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Organoid transplantation has a promising future in the treatment of liver disease, but a major limitation is the lack of guidance on the most appropriate method for transplantation that maximises organoid survival. Human induced pluripotent stem cell (hiPSC)-derived liver progenitor cell organoids were transplanted into four different transplantation sites in a mouse model of liver disease, using five organoid delivery methods. Organoids were transplanted into the vascularised chamber device established in the groin, or into the liver, spleen, and subcutaneous fat. For organoid transplantations into the liver, organoids were delivered either in Matrigel alone, or in Matrigel and a polyurethane scaffold. At 2 weeks post-transplantation, the vascularised chamber had the highest organoid survival, which was 5.1x higher than the site with second highest survival (p=0.0002), being the intra-hepatic scaffold approach. No organoid survival was observed when delivered into the liver without a scaffold, or when injected into the spleen. Very low survival occurred in transplantations into subcutaneous fat. Animals with the vascularised chamber also had the highest levels of human albumin (0.33 ± 0.09 ng/mL). This study provides strong evidence supporting the use of the vascularised chamber for future liver organoid transplantation studies, including its translation into clinical therapy.

Background Plastic and Reconstructive Surgery (PRS) can trace its origins as far back as 3000 BC. Despite this, it remained a relatively rare and unestablished branch of surgery until the devastating injuries of the World Wars necessitated reconstruction. Returning wartime surgeons used the skills they had learned on the battlefield to continue PRS in Australia and New Zealand. This article examines the significant contributions of Australian and New Zealand surgeons to the founding of PRS as a global specialty and provides an account of the strenuous dedicated competition that led to the development of microsurgery and advances in reconstruction. Methods A comprehensive review of medical, medical humanities, and history databases (PubMed; MEDLINE; Web of Knowledge; Anthropology; Encyclopaedia of ancient history) and non-digital printed texts was conducted using multiple search terms and filters including Reconstruction; Plastic Surgery; Burns; Flaps; and Microsurgery). The search was restricted to publications that focused on the period between 1818 CE to current. Results Significant contributions of surgeons from the Antipodes occurred during several periods including the Industrial era, World Wars, Post-war and in the modern age. Conclusions Stirred by their wartime experience, surgeons from Australia and New Zealand laid the foundations of the global success of Plastic Surgery in the modern age and helped establish it as a specialty in its own right.

This chapter reviews the history and indications of digital replantation and provides a thorough assessment of hand function to guide the surgeon. A technical guide to replantation is provided based on guiding principles of hand surgery and microsurgery. We provide a useful framework for the functional and cosmetic principles of microsurgical thumb reconstruction as well as pollicization and phalangization. We recommend a deep understanding of the hand's function prior to embarking on digital replantation and thumb reconstruction, as well as individualizing reconstruction to patient needs.

Tissue flaps are used to cover large/poorly healing wounds, but involve complex surgery and donor site morbidity. In this study a tissue flap is assembled using the mammalian body as a bioreactor to functionally connect an artery and vein to a human capillary network assembled from induced pluripotent stem cell-derived endothelial cells (hiPSC ECs). In vitro: Porous NovoSorb™ scaffolds (3 mm × 1.35 mm) were seeded with 200,000 hiPSC ECs ± 100,000 human vascular smooth muscle cells (hvSMC), and cultured for 1-3 days, with capillaries formed by 24 h which were CD31+, VE-Cadherin+, EphB4+, VEGFR2+ and Ki67+, whilst hvSMCs (calponin+) attached abluminally. In vivo: In SCID mice, bi-lateral epigastric vascular pedicles were isolated in a silicone chamber for a 3 week 'delay period' for pedicle capillary sprouting, then reopened, and two hiPSC EC ± hvSMCs seeded scaffolds transplanted over the pedicle. The chamber was either resealed (Group 1), or removed and surrounding tissue secured around the pedicle + scaffolds (Group 2), for 1 or 2 weeks. Human capillaries survived in vivo and were CD31+, VE-Cadherin+ and VEGFR2+. Human vSMCs remained attached, and host mesenchymal cells also attached abluminally. Systemically injected FITC-dextran present in human capillary lumens indicated inosculation to host capillaries. Human iPSC EC capillary morphometric parameters at one week in vivo were equal to or higher than the same parameters measured in human abdominal skin. This 'proof of concept' study has demonstrated that bio-engineering an autologous human tissue flap based on hiPSC EC could minimize the use of donor flaps and has potential applications for complex wound coverage. STATEMENT OF SIGNIFICANCE: Tissue flaps, used for surgical reconstruction of wounds, require complex surgery, often associated with morbidity. Bio-engineering a simpler alternative, we assembled a human induced pluripotent stem cell derived endothelial cell (hiPSC ECs) capillary network in a porous scaffold in vitro, which when transplanted over a mouse vascular pedicle in vivo formed a functional tissue flap with mouse blood flow in the human capillaries. Therefore it is feasible to form an autologous tissue flap derived from a hiPSC EC capillary network assembled in vitro, and functionally connect to a vascular pedicle in vivo that could be utilized in complex wound repair for chronic or acute wounds.

Background: Bacterial infection is a common and serious complication in orthopedic implants following traumatic injury, which is often associated with extensive soft tissue damage and contaminated wounds. Multidrug-resistant bacteria have been found in these infected wounds, especially in patients who have multi trauma and prolonged stay in intensive care units.Purpose: The objective of this study was to develop a coating on orthopedic implants that is effective against drug-resistant bacteria. Methods and results: We applied nanoparticles (30-70nm) of the trace element selenium (Se) as a coating through surface-induced nucleation-deposition on titanium implants and investigated the antimicrobial activity against drug resistant bacteria including Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-resistant Staphylococcus epidermidis (MRSE) in vitro and in an infected femur model in rats.The nanoparticles were shown in vitro to have antimicrobial activity at concentrations as low as 0.5ppm. The nanoparticle coatings strongly inhibited biofilm formation on the implants and reduced the number of viable bacteria in the surrounding tissue following inoculation of implants with biofilm forming doses of bacteria. Conclusion: This study shows a proof of concept for a selenium nanoparticle coatings as a potential anti-infective barrier for orthopedic medical devices in the setting of contamination with multi-resistant bacteria. It also represents one of the few (if only) in vivo assessment of selenium nanoparticle coatings on reducing antibiotic-resistant orthopedic implant infections.

Though the industrial era (1750 to 1914 AD) and the world wars (1914 to 1945 AD) were the catalysts for the establishment of Plastic and Reconstructive Surgery as a specialty in its own right, a careful examination of ancient texts and literature reveals that those conditions generally attributed to the craft were recognised and treated as early as 3000 BC. This article examines the remarkable medical documents of ancient civilisations as they pertain to our specialty and their legacies in the modern world. A comprehensive review of medical, medical humanities, and history databases (PubMed; MEDLINE; Web of Knowledge; Anthropology; Encyclopedia of ancient history) and non-digital printed texts was conducted using multiple search terms and filters including Reconstruction; Plastic Surgery; Ancient Surgery; Burns; Flaps; Sutures; and Dressings. The search was restricted to publications that focused on the period between 3100 BC and 476 AD. Additional information was sought from an eminent scholar of the Vedic period as well as the chief librarian of the Kaiser Library to aid in the identification of provenance. Five ancient periods and associated manuscripts were identified. Our specialty draws its earliest roots from antiquity. Reconstructive procedures used today were described in ancient eras indicating that the practice of plastic surgery is as old as surgery itself. Level of evidence: Not ratable.

A vast array of tumors can occur on the skin. Recent data demonstrated that more than two million of new cases of skin cancer are diagnosed annually [1]. Areas of sun exposure are more prone to develop numerous nonmelanoma skin cancers. The skin of the upper extremity and the hand is not an exception. It is known that the hand represents 1–2% of the total body surface. However, skin cancers involving this area account for 10–15% of all cases of skin malignancies [2, 3]. Unfortunately, there is a poor documentation of their natural history [4–6]. An explanation could be that either heterogeneous groups of soft tissue and cutaneous hand tumors or only specific skin cancers (e.g., squamous cell carcinomas) have been considered in the published series [7–12]. It is important to notice that the skin of the hand disposes a unique structure and is highly specialized allowing mobility for complex motor skills along with fine sensibility and resistance to withstand tearing and wearing forces. In addition the subcutaneous tissue is thin covering and protecting functionally important deeper structures. Subsequently, any therapeutic attempt necessitates a special consideration in this area counterbalancing sometimes two contradictory issues: tumor eradication and preservation or function restoration.

Background The survival of engineered cardiac muscle ‘grafts’ to the epicardium is limited by vascularization post‐transplantation in rat models. In this article, we describe the methodology of a novel rat model that allows for the transplantation of an engineered cardiac muscle flap (ECMF) onto the epicardium. Materials and Methods A total of 40 rats were used. Twenty‐four neonatal rats were used to harvest cardiomyocytes. At week 1, ECMF were generated by seeding cardiomyocytes into the arteriovenous loop (AVL) tissue engineering chamber implanted into the right groin of adult rats (n = 8). At week 6, the ECMF were harvested based on a pedicle along the femoral‐iliac‐abdominal vessel and anastomosed to the neck vessels of the recipient syngeneic adult rats (n = 8). The flaps were delivered into the thoracic cavity and onto the epicardium. The transplanted flaps were harvested at week 10. Survival of the flaps was assessed by the patency of anastomoses and viability of the cardiomyocytes through histological analysis (hematoxylin and eosin [H&E], desmin, and von Willebrand factor [vWF] immunostaining). Results Six out of 8 rats survived the transplantation procedure. These remaining 6 recipient rats survived until harvest time point at 4 weeks post‐transplantation. The mean area of the flap was 46.7mm2. Six out of 6 flaps harvested at week 10 showed viable cardiomyocytes using desmin immunostaining and vascular channels were seen at the interface between flap and epicardium. Conclusion This is a technically feasible model that will be useful for future assessment of different cardiac stem cell implants and their functional significance in rat heart models.

Thiol groups can undergo a large variety of chemical reactions and are used in solution phase to conjugate many bioactive molecules. Previous research on solid substrates with continuous phase glow discharge polymerization of thiol-containing monomers may have been compromised by oxidation. Thiol surface functionalization via glow discharge polymerization has been reported as requiring pulsing. Herein, continuous phase glow discharge polymerization of allyl mercaptan (2-propene-1-thiol) was used to generate significant densities of thiol groups on a mixed macrodiol polyurethane and tantalum. Three general classes of chemistry are used to conjugate proteins to thiol groups, with maleimide linkers being used most commonly. Here the pH specificity of maleimide reactions was used effectively to conjugate surface-bound thiol groups to amine groups in collagen. XPS demonstrated surface-bound thiol groups without evidence of oxidation, along with the subsequent presence of maleimide and collagen. Glow discharge reactor parameters were optimized by testing the resistance of bound collagen to degradation by 8 M urea. The nature of the chemical bonding of collagen to surface thiol groups was effectively assessed by colorimetric assay (ELISA) of residual collagen after incubation in 8 M urea over 8 days and after incubation with keratinocytes over 15 days. The facile creation of useable solid-supported thiol groups via continuous phase glow discharge polymerization of allyl mercaptan opens a route for attaching a vast array of bioactive molecules. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1940-1948, 2017.

Collagen 1 (C1) is commonly used to improve biological responses to implant surfaces. Here, the stability of C1 was compared with collagen 4 (C4) on a mixed macrodiol polyurethane, both adsorbed and covalently bound via acetaldehyde glow discharge polymerization and reductive amination. Substrate specimens were incubated in solutions of C1 and C4. The strength of conjugation was tested by incubation in 8 M urea followed by enzyme linked immunosorbent assays to measure residual C1 and C4. The basal lamina protein, laminin-332 (L332) was superimposed via adsorption on C4-treated specimens. Keratinocytes were grown on untreated, C1-treated, C4-treated, and C4 + L332-treated specimens, followed by measurement of cell area, proliferation, and focal adhesion density. Adsorbed C4 was shown to be significantly more stable than C1 and covalent conjugation conferred even greater stability, with no degradation of C4 over twenty days in 8 M urea. Cell growth was similar for C1 and C4, with no additional benefit conferred by superimposition of L332. The greater resistance of C4 to degradation may be consequent to cysteine residues and disulphide bonds in its non-collagenous domains. The use of C4 on implants, rather than C1, may improve their long-term stability in tissues. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1364-1373, 2017.

Supplemental Digital Content is available in the text., Background: Cancer patients often require radiotherapy (RTx) to enhance their survival. Unfortunately, RTx also damages nearby healthy non-cancer tissues, leading to progressive fibrotic soft-tissue injury, consisting of pain, contracture, tissue-breakdown, infection, and lymphoedema. Mechanisms underlying the clinically observed ability of fat grafting to ameliorate some of these effects, however, are poorly understood. It was hypothesized that RTx significantly alters fibroblast cell function and the paracrine secretome of adipose-derived stem cells (ADSC) may mitigate these changes. Methods: To investigate cellular changes resulting in the fibrotic side-effects of RTx, cultured normal human dermal fibroblasts (NHDF) were irradiated (10Gy), then studied using functional assays that reflect key fibroblast functions, and compared with unirradiated controls. RNA-Seq and targeted microarrays (with specific examination of TGFβ) were performed to elucidate altered gene pathways. Finally, conditioned-media from ADSC was used to treat irradiated fibroblasts and model fat graft surgery. Results: RTx altered NHDF morphology, with cellular functional changes reflecting transition into a more invasive phenotype: increased migration, adhesion, contractility, and disordered invasion. Changes in genes regulating collagen and MMP homeostasis and cell-cycle progression were also detected. However, TGFβ was not identified as a key intracellular regulator of the fibroblast response. Finally, treatment with ADSC-conditioned media reversed the RTx-induced hypermigratory state of NHDF. Conclusions: Our findings regarding cellular and molecular changes in irradiated fibroblasts help explain clinical manifestations of debilitating RTx-induced fibrosis. ADSC-secretome-mediated reversal indicated that these constituents may be used to combat the devastating side-effects of excessive unwanted fibrosis in RTx and other human fibrotic diseases.

For decades, plastic surgeons have spent considerable effort exploring anatomical regions for free flap design. More recently, tissue-engineering approaches have been utilised in an attempt to grow transplantable tissue flaps in vivo. The aim of this study was to engineer a fat flap with a vascular pedicle by combining autologous fat grafts and a novel acellular hydrogel (Adipogel) in an established tissue-engineering model comprising a chamber and blood vessel loop. An arteriovenous loop was created in the rat groin from the femoral vessels and positioned inside a perforated polycarbonate chamber. In Group 1, the chamber contained minced, centrifuged autologous fat; in Group 2, Adipogel was added to the graft; and in Group 3, Adipogel alone was used. Constructs were histologically examined at 6 and 12 weeks. In all groups, new tissue was generated. Adipocytes, although appearing viable in the graft at the time of insertion, were predominantly nonviable at 6 weeks. However, by 12 weeks, new fat had formed in all groups and was significantly greater in the combined fat/Adipogel group. No significant difference was seen in final construct total volume or construct neovascularisation between the groups. This study demonstrated that a pedicled adipose flap can be generated in rats by combining a blood vessel loop, an adipogenic hydrogel, and a lipoaspirate equivalent. Success appears to be based on adipogenesis rather than on adipocyte survival, and consistent with our previous work, this adipogenesis occurred subsequent to graft death and remodelling. The regenerative process was significantly enhanced in the presence of Adipogel.

Hair follicle cells contribute to wound healing, skin circulation, and skin diseases including skin cancer, and hair transplantation is a useful technique to study the participation of hair follicle cells in skin homeostasis and wound healing. Although hair follicle transplantation is a well-established human hair-restoration procedure, follicular transplantation techniques in animals have a number of shortcomings and have not been well described or optimized. To facilitate the study of follicular stem and progenitor cells and their interaction with surrounding skin, we have established a new murine transplantation model, similar to follicular unit transplantation in humans. Vibrissae from GFP transgenic mice were harvested, flip-side microdissected, and implanted individually into needle hole incisions in the back skin of immune-deficient nude mice. Grafts were evaluated histologically and the growth of transplanted vibrissae was observed. Transplanted follicles cycled spontaneously and newly formed hair shafts emerged from the skin after 2 weeks. Ninety percent of grafted vibrissae produced a hair shaft at 6 weeks. After pluck-induced follicle cycling, growth rates were equivalent to ungrafted vibrissae. Transplanted vibrissae with GFP-positive cells were easily identified in histological sections. We established a follicular vibrissa transplantation method that recapitulates human follicular unit transplantation. This method has several advantages over current protocols for animal hair transplantation. The method requires no suturing and minimizes the damage to donor follicles and recipient skin. Vibrissae are easier to microdissect and transplant than pelage follicles and, once transplanted, are readily distinguished from host pelage hair. This facilitates measurement of hair growth. Flip-side hair follicle microdissection precisely separates donor follicular tissue from interfollicular tissue and donor cells remain confined to hair follicles. This makes it possible to differentiate migration of hair follicle cells from interfollicular epidermis in lineage tracing wound experiments using genetically labeled donor follicles.

Background Adipose-derived stem cells (ASCs) have applications in regenerative medicine based on their therapeutic potential to repair and regenerate diseased and damaged tissue. They are commonly subject to oxidative stress during harvest and transplantation, which has detrimental effects on their subsequent viability. By functioning as an antioxidant against free radicals, melatonin may exert cytoprotective effects on ASCs. Methods We cultured human ASCs in the presence of varying dosages of hydrogen peroxide and/or melatonin for a period of 3 hours. Cell viability and apoptosis were determined with propidium iodide and Hoechst 33342 staining under fluorescence microscopy. Results Hydrogen peroxide (1–2.5 mM) treatment resulted in an incremental increase in cell death. 2 mM hydrogen peroxide was thereafter selected as the dose for co-treatment with melatonin. Melatonin alone had no adverse effects on ASCs. Co-treatment of ASCs with melatonin in the presence of hydrogen peroxide protected ASCs from cell death in a dose-dependent manner, and afforded maximal protection at 100 µM (n=4, one-way analysis of variance P Conclusions Melatonin possesses cytoprotective properties against oxidative stress in human ASCs and might be a useful adjunct in fat grafting and cell-assisted lipotransfer.

Avulsion, epidermal marsupialization, and infection cause failure at the skin-material interface. A robust interface would permit implantable robotics, prosthetics, and other medical devices; reconstruction of surgical defects, and long-term access to blood vessels and body cavities. Torus-shaped cap-scaffold structures were designed to work in conjunction with negative pressure to address the three causes of failure. Six wounds were made on the backs of each of four 3-month old pigs. Four unmodified (no caps) scaffolds were implanted along with 20 cap-scaffolds. Collagen type 4 was attached to 21 implants. Negative pressure then was applied. Structures were explanted and assessed histologically at day 7 and day 28. At day 28, there was close tissue apposition to scaffolds, without detectable reactions from defensive or interfering cells. Three cap-scaffolds explanted at day 28 showed likely attachment of epidermis to the cap or cap-scaffold junction, without deeper marsupialization. The combination of toric-shaped cap-scaffolds with negative pressure appears to be an intrinsically biocompatible system, enabling a robust skin-material interface. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1307-1318, 2017.

Obese and overweight patients represent a challenging surgical group for autologous free-flap breast reconstruction. There is a paucity of information regarding post-operative patient-reported satisfaction in this increasingly prevalent cohort. This retrospective study aimed to determine using BREAST-Q patient-reported satisfaction amongst normal, overweight and obese patients receiving autologous free-flap breast reconstruction.BREAST-Q (post-reconstruction) module was sent to 174 autologous breast free flap reconstruction patients between 2001 and 2012. Clinical data were collated for patients who returned questionnaires. Post-operative complications and satisfaction scores were compared between normal versus overweight and obese patients.A total of 101 patients (normal body mass index (BMI) = 27; overweight BMI = 48 and obese BMI = 25) completed BREAST-Q (response rate 66%). Obese and overweight patients are significantly more likely to develop major (44.8% and 31.1% vs. 29.6%) and minor (34.4% and 20% vs. 7.4%) complications in comparison to normal BMI patients (p0.02). Overweight and obese patients demonstrated similar levels of satisfaction compared with normal patients with the shape of their reconstructed breasts (73.2 and 72.1 vs. 71.2; p0.05) and overall surgical outcome (75.8 and 78.9 vs. 75.4; p0.05).Patient post-operative satisfaction is gaining increasing relevance in assessing the outcomes from autologous breast reconstruction. Overweight and obese women benefit from autologous breast reconstruction, despite being at increased risk of post-operative complications, and eventually achieve comparable levels of post-operative satisfaction. This should be reflected in pre-operative counselling.

The structural and physiological complexity of currently available liver organoids is limited, thereby reducing their relevance for drug studies, disease modelling, and regenerative therapy. In this study we combined mouse liver progenitor cells (LPCs) with mouse liver sinusoidal endothelial cells (LSECs) to generate hepatobiliary organoids with liver-specific vasculature. Organoids consisting of 5x103 cells were created from either LPCs, or a 1:1 combination of LPC/LSECs. LPC organoids demonstrated mild hepatobiliary differentiation in vitro with minimal morphological change; in contrast LPC/LSEC organoids developed clusters of polygonal hepatocyte-like cells and biliary ducts over a 7 day period. Hepatic (albumin, CPS1, CYP3A11) and biliary (GGT1) genes were significantly upregulated in LPC/LSEC organoids compared to LPC organoids over 7 days, as was albumin secretion. LPC/LSEC organoids also had significantly higher in vitro viability compared to LPC organoids. LPC and LPC/LSEC organoids were transplanted into vascularised chambers created in Fah−/−/Rag2−/−/Il2rg−/− mice (50 LPC organoids, containing 2.5x105 LPCs, and 100 LPC/LSEC organoids, containing 2.5x105 LPCs). At 2 weeks, minimal LPCs survived in chambers with LPC organoids, but robust hepatobiliary ductular tissue was present in LPC/LSEC organoids. Morphometric analysis demonstrated a 115-fold increase in HNF4α+ cells in LPC/LSEC organoid chambers (17.26 ± 4.34 cells/mm2 vs 0.15 ± 0.15 cells/mm2, p = 0.018), and 42-fold increase in Sox9+ cells in LPC/LSEC organoid chambers (28.29 ± 6.05 cells/mm2 vs 0.67 ± 0.67 cells/mm2, p = 0.011). This study presents a novel method to develop vascularised hepatobiliary organoids, with both in vitro and in vivo results confirming that incorporating LSECs with LPCs into organoids significantly increases the differentiation of hepatobiliary tissue within organoids and their survival post-transplantation.

Vascularisation is key to developing large transplantable tissue constructs capable of providing therapeutic benefits. The vascularised tissue engineering chamber originates from surgical concepts in tissue prefabrication and microsurgery. It serves as an in vivo bioreactor in the form of a closed, protected space surgically created and embedded within the body by fitting a noncollapsible chamber around major blood vessels. This creates a highly angiogenic environment which facilitates the engraftment and survival of transplanted cells and tissue constructs. This article outlines the chamber concept and explores its application in the context of recent advances in biomedical engineering, and how this can play a role in the future of cell therapies and regenerative medicine.

Porous polyester-ether hydrogel scaffolds (PEHs) were fabricated using acid chloride/alcohol chemistry and a salt templating approach. The PEHs were produced from readily available and cheap commercial reagents via the reaction of hydroxyl terminated poly(ethylene glycol) (PEG) derivatives with sebacoyl, succinyl, or trimesoyl chloride to afford ester cross-links between the PEG chains. Through variation of the acid chloride cross-linkers used in the synthesis and the incorporation of a hydrophobic modifier (poly(caprolactone) (PCL)), it was possible to tune the degradation rates and mechanical properties of the resulting hydrogels. Several of the hydrogel formulations displayed exceptional mechanical properties, remaining elastic without fracture at compressive strains of up to 80%, whilst still displaying degradation over a period of weeks to months. A subcutaneous rat model was used to study the scaffolds in vivo and revealed that the PEHs were infiltrated with well vascularised tissue within two weeks and had undergone significant degradation in 16 weeks without any signs of toxicity. Histological evaluation for immune responses revealed that the PEHs incite only a minor inflammatory response that is reduced over 16 weeks with no evidence of adverse effects. Refereed/Peer-reviewed

Background: Free fat grafting is popular, but it is still unclear how it works. Although focusing on graft survival seems an obvious direction for improving clinical results, the authors’ research suggests that long-term volume retention is in part attributable to new fat regeneration. Measures to facilitate adipogenesis may therefore be equally important. Methods: To investigate the relative roles of survival and regeneration of fat grafts, the authors measured the fate of human lipoaspirate implanted into the scalps of immunodeficient mice, with and without stromal vascular fraction and a porcine extracellular matrix (Adipogel). Specifically, the authors were interested in volume retention, and the composition of implanted or regenerated tissue at 6 and 12 weeks. Results: Free fat grafts exhibited poor volume retention and survival. Almost all of the injected human adipocytes died, but new mouse fat formed peripheral to the encapsulated fat graft. Adipogel and stromal vascular fraction improved proliferation of murine fat and human vasculature. Human CD34+ stromal cells were present but only in the periphery, and there was no evidence that these cells differentiated into adipocytes. Conclusions: In the authors’ model, most of the implanted tissue died, but unresorbed dead fat accounted substantially for the long-term, reduced volume. A layer of host-derived, regenerated adipose tissue was present at the periphery. This regeneration may be driven by the presence of dying fat, and it was enhanced by addition of the authors’ adipogenic adjuncts. Future research should perhaps focus not only on improving graft survival but also on enhancing the adipogenic environment conducive to fat regeneration.

Background The liver sinusoidal capillaries play a pivotal role in liver regeneration, suggesting they may be beneficial in liver bioengineering. This study isolated mouse liver sinusoidal endothelial cells (LSECs) and determined their ability to form capillary networks in vitro and in vivo for liver tissue engineering purposes. Methods and results In vitro LSECs were isolated from adult C57BL/6 mouse livers. Immunofluorescence labelling indicated they were LYVE-1+/CD32b+/FactorVIII+/CD31−. Scanning electron microscopy of LSECs revealed the presence of characteristic sieve plates at 2 days. LSECs formed tubes and sprouts in the tubulogenesis assay, similar to human microvascular endothelial cells (HMEC); and formed capillaries with lumens when implanted in a porous collagen scaffold in vitro. LSECs were able to form spheroids, and in the spheroid gel sandwich assay produced significantly increased numbers (p = 0.0011) of capillary-like sprouts at 24 h compared to HMEC spheroids. Supernatant from LSEC spheroids demonstrated significantly greater levels of vascular endothelial growth factor-A and C (VEGF-A, VEGF-C) and hepatocyte growth factor (HGF) compared to LSEC monolayers (p = 0.0167; p = 0.0017; and p

'Off-the-shelf' tissue-engineered skin alternatives for epidermal and dermal skin layers are available; however, no such alternative for the subdermal fat layer exists. Without this well-vascularized layer, skin graft take is variable and grafts may have reduced mobility, contracture and contour defects. In this study a novel adipose-derived acellular matrix (Adipogel) was investigated for its properties to generate subdermal fat in a rat model. In a dorsal thoracic site, a 1 × 1 cm Adipogel implant was inserted within a subdermal fat layer defect. In a dorsal lumbar site, an Adipogel implant was inserted in a subfascial pocket. Contralateral control defects remained empty. At 8 weeks wound/implant sites were evaluated histologically, immunohistochemically and morphometrically. Identifiable thoracic Adipogel implants lost volume in vivo over 8 weeks. Neovascularization and adipogenesis were evident within implants and adipocyte percentage volume was 33.07 ± 6.55% (mean ± SEM). A comparison of entire cross-sections of thoracic wounds demonstrated a significant increase in total wound fat in Adipogel-implanted wounds (37.19 ± 4.48%, mean ± SEM) compared to control (16.53 ± 4.60%; p = 0.0092), indicating that some Adipogel had been completely converted to normal fat. At the lumbar site, Adipogel also lost volume, appearing flattened, although fat generation and angiogenesis occurred. At both sites macrophage infiltration was mild, whilst many infiltrating cells were PDGFRβ-positive mesenchymal cells. Adipogel is adipogenic and angiogenic and is a promising candidate for subcutaneous fat regeneration; it has the potential to be a valuable adjunct to wound-healing therapy and reconstructive surgery practice. Copyright © 2015 John Wiley & Sons, Ltd.

As in vivo tissue engineering of complex tissues and organs progresses, there is a need for an independently vascularized, alterable, and recoverable model. Current models of islet cell transplantation (release into the portal venous system, placement under the renal capsule, and microencapsulation) lack these qualities. We have developed a model of angiogenesis and spontaneous tissue generation in the rat that lends itself as a potential platform for tissue engineering. In this experiment, we examined the effectiveness of such a model in addressing some of the shortcomings of endocrine pancreatic transplantation. An arteriovenous loop was created in the groins of five adult inbred Sprague-Dawley rats, and placed within polycarbonate chambers. Isolated pancreatic islet cell clusters were placed within the chambers, suspended in a matrix of Matrigel®. The chambers were recovered at 3 weeks, and the newly generated tissue was processed for histologic and immunohistochemical analysis. By 3 weeks, spontaneous generation of angiogenesis and collagen matrix and deposition of a collagen matrix was observed. Surviving islet cells were identified by histology and their viability was confirmed via immunohistochemistry for insulin and glucagon. This study demonstrates the ability to maintain viability and functionality of transplanted islet cells on a tissue-engineered platform with an independent vascular supply. The model provides the ability to alter the graft environment via matrix substitution, cellular coculture, and administration of growth factors. The transplanted tissues are recoverable without animal sacrifice and are microsurgically transferable. This model may provide an in vivo culture platform for the study of islet transplantation.

Background: Cell-assisted lipotransfer has been promisingly applied to restore soft-tissue defects in plastic surgery; however, the harvesting of stromal vascular fraction increases morbidity and poses potential safety hazards. The authors investigated whether adding indomethacin, an antiinflammatory proadipogenic drug, to the fat graft at the time of transplantation would enhance the final graft volume compared with cell-assisted lipotransfer. Methods: In vitro, human adipose-derived stem cells were cultured in conditioned growth media supplemented with various doses of indomethacin to investigate adipogenesis and the expression of the adipogenic genes. In vivo, lipoaspirate mixed with stromal vascular fractions or indomethacin was injected into the dorsum of mice. Tissues were harvested at weeks 2, 4, and 12 to evaluate histologic changes. Results: In vitro, polymerase chain reaction analysis revealed that increased up-regulation of adipogenic genes and activation of the peroxisome proliferator-activated receptor-[gamma] pathway. In vivo, the percentage volume of adipocytes in the indomethacin-assisted groups was higher than that in the lipoaspirate-alone (control) group at 12 weeks (p = 0.016), and was equivalent to the volume in the cell-assisted groups (p = 1.000). Indomethacin improved adipose volumes but had no effect on vascularity. A larger number of small adipocytes appeared in the treatment samples than in the controls at 2 weeks (p = 0.044) and 4 weeks (p = 0.021). Conclusions: Pretreating lipoaspirate with indomethacin enhances the final volume retention of engrafted fat. This result is explained in part by increased adipogenesis and possibly by the inhibition of inflammatory responses.

Hydrogels are an ideal medium for the expansion of cells in three dimensions. The ability to induce cell expansion and differentiation in a controlled manner is a key goal in tissue engineering. Here we describe a detailed method for producing hydrogels from soft tissues with an emphasis on adipose tissue. In this method, soluble, extractable proteins are recovered from the tissue and stored while the remaining insoluble tissue is processed and solubilised. Once the tissue has been sufficiently solubilised, the extracted proteins are added. The resulting product is a thermosensitive hydrogel with proteins representative of the native tissue. This method addresses common issues encountered when working with some biomaterials, such as high lipid content, DNA contamination, and finding an appropriate sterilisation method. Although the focus of this article is on adipose tissue, using this method we have produced hydrogels from other soft tissues including muscle, liver, and cardiac tissue.

Background There is a general consensus among reconstructive surgeons that preoperative radiotherapy is associated with a higher risk of flap failure and complications in head and neck surgery. Opinion is also divided regarding the effects of radiation dose on free flap outcomes and timing of preoperative radiation to minimize adverse outcomes. Our meta-analysis will attempt to address these issues. Method A systematic review of the literature was conducted in concordance to PRISMA protocol. Data were combined using STATA 12 and Open Meta-Analyst software programmes. Results Twenty-four studies were included comparing 2842 flaps performed in irradiated fields and 3491 flaps performed in non-irradiated fields. Meta-analysis yielded statistically significant risk ratios for flap failure (RR 1.48, P = 0.004), complications (RR 1.84, P 60 Gy radiation had a non-statistically significant higher risk of flap failure (RR 1.61, P = 0.145). Conclusion Preoperative radiation is associated with a statistically significant increased risk of flap complications, failure and fistula. Preoperative radiation in excess of 60 Gy after radiotherapy represents a potential risk factor for increased flap loss and should be avoided where possible.

This single case study discusses the various challenges and rehabilitation of Australia’s first hand transplant from the hand therapy perspective. The recipient was a 65-year-old quadrimembral amputee male following overwhelming pneumococcal sepsis. Key issues included cognitive relearning following the four-year loss of cortical feedback, extrinsic muscle retraining, poor coordination and functional limitations as a result of intrinsic dennervation, and an insensate hand. Distinct clinical differences from total hand replantation included consideration of immunosuppression drugs and potential rejection episodes. Although the patient was highly motivated, a client-centred approach was critical for ongoing commitment to the intensive long-term rehabilitation programme. The recipient’s progress in the first two and a half years has been impressive with achievement of many personal goals including eating, drinking, writing, driving and lawn bowls. His results, recorded on the International Hand Transplant Registry, showed superior measurements in areas of the DASH, grip strength, and TAM of forearm, wrist, and fingers.

The extracellular matrix (ECM) Matrigel™ has frequently and successfully been used to generate new adipose tissue experimentally, but is unsuitable for human application. This study sought to compare the adipogenic potential of a number of alternative, biologically derived or synthetic ECMs with potential for human application, with and without growth factors and a small fat autograft. Eight groups, with six severe combined immunodeficient (SCID) mice per group, were created with bilateral chambers (silicone tubes) implanted around the epigastric vascular pedicle, with one chamber/animal containing a 5mg fat autograft. Two animal groups were created for each of four ECMs (Matrigel™, Myogel, Cymetra® and PuraMatrix™) which filled the bilateral chambers. One group/ECM had no growth factors added to chambers whilst the other group had growth factors (GFs) (vascular endothelial growth factor-A (VEGF-A) plus fibroblast growth factor-2 (FGF-2) plus platelet-derived growth factor-BB (PDGF-BB)) added to both chambers. At 6weeks, chamber tissue was morphometrically assessed for percent and absolute adipose tissue volume. Overall, the triple GF regime significantly increased percent(∗) and absolute(#) adipose tissue volume (p

Poor vascularization remains a key limiting factor in translating advances in tissue engineering to clinical applications. Vascular pedicles (large arteries and veins) isolated in plastic chambers are known to sprout an extensive capillary network. This study examined the effect vascular pedicles and scaffold architecture have on vascularization and tissue integration of implanted silk scaffolds. Porous silk scaffolds with or without microchannels are manufactured to support implantation of a central vascular pedicle, without a chamber, implanted in the groin of Sprague Dawley rats, and assessed morphologically and morphometrically at 2 and 6 weeks. At both time points, blood vessels, connective tissue, and an inflammatory response infiltrate all scaffold pores externally, and centrally when a vascular pedicle is implanted. At week 2, vascular pedicles significantly increase the degree of scaffold tissue infiltration, and both the pedicle and the scaffold microchannels significantly increase vascular volume and vascular density. Interestingly, microchannels contribute to increased scaffold vascularity without affecting overall tissue infiltration, suggesting a direct effect of biomaterial architecture on vascularization. The inclusion of pedicles and microchannels are simple and effective proangiogenic techniques for engineering thick tissue constructs as both increase the speed of construct vascularization in the early weeks post in vivo implantation.

Tissue engineering and cell implantation therapies are gaining popularity because of their potential to repair and regenerate tissues and organs. To investigate the role of inflammatory cytokines in new tissue development in engineered tissues, we have characterized the nature and timing of cell populations forming new adipose tissue in a mouse tissue engineering chamber (TEC) and characterized the gene and protein expression of cytokines in the newly developing tissues. EGFP-labeled bone marrow transplant mice and MacGreen mice were implanted with TEC for periods ranging from 0.5 days to 6 weeks. Tissues were collected at various time points and assessed for cytokine expression through ELISA and mRNA analysis or labeled for specific cell populations in the TEC. Macrophage-derived factors, such as monocyte chemotactic protein-1 (MCP-1), appear to induce adipogenesis by recruiting macrophages and bone marrow-derived precursor cells to the TEC at early time points, with a second wave of nonbone marrow-derived progenitors. Gene expression analysis suggests that TNFα, LCN-2, and Interleukin 1β are important in early stages of neo-adipogenesis. Increasing platelet-derived growth factor and vascular endothelial cell growth factor expression at early time points correlates with preadipocyte proliferation and induction of angiogenesis. This study provides new information about key elements that are involved in early development of new adipose tissue.

The ability to generate controlled amounts of adipose tissue would greatly ease the burden on hospitals for reconstructive surgery. We have previously shown that a tissue engineering chamber containing a vascular pedicle was capable of forming new fat; however, further refinements are required to enhance fat formation. The development and maintenance of engineered adipose tissue requires a suitable source of growth factors and a suitable scaffold. A hydrogel derived from adipose tissue may fulfil this need. Subcutaneous fat was processed into a thermosensitive hydrogel we refer to as adipose-derived matrix (ADM). Protein analysis revealed high levels of basement membrane proteins, collagens and detectable levels of growth factors. Adipose-derived stem cells exposed to this hydrogel differentiated into adipocytes with >90% efficiency and in vivo testing in rats showed significant signs of adipogenesis after 8 weeks. ADM's adipogenic properties combined with its simple gelation, relatively long shelf life and its tolerance to multiple freeze-thaw cycles, makes it a promising candidate for adipose engineering applications.

Summary Introduction Options for breast reconstructions enclose autologous tissue transfers or implants. Fat grafting is gaining more interest in this specific field of breast surgery. This study concentrates on the technique and aesthetic results of breast reconstruction with fat grafts combined with implants, in women who have undergone total mastectomy. Methods Breast reconstructions (n = 23) was performed using a protocol of intratissular expansion with serial deflation-lipofilling. In order to achieve the best aesthetic outcome, an additional small implant was placed. A retrospective data analysis was performed. In all patients a tissue expander was placed at the time of mastectomy or after removal of a previous breast reconstruction. The mean of lipoaspirate material for the reconstruction was 333 mL (range 120–715 mL). To create an adequate volume of the reconstructed breast, a supplementary small implant was placed, with a mean volume of 222 mL (range 125–375 mL). The mean follow-up was 33 months (range 19–50 months). Results A MRI analysis was performed in eight patients at least 9 months after the last lipofilling procedure, demonstrating a mean of 171 mL (range 64–538 mL) of transferred fat, a mean fat survival of 53% and a volume ratio of fat graft/implant of 0.97 (range 0,3–3,8). Conclusion This composite technique of using autologous fat tissue and implants shows aesthetic pleasant results and must be considered as a valid alternative in a subset of patients. Further investigations to optimize the fat graft take must be encouraged.

Collagen 1 (C1) is commonly used to improve biological responses to implant surfaces. Here, the stability of C1 was compared with collagen 4 (C4) on a mixed macrodiol polyurethane, both adsorbed and covalently bound via acetaldehyde glow discharge polymerization and reductive amination. Substrate specimens were incubated in solutions of C1 and C4. The strength of conjugation was tested by incubation in 8 M urea followed by enzyme linked immunosorbent assays to measure residual C1 and C4. The basal lamina protein, laminin-332 (L332) was superimposed via adsorption on C4-treated specimens. Keratinocytes were grown on untreated, C1-treated, C4-treated, and C4 + L332-treated specimens, followed by measurement of cell area, proliferation, and focal adhesion density. Adsorbed C4 was shown to be significantly more stable than C1 and covalent conjugation conferred even greater stability, with no degradation of C4 over twenty days in 8 M urea. Cell growth was similar for C1 and C4, with no additional benefit conferred by superimposition of L332. The greater resistance of C4 to degradation may be consequent to cysteine residues and disulphide bonds in its non-collagenous domains. The use of C4 on implants, rather than C1, may improve their long-term stability in tissues. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1364-1373, 2017.

Thiol groups can undergo a large variety of chemical reactions and are used in solution phase to conjugate many bioactive molecules. Previous research on solid substrates with continuous phase glow discharge polymerization of thiol-containing monomers may have been compromised by oxidation. Thiol surface functionalization via glow discharge polymerization has been reported as requiring pulsing. Herein, continuous phase glow discharge polymerization of allyl mercaptan (2-propene-1-thiol) was used to generate significant densities of thiol groups on a mixed macrodiol polyurethane and tantalum. Three general classes of chemistry are used to conjugate proteins to thiol groups, with maleimide linkers being used most commonly. Here the pH specificity of maleimide reactions was used effectively to conjugate surface-bound thiol groups to amine groups in collagen. XPS demonstrated surface-bound thiol groups without evidence of oxidation, along with the subsequent presence of maleimide and collagen. Glow discharge reactor parameters were optimized by testing the resistance of bound collagen to degradation by 8 M urea. The nature of the chemical bonding of collagen to surface thiol groups was effectively assessed by colorimetric assay (ELISA) of residual collagen after incubation in 8 M urea over 8 days and after incubation with keratinocytes over 15 days. The facile creation of useable solid-supported thiol groups via continuous phase glow discharge polymerization of allyl mercaptan opens a route for attaching a vast array of bioactive molecules. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1940-1948, 2017.

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Vascularization is a major hurdle for growing three-dimensional tissue engineered constructs. This study investigated the mechanisms involved in hypoxic preconditioning of primary rat myoblasts in vitro and their influence on local angiogenesis postimplantation. Primary rat myoblast cultures were exposed to 90 min hypoxia at

Background Breast reconstruction involves the use of autologous tissues or implants. Occasionally, microsurgical reconstruction is not an option because of insufficient donor tissues. Fat grafting has become increasingly popular in breast surgery. The challenge with this technique is how to reconstruct a stable and living “scaffold” that resembles a breast. Methods Breast reconstruction (n=7) was performed using intratissular expansion with serial deflation–lipofilling sessions. Mean age of the patients was 41 years (22–53). The expander generated a vascularized capsule at 8 weeks, which demarcated a recipient site between the skin and the capsule itself, and functioned as a vascular source for angiogenesis. Serial sessions of deflation and lipofilling were initiated at 8 weeks with removal of the expander at the completion of the treatment. An average of 644ml (range, 415ml–950ml) of lipoaspirate material was injected to reconstruct the breast mound. An average of 4 (range, 3 to 5) fat-grafting sessions with a 3-month interval was needed to achieve symmetry with the contralateral breast. The average follow-up was 14 months (range, 9–29 months). MRI examination was performed at 8 months to analyze tissue survival and the residual volume. Results MRI examination retained tissue survival and the mean reconstructed breast volume was 386ml (range, 231ml–557ml). An aesthetically pleasant breast mound was created, with a high satisfaction rate. Conclusion We could reconstruct an aesthetically pleasant and stable breast mound in a selected group of patients by using intratissular expansion and fat grafting.