Introduction Up to one-fifth of breast cancer survivors will develop chronic breast cancer-related lymphoedema (BCRL). To date, complex physical decongestion therapy (CDT) is the gold standard of treatment. However, it is mainly symptomatic and often ineffective in preventing BCRL progression. Lymphovenous anastomosis (LVA) and vascularised lymph node transfer (VLNT) are microsurgical techniques that aim to restore lymphatic drainage. This international randomised trial aims to evaluate advantages of microsurgical interventions plus CDT versus CDT alone for BCRL treatment.Methods and analysis The effectiveness of LVA and/or VLNT in combination with CDT, which may be combined with liposuction, versus CDT alone will be evaluated in routine practice across the globe. Patients with BCRL will be randomly allocated to either surgical or conservative therapy. The primary end point of this trial is the patient-reported quality of life (QoL) outcome ‘lymphoedema-specific QoL’, which will be assessed 15 months after randomisation. Secondary end points are further patient-reported outcomes (PROs), arm volume measurements, economic evaluations and imaging at different time points. A long-term follow-up will be conducted up to 10 years after randomisation. A total of 280 patients will be recruited in over 20 sites worldwide.Ethics and dissemination This study will be conducted in compliance with the Declaration of Helsinki and the International Council for Harmonisation-Good Clinical Practice (ICH-GCP) E6 guideline. Ethical approval has been obtained by the lead ethics committee ‘Ethikkommission Nordwest- und Zentralschweiz’ (2023-00733, 22 May 2023). Ethical approval from local authorities will be sought for all participating sites. Regardless of outcomes, the findings will be published in a peer-reviewed medical journal. Metadata detailing the dataset’s type, size and content will be made available, along with the full study protocol and case report forms, in public repositories in compliance with the Findability, Accessibility, Interoperability and Reuse principles.Trial registration number NCT05890677.

Summary: Lower extremity reconstruction with free flaps in patients with only peroneal artery runoff remains a challenge. Here, we present a novel technique for reconstruction of medial defects in the distal leg using a medial approach to the peroneal artery and a short interposition vein graft anastomosed end to side to the peroneal artery. A retrospective, single-center study was performed including all patients who underwent lower extremity reconstruction with free flaps anastomosed to the peroneal artery using a mini vein graft from November 2020 to March 2022. The primary outcome measure was limb salvage. Secondary endpoints were flap survival and postoperative complications. Seven patients received lower extremity free flap reconstruction with a mini vein graft to the peroneal artery. Flap loss rate was 0%. Limb salvage was achieved in five patients (71%). At 6-month follow-up, all patients were ambulatory. One patient died 1 month after surgery due to heart failure. Mini vein graft to the peroneal artery allows reliable and safe free flap reconstruction of distal leg defects in patients with only peroneal artery runoff.

BACKGROUND:Total or subtotal glossectomy defects cause significant functional deficits in swallowing and speech and subsequently impair patients' quality of life (QOL). Recently, the profunda artery perforator (PAP) flap has emerged as a potential alternative for reconstructing extensive glossectomy defects. While previous studies assessing recovery of neurotized anterolateral thigh (ALT) flaps in head and neck reconstruction reported superior sensory recovery, improved swallow function, and improved overall patient satisfaction in patients with neurotized flaps vs. non-neurotized ALT flap reconstruction, PAP flap neurotization has not been described and systematically assessed in head and neck patients. METHODS:Six patients underwent subtotal tongue reconstruction with neurotized PAP flaps at the authors' institution from May 2022 until August 2023. A branch of the posterior femoral cutaneous nerve of the PAP flap was coaptated to the lingual nerve. Two-point discrimination, Semmes-Weinstein monofilament, pain, and temperature assessments were conducted at 3, 6, and 12 months postoperatively on the neo-tongue. The MD Anderson speech and deglutition scales and the EORTC-QLQ-H&N35 were used to record functional outcomes and QOL. RESULTS:The mean age was 69 ± 4 years, and the mean body mass index was 25 ± 7 kg/m2. Neo-tongue median 2-point discrimination at the tip improved from >10 mm at 3 months to 6 mm at 12 months. All patients had protective pain and temperature perception at the neo-tongue tip at the 6-month follow-up. Speech and swallowing functions were similar at the 12-month follow-up to data on neurotized ALT flaps from literature. No neuropathic pain was reported at the donor site at the 6-month follow-up. CONCLUSIONS:This is the first case series of PAP flap neurotization in head and neck patients, suggesting potential functional advantages with minimal donor-site morbidity. LEVEL OF EVIDENCE:V Case Series.

Zusammenfassung Der Anteil an Patienten jenseits der 7. Lebensdekade in der Bevölkerung steigt weltweit, insbesondere in den hochentwickelten Ländern. Der Bedarf an komplexen Rekonstruktionen der unteren Extremitäten nach Trauma, Tumor oder Infektion in dieser Altersgruppe nimmt zu. Die Rekonstruktion von Weichteildefekten an der unteren Extremität sollte nach dem Prinzip der plastisch-rekonstruktiven Stufenleiter resp. des Aufzugs erfolgen, um schmerzfreies und sicheres Stehen und Gehen zu erhalten. Insbesondere beim älteren Patienten sind sorgfältige multidisziplinäre Planung, gezielte präoperative Beurteilung und Optimierung von Komorbiditäten, wie z. B. Diabetes, Mangelernährung oder pathologischen Gefäßveränderungen, sowie ein altersentsprechendes perioperatives Management erforderlich. Durch die Umsetzung dieser Prinzipien können ältere und hochbetagte Patienten ihre Mobilität und ihre Autonomie, die für eine hohe Lebensqualität entscheidend sind, beibehalten.

The proportion of patients in the population beyond the 7th decade of life is increasing worldwide, especially in highly developed countries. Consequently, there is also an increasing need for complex lower extremity reconstructions after trauma, tumors, or infections in this age group. The reconstruction of soft tissue defects of the lower extremity should be performed according to the principle of the plastic-reconstructive ladder or elevator. The goal of reconstruction is to restore anatomy and function of the lower extremity to enable pain-free and stable standing and walking; however, for older patients in particular, a careful preoperative multidisciplinary planning, detailed preoperative assessment and optimization of comorbidities, such as diabetes, malnutrition or pathological vascular alterations, as well an age-adapted perioperative management are necessary. By implementing these principles, older and very old patients can maintain their mobility and autonomy, which are crucial for a high quality of life.

Pressure injuries (PI) are a common issue among individuals with spinal cord injury (SCI), especially in the sitting areas of the body. Considering the risk of infections occurring to PI during the wound healing process, the skin microbiome is likely to be a source of bacteria. We investigated the relationship between skin and PI microbiomes, and assessed any correlation with clinically relevant outcomes related to PI. Samples were isolated from SCI patients undergoing reconstructive surgery of PI, severity grades III and IV. DNA samples from skin and PI were analysed using 16S rRNA gene sequencing. Our results showed disparities in microbiome composition between skin and PI. The skin had lower diversity, while PI showed increased bacterial homogeneity as the severity grade progressed. The skin bacterial composition varied based on its location, influenced by Cutibacterium . Compositional differences were identified between PI grades III and IV, with clusters of bacteria colonizing PI, characterized by Pseudomonas , Proteus and Peptoniphilus . The skin and PI microbiomes were not affected by the level of the SCI. Our study highlights the differences in the microbiome of skin and PI in SCI patients. These findings could be used to target specific bacteria for PI treatment in clinical practice.

Replacement of damaged or lost tissue typically relies on the availability of living, functional substitutes and the rapid development of a stable and efficient vascularization upon transplantation, in order to guarantee their survival. These requirements challenge current surgical reconstruction techniques in the clinical practice.In the past decades, the field of tissue engineering has introduced the possibility to combine materials and living cells to generate functional substitutes, which can be tailored to specific requirements of the implantation site. At the same time, plastic and reconstructive surgery has developed a large armamentarium of grafting possibilities and flaps supporting vascularization of native tissues, especially through progress made in microsurgical techniques.In this chapter, we describe advances in the two fields and discuss how the principles and techniques independently developed could be combined towards the prefabrication of vascularized tissues. The resulting paradigm of “regenerative surgery,” here exemplified in the specific context of bone regeneration, could represent the future standard for the reconstruction of complex body parts.

The reconstruction of complex midface defects is a challenging clinical scenario considering the high anatomical, functional, and aesthetic requirements. In this study, we proposed a surgical treatment to achieve improved oral rehabilitation and anatomical and functional reconstruction of a complex defect of the maxilla with a vascularized, engineered composite graft. The patient was a 39-year-old female, postoperative after left hemimaxillectomy for ameloblastic carcinoma in 2010 and tumor-free at the 5-year oncological follow-up. The left hemimaxillary defect was restored in a two-step approach. First, a composite graft was ectopically engineered using autologous stromal vascular fraction (SVF) cells seeded on an allogenic devitalized bone matrix. The resulting construct was further loaded with bone morphogenic protein-2 (BMP-2), wrapped within the latissimus dorsi muscle, and pedicled with an arteriovenous (AV) bundle. Subsequently, the prefabricated graft was orthotopically transferred into the defect site and revascularized through microvascular surgical techniques. The prefabricated graft contained vascularized bone tissue embedded within muscular tissue. Despite unexpected resorption, its orthotopic transfer enabled restoration of the orbital floor, separation of the oral and nasal cavities, and midface symmetry and allowed the patient to return to normal diet as well as to restore normal speech and swallowing function. These results remained stable for the entire follow-up period of 2 years. This clinical case demonstrates the safety and the feasibility of composite graft engineering for the treatment of complex maxillary defects. As compared to the current gold standard of autologous tissue transfer, this patient’s benefits included decreased donor site morbidity and improved oral rehabilitation. Bone resorption of the construct at the ectopic prefabrication site still needs to be further addressed to preserve the designed graft size and shape.

Avascular necrosis of bone (AVN) leads to sclerosis and collapse of bone and joints. We have previously shown that axially vascularized osteogenic constructs, engineered by combining human stromal vascular fraction (SVF) cells and a ceramic scaffold, can revitalize necrotic bone of clinically relevant size in a rat model of AVN. For a clinical translation, the fetal bovine serum (FBS) used to generate such grafts should be substituted by a nonxenogeneic culture supplement. Human thrombin-activated platelet-rich plasma (tPRP) was evaluated in this context. SVF cells were cultured inside porous hydroxyapatite scaffolds with a perfusion-based bioreactor system for 5 days. The culture medium was supplemented with either 10% FBS or 10% tPRP. The resulting constructs were inserted into devitalized bovine bone cylinders to mimic the treatment of a necrotic bone. A ligated vascular bundle was inserted into the constructs upon subcutaneous implantation in the groin of nude rats. After 1 and 8 weeks, constructs were harvested, and vascularization, host cell recruitment, and bone formation were analyzed. After 1 week in vivo, constructs were densely vascularized, with no difference between tPRP- and FBS-based ones. After 8 weeks, bone formation and vascularization was found in both tPRP- and FBS-precultured constructs. However, the amount of bone and the vessel density were respectively 2.2- and 1.8-fold higher in the tPRP group. Interestingly, the density of M2, proregenerative macrophages was also significantly higher (6.9-fold) following graft preparation with tPRP than with FBS. Our findings indicate that tPRP is a suitable substitute for FBS to generate vascularized, osteogenic grafts from SVF cells and could thus be implemented in protocols for clinical translation of this strategy towards the treatment of bone loss and AVN.

Large and complex bone defects represent challenging clinical scenarios, typically requiring autologous vascularized bone transplants. In order to bypass the numerous associated limitations, here we aimed at ectopically prefabricating a bone graft surrogate with vascular pedicle. A hollow cylinder of devitalized cancellous bone was used to define the space of a large bone substitute. This space was filled with devitalized pellets of engineered hypertrophic cartilage as bone-inducing material, in combination or not with stromal vascular fraction (SVF) of adipose tissue as source of osteoprogenitors and endothelial cells. Vascularization of the space was targeted through axial insertion of an arterio-venous (AV) bundle. Constructs were subcutaneously implanted in nude rats for 12 weeks and analyzed for bone formation and vascularization by histology and microtomography. Retrieved constructs were extensively vascularized in all conditions, with vessels sprouting from the AV bundle and reaching a higher density in the axially central volume. Bone tissue was formed through remodeling of hypertrophic cartilage, and quantitatively correlated with de novo vascularization. Our study demonstrates feasibility to prefabricate large, pedicled bone grafts in predefined shapes. The combination of an AV bundle with engineered hypertrophic cartilage provided a germ for the coupled processes of vascularization and bone formation. The demonstrated osteoinductivity of devitalized hypertrophic cartilage offers the opportunity of implementing the proposed regenerative surgery strategy through off-the-shelf materials.

Despite the various reported approaches to generate osteochondral composites by combination of different cell types and materials, engineering of templates with the capacity to autonomously and orderly develop into cartilage-bone bi-layered structures remains an open challenge. Here, we hypothesized that the embedding of cells inducible to endochondral ossification (i.e. bone marrow derived mesenchymal stromal cells, BMSCs) and of cells capable of robust and stable chondrogenesis (i.e. nasal chondrocytes, NCs) adjacent to each other in bi-layered hydrogels would develop directly in vivo into osteochondral tissues. Poly(ethylene glycol) (PEG) hydrogels were functionalized with TGFβ3 or BMP-2, enzymatically polymerized encapsulating human BMSCs, combined with a hydrogel layer containing human NCs and ectopically implanted in nude mice without pre-culture. The BMSC-loaded layers reproducibly underwent endochondral ossification and generated ossicles containing bone and marrow. The NC-loaded layers formed cartilage tissues, which (under the influence of BMP-2 but not of TGFβ3 from the neighbouring layer) remained phenotypically stable. The proposed strategy, resulting in orderly connected osteochondral composites, should be further assessed for the repair of osteoarticular defects and will be useful to model developmental processes leading to cartilage-bone interfaces.

Bone tissue has a strong intrinsic regenerative capacity, thanks to a delicate and complex interplay of cellular and molecular processes, which tightly involve the immune system. Pathological settings of anatomical, biomechanical or inflammatory nature may lead to impaired bone healing. Innovative strategies to enhance bone repair, including the delivery of osteoprogenitor cells or of potent cytokines/morphogens, indicate the potential of ‘orthobiologics’, but are not fully satisfactory. Here, we review different approaches based on the delivery of regenerative cues produced by cells but in cell-free, possibly off-the-shelf configurations. Such strategies exploit the paracrine effect of the secretome of mesenchymal stem/stromal cells, presented in soluble form, shuttled through extracellular vesicles, or embedded within the network of extracellular matrix molecules. In addition to osteoinductive molecules, attention is given to factors targeting the resident immune cells, to reshape inflammatory and immunity processes from scarring to regenerative patterns.

INTRODUCTION: Avascular necrosis of bone (AVN) leads to sclerosis and collapse of bone and joints. Engineered, axially vascularized, human adipose-derived stromal vascular fraction (SVF) cell based osteogenic constructs have been shown to revitalize necrotic bone of clinically-relevant size in a challenging rat model of AVN. To reduce regulatory difficulties towards clinical translation, fetal bovine serum (FBS) was substituted by thrombin-activated platelet-rich plasma (tPRP). METHODS: SVF cells from 5 lipoaspirate were isolated and cultured onto porous hydroxyapatite scaffolds within a perfusion-based bioreactor system for 5 days. The medium was supplemented either with 10% FBS or 10% tPRP. The resulting constructs were inserted into devitalized bone cylinders mimicking AVN-affected bone. A ligated vascular bundle was inserted upon subcutaneous implantation of constructs in nude rats. After 1 and 8 weeks, vascularization and bone formation were analyzed. RESULTS: After 1 week, neither maximal distance of vessels from the bundle nor vascular density was significantly different. However, FBS cultured grafts revealed significantly more human vessel (hCD34) and osteoclasts compared to tPRP cultured grafts. Overall macrophages and M2 macrophages were not different between FBS and tPRP cultured grafts. After 8 weeks in vivo, 3/5 samples from FBS culture and 1/5 sample from tPRP culture showed bone formation. Roughly 40% of the osteocytes were of human origin (36% from FBS and 42% from tPRP culture). DISCUSSION: Despite promising results in literature, replacement of FBS by tPRP resulted in a reduced capacity of bone formation in our series. Further studies are necessary to analyze and improve the low bone formation capacity, possibly caused by the heterogeneity of the SVF donors and tPRP samples.

Background: Avascular necrosis of bone (AVN) leads to sclerosis and collapse of bone and joints. The standard of care, vascularized bone grafts, is limited by donor site morbidity and restricted availability. The aim of this study was to generate and test engineered, axially vascularized SVF cells-based bone substitutes in a rat model of AVN. Methods: SVF cells were isolated from lipoaspirates and cultured onto porous hydroxyapatite scaffolds within a perfusion-based bioreactor system for 5 days. The resulting constructs were inserted into devitalized bone cylinders mimicking AVN-affected bone. A ligated vascular bundle was inserted upon subcutaneous implantation of constructs in nude rats. After 1 and 8 weeks in vivo, bone formation and vascularization were analyzed. Results: Newly-formed bone was found in 80% of SVF-seeded scaffolds after 8 weeks but not in unseeded controls. Human ALU + cells in the bone structures evidenced a direct contribution of SVF cells to bone formation. A higher density of regenerative, M2 macrophages was observed in SVF-seeded constructs. In both experimental groups, devitalized bone was revitalized by vascularized tissue after 8 weeks. Conclusion: SVF cells-based osteogenic constructs revitalized fully necrotic bone in a challenging AVN rat model of clinically-relevant size. SVF cells contributed to accelerated initial vascularization, to bone formation and to recruitment of pro-regenerative endogenous cells. Statement of significance Avascular necrosis (AVN) of bone often requires surgical treatment with autologous bone grafts, which is surgically demanding and restricted by significant donor site morbidity and limited availability. This paper describes a de novo engineered axially-vascularized bone graft substitute and tests the potential to revitalize dead bone and provide efficient new bone formation in a rat model. The engineering of an osteogenic/vasculogenic construct of clinically-relevant size with stromal vascular fraction of human adipose, combined to an arteriovenous bundle is described. This construct revitalized and generated new bone tissue. This successful approach proposes a novel paradigm in the treatment of AVN, in which an engineered, vascularized osteogenic graft would be used as a germ to revitalize large volumes of necrotic bone. (C) 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Objectives . Bone ischemia and necrosis are challenging to treat, requiring investigation of native and engineered bone revascularisation processes through advanced imaging techniques. This study demonstrates an experimental two-step method for precise bone and vessel analysis in native bones or vascularised bone grafts using X-ray microtomography ( μ CT), without interfering with further histological processing. Methods . Distally ligated epigastric arteries or veins of 6 nude rats were inserted in central channels of porous hydroxyapatite cylinders and these pedicled grafts were implanted subcutaneously. One week later, the rats were perfused with ink-gelatin and euthanised and the femurs, tibias, and grafts were explanted. Samples were scanned using μ CT, decalcified, incubated with phosphotungstic acid (PTA) for contrast enhancement, rescanned, and processed histologically. Results. Contrast-enhanced μ CT displayed the course and branching of native bone vessels. Histologically, both central (−17%) and epiphyseal vessels (−58%) appeared smaller than in μ CT scans. Hydroxyapatite cylinders were thoroughly vascularised but did not display bone formation. Grafts with a central artery had more (+58%) and smaller (−52%) vessel branches compared to grafts with a vein. Conclusions . We present a relatively inexpensive and easy-to-perform two-step method to analyse bone and vessels by μ CT, suitable to assess a variety of bone-regenerative strategies.

Bone-derived mesenchymal stromal cells (MSCs) differentiate into multiple lineages including chondro- and osteogenic fates and function in establishing the hematopoietic compartment of the bone marrow. Here, we analyze the emergence of different MSC types during mouse limb and long bone development. In particular, PDGFRαposSCA-1pos (PαS) cells and mouse skeletal stem cells (mSSCs) are detected within the PDGFRαposCD51pos (PαCD51) mesenchymal progenitors, which are the most abundant progenitors in early limb buds and developing long bones until birth. Long-bone-derived PαS cells and mSSCs are most prevalent in newborn mice, and molecular analysis shows that they constitute distinct progenitor populations from the earliest stages onward. Differential expression of CD90 and CD73 identifies four PαS subpopulations that display distinct chondro- and osteogenic differentiation potentials. Finally, we show that cartilage constructs generated from CD90pos PαS cells are remodeled into bone organoids encompassing functional endothelial and hematopoietic compartments, which makes these cells suited for bone tissue engineering.

: Engineered and devitalized hypertrophic cartilage (HC) has been proposed as bone substitute material, potentially combining the features of osteoinductivity, resistance to hypoxia, capacity to attract blood vessels, and customization potential for specific indications. However, in comparison with vital tissues, devitalized HC grafts have reduced efficiency of bone formation and longer remodeling times. We tested the hypothesis that freshly harvested stromal vascular fraction (SVF) cells from human adipose tissue-which include mesenchymal, endothelial, and osteoclastic progenitors-enhance devitalized HC remodeling into bone tissue. Human SVF cells isolated from abdominal lipoaspirates were characterized cytofluorimetrically. HC pellets, previously generated by human bone marrow-derived stromal cells and devitalized by freeze/thaw, were embedded in fibrin gel with or without different amounts of SVF cells and implanted either ectopically in nude mice or in 4-mm-diameter calvarial defects in nude rats. In the ectopic model, SVF cells added to devitalized HC directly contributed to endothelial, osteoblastic, and osteoclastic populations. After 12 weeks, the extent of graft vascularization and amount of bone formation increased in a cell-number-dependent fashion (up to, respectively, 2.0-fold and 2.9-fold using 12 million cells per milliliter of gel). Mineralized tissue volume correlated with the number of implanted, SVF-derived endothelial cells (CD31+ CD34+ CD146+). In the calvarial model, SVF activation of HC using 12 million cells per milliliter of gel induced efficient merging among implanted pellets and strongly enhanced (7.3-fold) de novo bone tissue formation within the defects. Our findings outline a bone augmentation strategy based on off-the-shelf devitalized allogeneic HC, intraoperatively activated with autologous SVF cells.SIGNIFICANCE:This study validates an innovative bone substitute material based on allogeneic hypertrophic cartilage that is engineered, devitalized, stored, and clinically used, together with autologous cells, intraoperatively derived from a lipoaspirate. The strategy was tested using human cells in an ectopic model and an orthotopic implantation model, in immunocompromised animals.

In this report, we present two cases of the bony reconstruction with the medial trochlea (MFT) flap including a skin island that was used to monitor the perfusion of flap in the postoperative period. Between March 2013 and April 2015, we performed surgery on two patients who suffered from scaphoid and talus non‐union after trauma and initial treatment by osteosynthesis. A skin island (1 cm × 1 cm and 3 cm × 1 cm, respectively) was included with the osseous flap (1.6 cm × 1 cm × 1 cm and 2 cm × 3 cm × 2 cm, respectively) to assess the perfusion of the flap. The design of the skin island was based on either the saphenous artery perforator or a cutaneous perforator of the descending genicular artery. Both flaps remained viable throughout the postoperative period, and there were no donor site complications. After a follow‐up of 36 and 11 months, bony union was observed in both patients with a high degree of satisfaction. Thus, a MFT flap with a skin island could be a tool to assess the perfusion of the flap in the early postoperative period. © 2016 Wiley Periodicals, Inc. Microsurgery 37:431–435, 2017.