. Destruction of nerve fibers results in denervation of targeted muscles, which, subsequently, undergo progressive atrophy and loss of function. Timely restoration of neural innervation to muscle fibers is crucial to the preservation of muscle homeostasis and function. The goal of this study was to evaluate the impact of addition of adipose stem cells (ASCs) to polycaprolactone (PCL) nerve conduit guides on peripheral nerve repair and functional muscle recovery in the setting of a critical size nerve defect. To this end, peripheral nerve injury was created by surgically ablating 6 mm of the common peroneal nerve in a rat model. A PCL nerve guide, filled with ASCs and/or poloxamer hydrogel, was sutured to the nerve ends. Negative and positive controls included nerve ablation only (no repair), and reversed polarity autograft nerve implant, respectively. Tibialis anterior (TA) muscle function was assessed at 4, 8, and 12 weeks postinjury, and nerve and muscle tissue was retrieved at the 12-week terminal time point. Inclusion of ASCs in the PCL nerve guide elicited statistically significant time-dependent increases in functional recovery (contraction) after denervation; ∼25% higher than observed in acellular (poloxamer-filled) implants and indistinguishable from autograft implants, respectively, at 12 weeks postinjury (p < 0.05, n = 7-8 in each group). Analysis of single muscle fiber cross-sectional area (CSA) revealed that ASC-based treatment of nerve injury provided a better recapitulation of the overall distribution of muscle fiber CSAs observed in the contralateral TA muscle of uninjured limbs. In addition, the presence of ASCs was associated with improved features of re-innervation distal to the defect, with respect to neurofilament and S100 (Schwann cell marker) expression. In conclusion, these initial studies indicate significant benefits of inclusion of ASCs to the rate and magnitude of both peripheral nerve regeneration and functional recovery of muscle contraction, to levels equivalent to autograft implantation. These findings have important implications to improved nerve repair, and they provide input for future work directed to restoration of nerve and muscle function after polytraumatic injury. Impact Statement This works explores the application of adipose stem cells (ASCs) for peripheral nerve regeneration in a rat model. Herein, we demonstrate that the addition of ASCs in poloxamer-filled PCL nerve guide conduits impacts nerve regeneration and recovery of muscle function, to levels equivalent to autograft implantation, which is considered to be the current gold standard treatment. This study builds on the importance of a timely restoration of innervation to muscle fibers for preservation of muscle homeostasis, and it will provide input for future work aiming at restoring nerve and muscle function after polytraumatic injury.

Clinical Evaluation of an Off-the-Shelf Allogeneic Adipose Matrix for Soft Tissue Reconstruction.

Biomaterials derived from human adipose extracellular matrix have shown promise in vitro and in animal studies as an off-the-shelf adipogenic matrix for sustained volume replacement. Herein, we report the results of a randomized prospective study conducted with allograft adipose matrix (AAM) grafted into the pannus of presurgical abdominoplasty patients 3 or 6 months before scheduled surgery. This is the first report of a longitudinal histologic analysis of AAM in clinical use. METHODS: Ten healthy patients undergoing elective abdominoplasty were recruited to receive AAM before surgery. Enrolled subjects were randomized into either a 3-month follow-up cohort or a 6-month follow-up cohort. Subjects were monitored for adverse events associated with AAM grafting in addition to undergoing serial biopsy. Following surgical excision of the pannus, representative samples from the AAM surgical sites were stained and evaluated with hematoxylin and eosin for tissue morphology, Masson's trichrome for collagen, and perilipin for adipocytes. RESULTS: All subjects tolerated AAM with no severe adverse events reported. At 3 months following implantation, AAM remained visible within the confines of the subjects' native surrounding adipose tissue with sparse adipocytes apparent within the matrix. By 6 months, AAM had remodeled and was primarily composed of perilipin-positive adipocytes. Histologic analysis confirmed tissue remodeling (hematoxylin and eosin), adipogenesis (perilipin), and angiogenesis (Masson's trichrome) occurred with the presence of AAM. CONCLUSIONS: AAM is a safe, allogeneic, off-the-shelf regenerative matrix that is adipogenic and noninflammatory and promotes angiogenesis.

Long-gap peripheral nerve repair through sustained release of a neurotrophic factor in nonhuman primates.

Severe injuries to peripheral nerves are challenging to repair. Standard-of-care treatment for nerve gaps >2 to 3 centimeters is autografting; however, autografting can result in neuroma formation, loss of sensory function at the donor site, and increased operative time. To address the need for a synthetic nerve conduit to treat large nerve gaps, we investigated a biodegradable poly(caprolactone) (PCL) conduit with embedded double-walled polymeric microspheres encapsulating glial cell line-derived neurotrophic factor (GDNF) capable of providing a sustained release of GDNF for >50 days in a 5-centimeter nerve defect in a rhesus macaque model. The GDNF-eluting conduit (PCL/GDNF) was compared to a median nerve autograft and a PCL conduit containing empty microspheres (PCL/Empty). Functional testing demonstrated similar functional recovery between the PCL/GDNF-treated group (75.64 ± 10.28%) and the autograft-treated group (77.49 ± 19.28%); both groups were statistically improved compared to PCL/Empty-treated group (44.95 ± 26.94%). Nerve conduction velocity 1 year after surgery was increased in the PCL/GDNF-treated macaques (31.41 ± 15.34 meters/second) compared to autograft (25.45 ± 3.96 meters/second) and PCL/Empty (12.60 ± 3.89 meters/second) treatment. Histological analyses included assessment of Schwann cell presence, myelination of axons, nerve fiber density, and g-ratio. PCL/GDNF group exhibited a statistically greater average area occupied by individual Schwann cells at the distal nerve (11.60 ± 33.01 µm2) compared to autograft (4.62 ± 3.99 µm2) and PCL/Empty (4.52 ± 5.16 µm2) treatment groups. This study demonstrates the efficacious bridging of a long peripheral nerve gap in a nonhuman primate model using an acellular, biodegradable nerve conduit.

Design and Fabrication of an Automatable, 3D Printed Perfusion Device for Tissue Infusion and Perfusion Engineering.

Tissue decellularization for generating extracellular matrices has become a staple of regenerative medicine in the recent decades, extending from the research setting to clinical usage. Although methods and protocols for tissue decellularization are abundant throughout the literature, they can be time intensive and typically require specific overhead in terms of equipment. To reduce these barriers to entry, a functional and reproducible prototype of a tissue infusion/perfusion device (TIPD) has been designed and fabricated using three-dimensional printed parts in conjunction with commercially available components. This TIPD forms a system composed of two peristaltic pumps, two 3-way valves, and a chamber in which tissue is contained, and is controlled by user-customizable software. To increase repeatability among decellularization protocols, an automation function has been integrated into the software, which is able to specify fluid flow rates and define specific valve locations enabling selection of solutions to be introduced into a scaffold over the course of a decellularization process. The prototype has been tested for proof of concept through infusion and perfusion decellularization of skeletal muscle and intact kidneys, respectively, and has shown successful removal of cellular content while maintaining an intact ultrastructure. In an effort to increase the reproducibility of experimental designs and to promote an open source hardware initiative in the field of tissue engineering, a novel device was conceptualized and prototyped with printable part files made available for its fabrication in tandem with instructions for assembly. Impact Statement Repeatable methods for decellularization are essential for achieving consistent substrates between batches, laboratories, and facilities. To meet this end, an automatable tissue infusion/perfusion device composed of three-dimensional printed parts and commercially available components has been prototyped and tested. Materials and instructions for its assembly have been made available in an effort to reduce variability among equipment as well as to provide a platform on which to iterate open-source hardware in tissue engineering.

The Impact of Plastic Surgery Training on Family Planning and Prenatal Health.

BACKGROUND: Plastic surgery trainees who wish to start a family face challenges. This is the first study to collect data directly from residents and fellows to understand issues surrounding childbearing and to propose solutions. METHODS: Following institutional review board approval, an anonymous survey was distributed to all current plastic surgery residents and fellows in the United States. Data regarding demographics, obstetrical complications, parental leave, breastfeeding, and use of assisted reproductive technology were collected. RESULTS: The survey was completed by 307 trainees, for a resident response rate of 27.0 percent. Mean age of the respondents was 31.7 ± 3.8 years, 58.6 percent were married, and 35.3 percent reported at least one pregnancy for themselves or for their partner. Both male (67.4 percent) and female (76.5 percent) respondents intentionally postponed having children because of career. Women were significantly more likely to report negative stigma attached to pregnancy (70.4 percent versus 51.1 percent; p = 0.003) and plan to delay childbearing until after training. Fifty-six percent of female trainees reported an obstetrical complication. Assisted reproductive technology was used by 19.6 percent of trainees. Mean maternity leave was 5.5 weeks, with 44.4 percent taking less than 6 weeks. Mean paternity leave was 1.2 weeks. Sixty-two percent of women and 51.4 percent of men reported dissatisfaction with leave. Sixty-one percent of female trainees breastfed for 6 months and 19.5 percent continued for 12 months. Lactation facilities were available near operating rooms for 29.4 percent of respondents. CONCLUSIONS: Plastic surgery training may negatively impact fertility, obstetrical health, and breastfeeding practices. The data presented in this article provide the groundwork for identifying areas of concern and potential solutions.

Adipose-derived stem cells delay muscle atrophy after peripheral nerve injury in the rodent model.

INTRODUCTION: Injuries to peripheral nerves cause distal muscle atrophy. The effects of adipose-derived stem cell (ASC) injections into a muscle after injury were examined. METHODS: A 1.5 cm defect in the rat sciatic nerve was created, resulting in gastrocnemius muscle atrophy. The nerve defect was repaired with autograft; DiR-labeled ASCs were injected into the gastrocnemius immediately postoperatively. Quantitation of gross musculature and muscle fiber area, cell survival, fibrosis, lipid deposition, inflammation, and reconstructive responses were investigated. RESULTS: ASCs were identified in the muscle at 6 weeks, where injections showed increased muscle mass percentage retained, larger average fiber area, and less overall lipid content accumulated throughout the musculature. Muscles having received ASCs showed increased presence of interlukin-10 and Ki67, and decreased inducible nitric oxide synthase (iNOS). DISCUSSION: This investigation is suggestive that an ASC injection into denervated muscle post-operatively is able to delay the onset of atrophy. Muscle Nerve 59:603-603, 2019.

Injectable Allograft Adipose Matrix Supports Adipogenic Tissue Remodeling in the Nude Mouse and Human.

BACKGROUND: Adipose tissue reaches cellular stasis after puberty, leaving adipocytes unable to significantly expand or renew under normal physiologic conditions. This is problematic in progressive lipodystrophies, in instances of scarring, and in soft-tissue damage resulting from lumpectomy and traumatic deformities, because adipose tissue will not self-renew once damaged. This yields significant clinical necessity for an off-the-shelf de novo soft-tissue replacement mechanism. METHODS: A process comprising separate steps of removing lipid and cellular materials from adipose tissue has been developed, creating an ambient temperature-stable allograft adipose matrix. Growth factors and matrix proteins relevant to angiogenesis and adipogenesis were identified by enzyme-linked immunosorbent assay and immunohistochemistry, and subcutaneous soft-tissue integration of the allograft adipose matrix was investigated in vivo in both the athymic mouse and the dorsum of the human wrist. RESULTS: Allograft adipose matrix maintained structural components and endogenous growth factors. In vitro, adipose-derived stem cells cultured on allograft adipose matrix underwent adipogenesis in the absence of media-based cues. In vivo, animal modeling showed vasculature formation followed by perilipin A-positive tissue segments. Allograft adipose matrix maintained soft-tissue volume in the dorsal wrist in a 4-month investigation with no severe adverse events, becoming palpably consistent with subcutaneous adipose. CONCLUSIONS: Subcutaneous implantation of allograft adipose matrix laden with retained angiogenic and adipogenic factors served as an inductive scaffold for sustaining adipogenesis. Tissue incorporation assessed histologically from both the subcutaneous injection site of the athymic nude mouse over 6 months and human dorsal wrist presented adipocyte morphology residing within the injected scaffold.