Free Flaps in Patients with Advanced Arteriosclerosis of the Tibial Vessels: Mini Vein Graft to the Peroneal Artery.

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.

Impact of adjuvant radiochemotherapy on free flap volume in head and neck reconstruction: A systematic review and meta-analysis.

BACKGROUND: Limited data exist regarding the effect of adjuvant radiochemotherapy on free flap volume in head and neck reconstruction. However, an adequate free flap volume is an important predictor of functional and patient-reported outcomes in head and neck reconstruction. METHODS: A systematic review of Medline, Embase, and the Cochrane Central Register of Controlled Trials was conducted using the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) guidelines. A total of 6710 abstracts were screened, and 36 full-text papers were reviewed. Nineteen studies met the inclusion criteria and were used to extract data for this analysis. RESULTS: A meta-analysis of 14 two-arm studies comparing the impact of adjuvant radiotherapy versus no adjuvant radiotherapy was performed. The main analysis revealed that 6 months postoperatively, irradiated flaps showed a significant reduction of volume (average, 9.4%) compared to nonirradiated flaps. The average interpolated pooled flap volumes 6 months postoperatively were 76.4% in irradiated flaps and 81.8% in nonirradiated flaps. After a median postoperative follow-up of 12 months, the total flap volume was 62.6% for irradiated flaps and 76% for nonirradiated flaps. Four studies reported that chemotherapy had no significant impact on free flap volume. CONCLUSIONS: Compared to nonirradiated flaps, irradiated flaps were significantly reduced in volume (range, 5% to 15.5%). Clinicians should take this into account when planning the surgical reconstruction of head and neck defects. Conducting large-scale prospective studies with standardized protocols and well-defined follow-up measurements could contribute to defining the ideal, personalized free flap volume for optimal function and patient-reported outcomes.

Harnessing human adipose-derived stromal cell chondrogenesis in vitro for enhanced endochondral ossification.

Endochondral ossification (ECO), the major ossification process during embryogenesis and bone repair, involves the formation of a cartilaginous template remodelled into a functional bone organ. Adipose-derived stromal cells (ASC), non-skeletal multipotent progenitors from the stromal vascular fraction (SVF) of human adipose tissue, were shown to recapitulate ECO and generate bone organs in vivo when primed into a hypertrophic cartilage tissue (HCT) in vitro. However, the reproducibility of ECO was limited and the major triggers remain unknown. We studied the effect of the expansion of cells and maturation of HCT on the induction of the ECO process. SVF cells or expanded ASC were seeded onto collagen sponges, cultured in chondrogenic medium for 3-6 weeks and implanted ectopically in nude mice to evaluate their bone-forming capacities. SVF cells from all tested donors formed mature HCT in 3 weeks whereas ASC needed 4-5 weeks. A longer induction increased the degree of maturation of the HCT, with a gradually denser cartilaginous matrix and increased mineralization. This degree of maturation was highly predictive of their bone-forming capacity in vivo, with ECO achieved only for an intermediate maturation degree. In parallel, expanding ASC also resulted in an enrichment of the stromal fraction characterized by a rapid change of their proteomic profile from a quiescent to a proliferative state. Inducing quiescence rescued their chondrogenic potential. Our findings emphasize the role of monolayer expansion and chondrogenic maturation degree of ASC on ECO and provides a simple, yet reproducible and effective approach for bone formation to be tested in specific clinical models.

Innovative Strategies in Microvascular Head and Neck Reconstruction.

The field of reconstructive microsurgery has witnessed considerable advancements over the years, driven by improvements in technology, imaging, surgical instruments, increased understanding of perforator anatomy, and experience with microsurgery. However, within the subset of microvascular head and neck reconstruction, novel strategies are needed to improve and optimize both patient aesthetics and post-operative function. Given the disfiguring defects that are encountered following trauma or oncologic resections, the reconstructive microsurgeon must always aim to innovate new approaches, reject historic premises, and challenge established paradigms to further achieve improvement in both aesthetic and functional outcomes. The authors aim to provide an up-to-date review of innovations in head and neck reconstruction for oncologic defects.

Profunda Artery Perforator Flap Tongue Reconstruction: An Effective and Safe Alternative to the Anterolateral Thigh Flap.

BACKGROUND: The anterolateral thigh (ALT) perforator flap is a workhorse flap for tongue reconstruction. The authors present an alternative option using the profunda artery perforator (PAP) flap for glossectomy reconstruction compared to the ALT flap. METHODS: A retrospective review was conducted of 65 patients who underwent subtotal or total glossectomy reconstruction between 2016 and 2020 (46 ALT vs. 19 PAP flaps). Flap volume was assessed using CT scans at two different time points. Quality of life and functional outcomes were measured using the MD Anderson Symptom Inventory for head and neck cancer (MDASI-HN). RESULTS: Patients undergoing a PAP flap had significantly lower BMI compared to ALT flaps (22.7±5.0 vs. 25.8±5.1; p=0.014). Donor site and recipient site complications were similar as was the mean flap volume seven months after surgery (30.9% for ALT vs. 28.1% for PAP; p=0.93). Radiation and chemotherapy did not appear to have a significant effect on flap volume change over time. The most frequently reported high-severity items in MDASI-HN were swallowing/chewing and voice/speech for both cohorts. Patients who had reconstruction with a PAP flap had significantly better swallowing function (p=0.034). CONCLUSION: Both the PAP and ALT flaps appear to be safe and effective choices for subtotal and total tongue reconstruction. The PAP flap can serve as an alternative donor site, especially in the setting of malnourished patients with thin lateral thigh thickness undergoing reconstruction of extensive glossectomy defects.

In vitro antibacterial activity and synergetic effect of crude extract of the Wohlfahrtia nuba (Diptera: Sarcophagidae) flesh fly larvae.

Multidrug-resistant pathogens have become ubiquitous, and effective treatment alternatives are urgently required. Maggot therapy is a promising agent that is being studied to overcome antibiotic-resistant pathogens. This study evaluated the antibacterial activity of the larvae extract of the Wohlfahrtia nuba (wiedmann) (Diptera: Sarcophagidae) flesh fly on the growth of five pathogenic bacterial species (methicillin-sensitive Staphylococcus aureus [ATCC 29213], methicillin-resistant Staphylococcus aureus [ATCC BAA-1680], Pseudomonas aeruginosa [ATCC 27853], Escherichia coli [ATCC 25922], and Salmonella typhi [ATCC 19430]) in vitro by using different techniques. Resazurin-based turbidimetric assay demonstrated that the W. nuba maggot exosecretion (ES) was potent against all the bacterial species tested, and according to the determined minimum inhibitory concentration (MIC) for each bacterium, gram-negative bacteria were more sensitive than gram-positive bacteria. Additionally, colony-forming unit assay showed that maggot ES was able to inhibit bacterial growth rate for all bacterial species tested, where the highest bacterial reduction was observed with methicillin-sensitive S. aureus (MSSA) followed by S. typhi. Moreover, maggot ES was shown to be concentration-dependent, where 100 µL of ES at 200 mg/mL was bactericidal towards methicillin-resistant S. aureus (MRSA) and P. aeruginosa compared with 100 µL at the MIC of the ES. Moreover, based on the result of agar disc diffusion assay, maggot extract was more efficient against P. aeruginosa and E. coli than the remaining reference strains tested. Furthermore, the combination between regular antibiotics with maggot ES at different concentrations indicated that ES acts synergistically with the tested antibiotics against the five bacterial models.

In Vitro Propagation of Three Date Palm (Phoenix dactylifera L.) Varieties Using Immature Female Inflorescences.

Immature female inflorescences are promising materials for use as explants for the tissue culture of date palm. Four types of MS media were used in this study during the four micropropagation stages-starting media (SM), maturation media (MM), multiplication media (PM) and rooting media (RM)-to micropropagate three elite date palm varieties, Amri, Magdoul and Barhy using the immature female inflorescences as explant. The highest percentage of callus induction in all the varieties studied was obtained on the SM1 (9 µM 2,4-D + 5.7 µM IAA + 10 µM NAA). Culturing on the MM1 (4.5 µM 2,4-D + 9.8 µM 2-iP + 1.5 AC) allowed us to obtain the best value in terms of callus weight. After culturing on the PM1 (4.4 µM BA + 9.8 µM 2-iP) produced the highest numbers of somatic embryos and shoots. The explants on RM2 (0.5 µM NAA + 1.25 µM IBA + 3 g AC) showed the highest root numbers and root lengths, while the highest shoot length was achieved on RM3 (0.5 µM NAA + 0.5 µM IBA + 3 g AC). The Amri variety presented the best response among the three varieties in all parameters, followed by the Magdoul and Barhy varieties. In all the stages of micropropagation, the analysis of variance revealed highly significant variations among varieties and culture media, and a significant difference in the number of roots during the rooting stage. The results also showed non-significant differences in the interaction between varieties and culture media, except for shoot length in the rooting stage. The results also reveal the broad sense heritability ranging from low to high for the measured parameters. It can be concluded that the immature female inflorescences can be used as a productive explant source for successful date palm micropropagation using the SM1, MM1, PM1 and RM2 culture media. It can also be concluded that the success of date palm micropropagation not only depends on the concentrations of growth regulators, but also on their types.

The Medial Femoral Condyle Flap: A Novel Versatile Tool for Complex Microvascular Maxillofacial Reconstruction.

SUMMARY: The medial femoral condyle flap is well-described for reconstruction of small bone defects of the upper and lower extremities. There are limited case reports of its use in other anatomic sites, particularly for reconstruction of complex head and neck defects. In the setting of previous radiation and contaminated fields, vascularized bone is generally preferred to bone grafts, cadaveric allografts, or synthetic implants. The authors present a case series of complex craniofacial defects involving the midface that were reconstructed using medial femoral condyle flaps, focusing on the type of defect and lessons learned from their early experience to promote awareness of this flap among microsurgeons, who may wish to consider the potential of this flap and incorporate its use into their armamentarium. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Impact of Adjuvant Radiotherapy on Free Flap Volume in Autologous Breast Reconstruction: A Scoping Review.

In autologous breast reconstruction, a sufficient flap volume is fundamental to restore breast shape and ensure an aesthetic outcome. After mastectomy, postoperative irradiation is regularly indicated in the oncological treatment algorithm. When administering radiation therapy after autologous reconstruction, the tissue transferred is inherently irradiated. Although there is evidence that points to a reduction in flap volume after adjuvant radiotherapy, the data have been contradicting and inconclusive. To address this anecdotal evidence, we performed a scoping review of the current literature that addresses the effect of radiotherapy on breast flap volume. Six two-armed studies, comprising a total of 462 patients, reported on the effect of adjuvant radiotherapy on free flap volume changes. Of those, two studies found a significant negative impact of radiotherapy on free flap volume, while the other four studies did not. Reported flap volume changes ranged from no change to a reduction of 26.2%, measured up to two years postoperatively. The selected studies contain varying patient numbers, follow-up timepoints, types of flaps, and measuring methods, contributing to a relatively high heterogeneity. While we present some evidence suggesting a significant impact of adjuvant radiotherapy on breast flap volume, future studies are needed to further investigate this potential correlation.

Prophylactic cryoprecipitate transfusion in patients undergoing scoliosis surgery: A randomised-controlled trial.

BACKGROUND: Scoliosis surgeries in adults often have a high risk of massive blood loss and significant transfusion of blood products during and after surgery. It is not known whether early cryoprecipitate therapy is useful in reducing blood loss and transfusion requirements. The objective of this randomised, prospective placebo control study was to evaluate whether prophylactic administration of cryoprecipitate would reduce blood loss and transfusion requirements during scoliosis surgery. METHODS: Eighty adult patients scheduled to undergo elective scoliosis correction were randomly assigned to receive either ten units of cryoprecipitate before incision (cryo group) or an equivalent volume of 0.9% saline (placebo group). Blood loss, transfusion requirements, coagulation parameters and complications were assessed. RESULTS: No significant differences were found in the volume of transfused blood products, intraoperative estimated blood loss between the intervention and placebo groups. Postoperative blood loss was significantly lower in the cry group when compared to the other group. During adult surgical correction of scoliosis, prophylactic administration of cryoprecipitate did not diminish the amount of transfused blood products or decrease intraoperative blood loss. CONCLUSION: It could be concluded that the prophylactic administration of cryoprecipitate shows no differences in intraoperative blood loss and transfusion requirements during scoliosis surgery.

Improvement of German Chamomile (Matricaria recutita L.) for Mechanical Harvesting, High Flower Yield and Essential Oil Content Using Physical and Chemical Mutagenesis.

Chamomile (Matricariarecutita L.) is one of the most important medicinal plants with various applications. The flowers and flower heads are the main organs inthe production of essential oil. The essential improvement goals of chamomile are considered to be high flower yield and oil content, as well asthe suitability for mechanical harvesting. The present study aimed to improve the flower yield, oil content and mechanical harvestability of German chamomile via chemical and physical mutagens. Three German chamomile populations (Fayum, Benysuif and Menia) were irradiated with 100, 200, 300 and 400 Gray doses of gamma rays, as well as chemically mutagenized using 0.001, 0.002 and 0.003 mol/mL of sodium azide for 4 h. The two mutagens produced a wide range of changes in the flowers' shape and size. At M3 generation, 18 mutants (11 from gamma irradiation and 7 from sodium azide mutagenization) were selected and morphologically characterized. Five out of eighteen mutants were selected for morphological and chemical characterization for oil content, oil composition and oil quality in M4 generation. Two promising mutants, F/LF5-2-1 and B/HNOF 8-4-2, were selected based on their performance in most studied traits during three generations, as well as the high percentage of cut efficiency and a homogenous flower horizon, which qualify them as suitable candidates for mechanical harvesting. The two mutants are late flowering elite mutants; the F/LF5-2-1 mutant possessed the highest oil content (1.77%) and number of flowers/plant (1595), while the second promising B/HNOF 8-4-2 mutant hada high oil content (1.29%) and chamazulene percentage (13.98%) compared to control plants. These results suggest that the B/HNOF 8-4-2 and F/LF5-2-1 mutants could be integrated as potential parents into breeding programs for a high number of flowers, high oil content, oil composition and oil color traits for German chamomile improvement.

Powerful Antioxidants and Cytotoxic Activities of the Methanol Extracts from Eight Soybean Cultivars.

In the present study, the chemical composition and total phenolic (TPC) and total flavonoid contents (TFC) of eight soybean cultivars (Giza 21, Giza 22, Giza 35, Giza 111, Giza 82, Giza 83, Crawford, and Holliday) were estimated. Moreover, antioxidant activity and in vitro cytotoxic activities against HepG-2 and MCF-7 were evaluated. Giza 21, Giza 111, and Crawford cultivars recorded higher than 40% crude protein. The analysis revealed that TPC values in seed extracts ranged from 10.5 mg GAE/g extract in Giza 35 to 6.4 mg GAE/g extract in Giza 22. TFC varied from 1.20 mg QE/g extract in Giza 111 to 0.55 mg QE/g extract in Crawford. Giza 35 exhibited the highest content of genistein and daidzein and the highest free radical scavenging activity (61.833%). The results of the MTT assay demonstrated that the soybean methanolic extracts inhibited the proliferation of HepG-2 and MCF-7 cells in a dose-dependent manner. Giza 35 exhibited the highest cytotoxic activity. In conclusion, Giza 35 cultivar recorded the highest TPC and TFC values and antioxidant and cytotoxic activities. Therefore, this cultivar can be used as a source for the production of pharmaceutical and medicinal products rather than as a nutritional source of protein.

Synergistic activity and molecular modelling of fosfomycin combinations with some antibiotics against multidrug resistant Helicobacter pylori.

Antibiotic resistance represents the main challenge of Helicobacter pylori infection worldwide. This study investigates the potential bactericidal effects of fosfomycin combinations with clarithromycin, metronidazole, ciprofloxacin, amoxicillin, rifampicin, and doxycycline against thirty-six H. pylori strains using the checkerboard and time-kill assay methods. The results showed that ≥ 50% of the strains were resistant to the six antibiotics. Remarkably, only six strains exerted resistance to these antibiotics, with the minimum inhibitory concentrations (MICs) ranges of (3.2-12.8 mg/l), (32-256 mg/l), (3.2-51.2 mg/l), (3.2-25.6 mg/l), (1.6-3.2 mg/l), and (25.6 > 51.2 mg/l), respectively. The seven antibiotics were evaluated through in silico studies for their permeability and ability to bind UDP-N-acetylglucosamine1-carboxyvinyltransferase (MurA) of H. pylori. The results indicated that fosfomycin exhibited the highest predicted membrane permeability (membrane ∆G insert = - 37.54 kcal/mol) and binding affinity (docking score = - 5.310 kcal/mol) for H. pylori MurA, compared to other tested antibiotics. The combinations of fosfomycin with these antibiotics exerted synergistic interactions (Fractional inhibitory concentration, FIC index < 1) against the six strains. Importantly, the combinations of fosfomycin with clarithromycin, doxycycline and rifampicin achieved bactericidal effects (reduction ≥ 3.0 Log10 cfu/ml) against the most resistant H. pylori strain. Notably, these effects increased with presence of metronidazole, which enhanced the activity of the fosfomycin combination with amoxicillin from a weak inhibition to bactericidal effect. This study provides evidence that the combination of fosfomycin with either clarithromycin, amoxicillin, doxycycline, or rifampicin (especially with the presence of metronidazole) could be a promising option for treating MDR H. pylori infection.

T-cadherin Expressing Cells in the Stromal Vascular Fraction of Human Adipose Tissue: Role in Osteogenesis and Angiogenesis.

Cells of the stromal vascular fraction (SVF) of human adipose tissue have the capacity to generate osteogenic grafts with intrinsic vasculogenic properties. However, cultured adipose-derived stromal cells (ASCs), even after minimal monolayer expansion, lose osteogenic capacity in vivo. Communication between endothelial and stromal/mesenchymal cell lineages has been suggested to improve bone formation and vascularization by engineered tissues. Here, we investigated the specific role of a subpopulation of SVF cells positive for T-cadherin (T-cad), a putative endothelial marker. We found that maintenance during monolayer expansion of a T-cad-positive cell population, composed of endothelial lineage cells (ECs), is mandatory to preserve the osteogenic capacity of SVF cells in vivo and strongly supports their vasculogenic properties. Depletion of T-cad-positive cells from the SVF totally impaired bone formation in vivo and strongly reduced vascularization by SVF cells in association with decreased VEGF and Adiponectin expression. The osteogenic potential of T-cad-depleted SVF cells was fully rescued by co-culture with ECs from a human umbilical vein (HUVECs), constitutively expressing T-cad. Ectopic expression of T-cad in ASCs stimulated mineralization in vitro but failed to rescue osteogenic potential in vivo, indicating that the endothelial nature of the T-cad-positive cells is the key factor for induction of osteogenesis in engineered grafts based on SVF cells. This study demonstrates that crosstalk between stromal and T-cad expressing endothelial cells within adipose tissue critically regulates osteogenesis, with VEGF and adiponectin as associated molecular mediators.

Evolution of a concept with enzymatic debridement and autologous in situ cell and platelet-rich fibrin therapy (BroKerF).

BACKGROUND: Deep partial-thickness burns are traditionally treated by tangential excision and split thickness skin graft (STSG) coverage. STSGs create donor site morbidity and increase the wound surface in burn patients. Herein, we present a novel concept consisting of enzymatic debridement of deep partial-thickness burns followed by co-delivery of autologous keratinocyte suspension and plated-rich fibrin (PRF) or fibrin glue. MATERIAL AND METHODS: In a retrospective case study, patients with deep partial-thickness burns treated with enzymatic debridement and autologous cell therapy combined with PRF or fibrin glue (BroKerF) between 2017 and 2018 were analysed. BroKerF was applied to up to 15% total body surface area (TBSA); larger injuries were combined with surgical excision and skin grafting. Exclusion criteria were age <18 or >70 years, I°, IIa°-only, III° burns and loss of follow-up. RESULTS: A total of 20 patients with burn injuries of 16.8% ± 10.3% TBSA and mean Abbreviated Burn Severity Score 5.45 ± 1.8 were identified. Of the patients, 65% (n = 13) were treated with PRF, while 35% (n = 7) were treated with fibrin glue. The mean area treated with BroKerF was 7.5% ± 0.05% TBSA, mean time to full epithelialization was 21.06 ± 9.2 days and mean hospitalization time was 24.7 ± 14.4 days. Of the patients, 35% (n = 7) needed additional STSG, 43% (n = 3) of whom had biopsy-proven wound infections. CONCLUSION: BroKerF is an innovative treatment strategy, which, in our opinion, will show its efficacy when higher standardization is achieved. The combination of selective debridement and autologous skin cells in a fibrin matrix combines regenerative measures for burn treatment. LAY SUMMARY: Patients suffering from large burn wounds often require the use of large skin grafts to bring burned areas to heal. Before the application of skin grafts, the burned skin must be removed either by surgery or using enzymatic agents. In this article, we describe a method where small areas of skin are taken and skin cells are extracted and sprayed on wound areas that were treated with an enzymatic agent. The cells are held in place by a substance extracted from patients' blood (PRF) that is sprayed on the wound together with the skin cells. We believe this technique can be helpful to reduce the need of skin grafts in burned patients and improve the healing process.

Case Report: Reconstruction of a Large Maxillary Defect With an Engineered, Vascularized, Prefabricated Bone Graft.

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.

INHALEd nebulised unfractionated HEParin for the treatment of hospitalised patients with COVID-19 (INHALE-HEP): Protocol and statistical analysis plan for an investigator-initiated international metatrial of randomised studies.

AIMS: Inhaled nebulised unfractionated heparin (UFH) has a strong scientific and biological rationale that warrants urgent investigation of its therapeutic potential in patients with COVID-19. UFH has antiviral effects and prevents the SARS-CoV-2 virus' entry into mammalian cells. In addition, UFH has significant anti-inflammatory and anticoagulant properties, which limit progression of lung injury and vascular pulmonary thrombosis. METHODS: The INHALEd nebulised unfractionated HEParin for the treatment of hospitalised patients with COVID-19 (INHALE-HEP) metatrial is a prospective individual patient data analysis of on-going randomised controlled trials and early phase studies. Individual studies are being conducted in multiple countries. Participating studies randomise adult patients admitted to the hospital with confirmed SARS-CoV-2 infection, who do not require immediate mechanical ventilation, to inhaled nebulised UFH or standard care. All studies collect a minimum core dataset. The primary outcome for the metatrial is intubation (or death, for patients who died before intubation) at day 28. The secondary outcomes are oxygenation, clinical worsening and mortality, assessed in time-to-event analyses. Individual studies may have additional outcomes. ANALYSIS: We use a Bayesian approach to monitoring, followed by analysing individual patient data, outcomes and adverse events. All analyses will follow the intention-to-treat principle, considering all participants in the treatment group to which they were assigned, except for cases lost to follow-up or withdrawn. TRIAL REGISTRATION, ETHICS AND DISSEMINATION: The metatrial is registered at ClinicalTrials.gov ID NCT04635241. Each contributing study is individually registered and has received approval of the relevant ethics committee or institutional review board. Results of this study will be shared with the World Health Organisation, published in scientific journals and presented at scientific meetings.

Platelet-rich plasma and stromal vascular fraction cells for the engineering of axially vascularized osteogenic grafts.

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.

Improved Adipocyte Viability in Autologous Fat Grafting With Ascorbic Acid-Supplemented Tumescent Solution.

INTRODUCTION: In reconstructive surgery, fat volume augmentation is often necessary for esthetic or functional reasons. As an alternative to synthetic and xenogeneic materials, autologous fat grafting (AFG) based on liposuction is gaining popularity, yet successful transplantation and long-term volume maintenance are difficult. Standard tumescent solution formulations neglect adipocyte and stromal vascular fraction (SVF) cell survival during extraction, as well as SVF differentiation into adipocytes thereafter, all of which are crucial for the success of AFG. Here we hypothesized that addition of ascorbic acid (AA) to the tumescent solution could prevent liposuction-induced cell damage. MATERIALS AND METHODS: The effect of 0.1 mmol/L AA in tumescent solution was investigated in a previously described ex vivo model of AFG. Briefly, excision fat was infiltrated with tumescent solution, with or without AA, and incubated for 20 minutes at 37°C. Hand-assisted liposuction was then performed with a blunt cannula. Total cell viability, clonogenicity, and differentiation capacity of the SVF cells were assessed. RESULTS: With AA, 10.3% more cells and in particular 14.9% more adipocytes survived liposuction. Clonogenicity, adipocyte and osteoblast differentiation by SVF cells remained unchanged. CONCLUSIONS: Addition of AA successfully improved survival of adipocytes during liposuction without affecting SVF growth and differentiation. This study therefore identified a useful supplement to the tumescent solution which may lead to improving AFG success.