Prevascularized spongy-like hydrogels maintain their angiogenic potential after prolonged hypothermic storage.

The chronic shortage of organs and tissues for transplantation represents a dramatic burden on healthcare systems worldwide. Tissue engineering offers a potential solution to address these shortages, but several challenges remain, with prevascularization being a critical factor for in vivo survival and integration of tissue engineering products. Concurrently, a different challenge hindering the clinical implementation of such products, regards their efficient preservation from the fabrication site to the bedside. Hypothermia has emerged as a potential solution for this issue due to its milder effects on biologic systems in comparison with other cold preservation methodologies. Its impact on prevascularization, however, has not been well studied. In this work, 3D prevascularized constructs were fabricated using adipose-derived stromal vascular fraction cells and preserved at 4 °C using Hypothermosol or basal culture media (α-MEM). Hypothermosol efficiently preserved the structural and cellular integrity of prevascular networks as compared to constructs before preservation. In contrast, the use of α-MEM led to a clear reduction in prevascular structures, with concurrent induction of high levels of apoptosis and autophagy at the cellular level. In vivo evaluation using a chorioallantoic membrane model demonstrated that, in opposition to α-MEM, Hypothermosol preservation retained the angiogenic potential of constructs before preservation by recruiting a similar number of blood vessels from the host and presenting similar integration with host tissue. These results emphasize the need of studying the impact of preservation techniques on key properties of tissue engineering constructs such as prevascularization, in order to validate and streamline their clinical application.

Generation of 3D melanoma models using an assembloid-based approach.

While 3D tumor models have greatly evolved over the past years, there is still a strong requirement for more biosimilar models which are capable of recapitulating cellular crosstalk within the tumor microenvironment while equally displaying representative levels of tumor aggressiveness and invasion. Herein, we disclose an assembloid melanoma model based on the fusion of individual stromal multicellular spheroids (MCSs). In contrast to more traditional tumor models, we show that it is possible to develop self-organizing, heterotypic melanoma models where tumor cells present stem-cell like features like up-regulated pluripotency master regulators SOX2, POU5F1 and NANOG. Additionally, these assembloids display high levels of invasiveness while embedded in 3D matrices as evidenced by stromal cell promotion of melanoma cell invasion via metalloproteinase production. Furthermore, sensitivity to anticancer drug doxorubicin was demonstrated for the melanoma assembloid model. These findings suggest that melanoma assembloids may play a significant role in the field of 3D cancer models as they more closely mimic the tumor microenvironment when compared to more traditional MCSs, opening the doors to a better understanding of the role of tumor microenvironment in supporting tumor progression. STATEMENT OF SIGNIFICANCE: The development of complex 3D tumor models that better recapitulate the tumor microenvironment is crucial for both an improved comprehension of intercellular crosstalk and for more efficient drug screening. We have herein developed a self-organizing heterotypic assembloid-based melanoma model capable of closely mimicking the tumor microenvironment. Key features recapitulated were the preservation of cancer cell stemness, sensitivity to anti-cancer agents and tumor cell invasion promoted by stromal cells. The approach of pre-establishing distinct stromal domains for subsequent combination into more complex tumor constructs provides a route for developing superior tumor models with a higher degree of similarity to native cancer tissues.

Pre-selection of fibroblast subsets prompts prevascularization of tissue engineered skin analogues.

The papillary and reticular dermis harbors phenotypically distinct fibroblasts, whose functions such as maintenance of skin's microvasculature are also distinct. Thus, we hypothesized that pre-selection of the subpopulations of fibroblasts would benefit the generation of skin tissue engineered (TE) constructs, promoting their prevascularization in vitro. We first isolated papillary and reticular fibroblasts using fluorescence-activated cell sorting and studied the effect of their secretome and extracellular matrix (ECM) on human dermal microvascular endothelial cell (hDMEC) organization. Subsequently, we developed a bilayered 3D polymeric structure with distinct layer-associated features to house the subpopulations of fibroblasts, to generate a skin analogue. Both papillary and reticular fibroblasts were able to stimulate capillary-like network formation in a Matrigel assay. However, the secretome of the two subpopulations was substantially different, being enriched in VEGF, IGF-1, and Angio-1 in the case of papillary fibroblasts and in HGF and FGF-2 for the reticular subset. In addition, the fibroblast subpopulations deposited varied levels of ECM proteins, more collagen I and laminin was produced by the reticular subset, but these differences did not impact hDMEC organization. Vessel-like structures with lumens were observed earlier in the 3D skin analogue prepared with the sorted fibroblasts, although ECM deposition was not affected by the cell's pre-selection. Moreover, a more differentiated epidermal layer was obtained in the skin analogue formed by the sorted fibroblasts, confirming that its whole structure was not affected. Overall, we provide evidence that pre-selection of papillary and reticular fibroblasts is relevant for promoting the in vitro prevascularization of skin TE constructs.

Stromal Vascular Fraction Obtained From Subcutaneous Adipose Tissue: Ex-Obese and Older Population as Main Clinical Targets.

INTRODUCTION: Human adipose tissue contains a heterogeneous and synergistic mixture of cells called stromal vascular fraction (SVF) with highly proliferative and angiogenic properties, conferring promising applicability in the field of regenerative medicine. This study aims to investigate if age, body mass index (BMI), history of obesity and massive weight loss, and harvest site are related to SVF cell marker expression. METHODS: A total of 26 samples of subcutaneous adipose tissue were harvested from patients admitted to the Plastic and Reconstructive department in University Hospital Center of São João, Porto, Portugal, for body contouring surgery. The percentage of cells expressing CD31, CD34, CD45, CD73, CD90, and CD105 was assessed and compared with patient's age, BMI, history of obesity and massive weight loss (ex-obese group), and harvest site. RESULTS: In the ex-obese group, a significantly higher number of cells expressing CD90 (P = 0.002) was found. BMI, harvest site, and age appear to have no association with SVF subpopulations. CONCLUSIONS: This study suggests that ex-obese patients have a higher percentage of SVF cells expressing CD90, which correlates with higher proliferative and angiogenic rates. The effect of former obesity and massive weight loss on the expression of CD90 is a new and relevant finding because it makes this population a suitable candidate for reconstructive and aesthetic surgery and other fields of regenerative medicine. The use of SVF appears also promising in older patients because no negative correlation between increasing age and different cell markers expression was found.

Successful Surgical Resection and Reconstruction of a Giant Facial Vascular Malformation in an Elderly Patient.

Venous vascular malformations can be challenging, especially in an elderly patient. As these lesions can present with ulceration, deformity, pain, and swelling resulting from thrombi formed due to low flow (palpable phleboliths), removing them can be important for the well-being of the patient. A 79-year-old patient presented with a giant venous malformations occupying the left hemiface and a deprivation amblyopia in his left eye. Successful surgical resection and reconstruction was achieved and the patient was very pleased with his new appearance. Despite his age and surgical risks, we consider that never is late to improve a disfigured face.

Role of the Plastic Surgeon in Tuberculosis Treatment.

Background: Tuberculous empyema is rare. Its treatment requires oral antituberculous drugs, empyema drainage, and in severe cases, decortication and pneumectomy. In the presence of tuberculosis, lung resection has a high risk of postoperative bronchopleural fistula (BPF) and empyema. Treatment includes drainage, fistula occlusion, dead space obliteration, and infection control. Muscle flap transfer allows BPF occlusion and dead space obliteration. Methods: This report presents a case of a 63-year-old man with tuberculosis and postoperative BPF with empyema after pleural decortication and left lower lobe resection. The empyema was drained, and antituberculous drugs were started. The BPF was occluded with a latissimus dorsi and serratus anterior chimeric muscle flap, and the remaining thoracic dead space and chest wall defect were reconstructed with a pedicled pectoralis major myocutaneous flap. Results: Healing occurred uneventfully, and the patient was discharged from the hospital after 2 weeks. Conclusions: This type of thoracic defect is rare nowadays, especially in the setting of tuberculous infections. Although workhorse flaps like latissimus dorsi or pectoralis major flaps have been progressively surpassed by more elegant solutions like fasciocutaneous pedicled flaps and free flaps, they must still be considerations in the decision-making process of a reconstructive surgeon, and flap choice must be made on a case-by-case basis.

Spongy-like hydrogels prevascularization with the adipose tissue vascular fraction delays cutaneous wound healing by sustaining inflammatory cell influx.

In vitro prevascularization is one of the most explored approaches to foster engineered tissue vascularization. We previously demonstrated a benefit in tissue neovascularization by using integrin-specific biomaterials prevascularized by stromal vascular fraction (SVF) cells, which triggered vasculogenesis in the absence of extrinsic growth factors. SVF cells are also associated to biological processes important in cutaneous wound healing. Thus, we aimed to investigate whether in vitro construct prevascularization with SVF accelerates the healing cascade by fostering early vascularization vis-à-vis SVF seeding prior to implantation. Prevascularized constructs delayed re-epithelization of full-thickness mice wounds compared to both non-prevascularized and control (no SVF) groups. Our results suggest this delay is due to a persistent inflammation as indicated by a significantly lower M2(CD163+)/M1(CD86+) macrophage subtype ratio. Moreover, a slower transition from the inflammatory to the proliferative phase of the healing was confirmed by reduced extracellular matrix deposition and increased presence of thick collagen fibers from early time-points, suggesting the prevalence of fiber crosslinking in relation to neodeposition. Overall, while prevascularization potentiates inflammatory cell influx, which negatively impacts the cutaneous wound healing cascade, an effective wound healing was guaranteed in non-prevascularized SVF cell-containing spongy-like hydrogels confirming that the SVF can have enhanced efficacy.

Growth Factor-Free Vascularization of Marine-Origin Collagen Sponges Using Cryopreserved Stromal Vascular Fractions from Human Adipose Tissue.

The successful integration of transplanted three-dimensional tissue engineering (TE) constructs depends greatly on their rapid vascularization. Therefore, it is essential to address this vascularization issue in the initial design of constructs for perfused tissues. Two of the most important variables in this regard are scaffold composition and cell sourcing. Collagens with marine origins overcome some issues associated with mammal-derived collagen while maintaining their advantages in terms of biocompatibility. Concurrently, the freshly isolated stromal vascular fraction (SVF) of adipose tissue has been proposed as an advantageous cell fraction for vascularization purposes due to its highly angiogenic properties, allowing extrinsic angiogenic growth factor-free vascularization strategies for TE applications. In this study, we aimed at understanding whether marine collagen 3D matrices could support cryopreserved human SVF in maintaining intrinsic angiogenic properties observed for fresh SVF. For this, cryopreserved human SVF was seeded on blue shark collagen sponges and cultured up to 7 days in a basal medium. The secretome profile of several angiogenesis-related factors was studied throughout culture times and correlated with the expression pattern of CD31 and CD146, which showed the formation of a prevascular network. Upon in ovo implantation, increased vessel recruitment was observed in prevascularized sponges when compared with sponges without SVF cells. Immunohistochemistry for CD31 demonstrated the improved integration of prevascularized sponges within chick chorioalantoic membrane (CAM) tissues, while in situ hybridization showed human cells lining blood vessels. These results demonstrate the potential of using cryopreserved SVF combined with marine collagen as a streamlined approach to improve the vascularization of TE constructs.

Integrin-specific hydrogels for growth factor-free vasculogenesis.

Integrin-binding biomaterials have been extensively evaluated for their capacity to enable de novo formation of capillary-like structures/vessels, ultimately supporting neovascularization in vivo. Yet, the role of integrins as vascular initiators in engineered materials is still not well understood. Here, we show that αvß3 integrin-specific 3D matrices were able to retain PECAM1+ cells from the stromal vascular fraction (SVF) of adipose tissue, triggering vasculogenesis in vitro in the absence of extrinsic growth factors. Our results suggest that αvß3-RGD-driven signaling in the formation of capillary-like structures prevents the activation of the caspase 8 pathway and activates the FAK/paxillin pathway, both responsible for endothelial cells (ECs) survival and migration. We also show that prevascularized αvß3 integrin-specific constructs inosculate with the host vascular system fostering in vivo neovascularization. Overall, this work demonstrates the ability of the biomaterial to trigger vasculogenesis in an integrin-specific manner, by activating essential pathways for EC survival and migration within a self-regulatory growth factor microenvironment. This strategy represents an improvement to current vascularization routes for Tissue Engineering constructs, potentially enhancing their clinical applicability.

Salvage of a Near-Total Penile Amputation following Urinary Fistulization and Carbapenemase-Producing Klebsiella pneumoniae Infection with a Composite ALT Flap and Vascularized Fascia Lata.

Reconstruction of complex penile defects is always challenging, as some defects are not possible to reconstruct with skin or mucosa grafts, and even local flaps may be precluded in complex wounds. We present a case of a 63-year-old otherwise healthy man, who underwent transurethral resection of the prostate for benign prostatic hyperplasia. After the procedure, he developed panurethral necrosis with consequent stricture. Three urethroplasties for reconstruction of the bulbar and distal urethra using buccal mucosa grafts, a preputial flap, and penile skin were performed by urology team in different institutions, but serious urinary fistulization and carbapenemase-producing Klebsiella pneumoniae (KPC) infection translated in a chronic wound, urethra necrosis, and near-total penile amputation. A composite anterolateral thigh flap and vascularized fascia lata were used with success together with a perineal urethroplasty in different stages, improving the ischemic wound condition. The extended segment of fascia lata was used for Buck's fascia replacement and circumferential reinforcement to cover the erectile bodies of the penis. The postoperative period was uneventful and after 12 months, there were no signs of recurrence or wound dehiscence. He was able and easily adapted to void in a seated position through the perineal urethrostomy that was made. To the best of our knowledge, this procedure has not been reported previously as a salvage procedure in a fistulizated and KPC infected penis, but it may be considered to avoid penile amputation in chronic infected and intractable wounds.

Facial Reconstruction Based on Combined Three-Dimensional Printing and Microsurgical Free Transfer.

There are patients with craniofacial deformity that can lead to extensive bone loss and severe disfigurement. Autologous reconstruction may be challenging in these patients, and it is usually associated with flap donor area morbidity and unfavorable aesthetic and functional results. A 51-year-old patient with human immunodeficiency virus infection, developed in the context of immunosuppression a fulminant fungal rhino-sinusitis with the need for surgical debridement, and in consequence extensive destruction of the nasal cavity and upper jaw, resulting in severe disfigurement due to nasal deformity and maxillary collapse. Human immunodeficiency virus disease was controlled and the complex craniofacial defect was posteriorly reconstructed with direct 3-dimensional (3D) printing combined with microsurgical free tissue transfer. The 3D facial implant, in titanium, was individualized and fabricated based on computed tomography images of the patient. A radial forearm free flap was used since a soft-tissue defect was anticipated after scar release and implant placement. It allowed simultaneous coverage of the palate, the anterior surface of maxilla and intranasal lining. The flap survived despite flap venous congestion in the postoperative period probably facilitated because of the complex 3D flap configuration and pedicle tunneling into the neck. After 9 months, the patient showed a tremendous aesthetic and functional improvement. The 3D printing was useful in our patient with craniofacial reconstruction. Its combination with free tissue transfer may improve the surgeon's armamentarium when dealing with complex patients.

Antero-cervical thermophysiological characterization of obstructive sleep apnea patients.

PURPOSE: Obstructive sleep apnea syndrome (OSAS) is characterized by upper airway inflammation. The aim of this study was to characterize thermal profile of the antero-cervical region in OSAS patients through medical thermal imaging and to compare the respective subjects with non-OSAS individuals. METHODS: Image capture followed the Glamorgan Protocol. A dynamic thermographic examination of the anterior cervical region (at baseline and after a cold stimulus) was conducted in 26 patients diagnosed with overnight polysomnography (PSG). PSG results stratified the subjects into OSAS and non-OSAS groups and their thermograms were compared. RESULTS: Eleven non-OSAS and 15 OSAS subjects were evaluated. Antero-cervical right side (RS) temperature was higher in OSAS group at baseline (p = 0.014). Right side index (RSI) temperature-the difference between RS and submental region, considered as control-was lower in OSAS subjects at baseline (p = 0.020) and 10 min after the cold stimuli was applied (p = 0.008), indicating higher absolute temperatures in this group. Left side index (LSI) was also lower at 10 min in OSAS group (p = 0.021). Statistical correlation was found between apnea-hypopnea index and RS at baseline (r = 0.424, p = 0.031) and at 10 min (r = 0.403, p = 0.041) and RSI at baseline (r = - 0.458, p = 0.019) and 10 min after cold provocation was applied (r = - 0.435, p = 0.025). CONCLUSIONS: OSAS patients have shown higher antero-cervical temperatures compared with non-OSAS counterparts and temperature was associated with severity of the condition. Medical thermography may be a suitable tool in the setting of OSAS suspicion.