Summary:. Despite its first descriptions in the previous decade, the use of the hemostatic net has experienced a recent resurgence of enthusiasm in cervicofacial rhytidectomy. Sizeable studies demonstrate a reduction of postoperative hematoma and better skin retraction thanks to external quilting suture techniques. Recent evidence supports this technique’s safety for skin and soft tissue vascularization. We describe an innovative extension of transcutaneous suture indications to postbariatric and body-contouring surgery. By extending the hemostatic net, described for cervical and thoracic surgery, to postbariatric and body-contouring surgery, we aim to propose a safe, reproducible, and innovative technique to enhance contour shaping of the lumbar region. We present a series of 8 massive weight loss patients undergoing liposuction followed by hemostatic net sutures to improve contouring while minimizing scarring. Stitches were performed with 1/0 braided sutures and removed after 2 days. The follow-up ranged from 6 to 20 months. The average lipoaspirated volume was 1431 mL (range, 450–2300 mL). No complications were reported, and no subsequent procedures were needed. Lower back contouring improvement was evaluated using pre- and postoperative photograph comparisons by the patient and the surgeon. Skin marks partially faded at 6 months and disappeared at 12 months. This study primarily emphasized the safety and feasibility of external quilting sutures as an adjunct in postbariatric contouring rather than demonstrating definitive superiority over existing methods. This technique offered an additional option in the plastic surgeon’s arsenal to address massive weight-loss sequelae despite poor skin tone.

PURPOSE:The localization of the human spinal cord (SC) segments remains a challenge without thorough post-mortem analysis and their relationship with the vertebrae has long been investigated. In this post-mortem study, we assessed the relationship between the cervical, thoracic, lumbar and sacral lengths of the SC and of the vertebral column (VC). METHODS:Twenty human SCs were extracted from the vertebral canal. The cervical, thoracic, lumbar and sacral regional lengths of SC and VC were measured by two examiners. Statistical analyses included t-test, ANOVA, Fisher's exact test, Kruskal-Wallis H-test and Pearson's correlation test. RESULTS:No significant relationship was found between the sex of the subjects or the total SC length and its caudal limit in the vertebral canal. The length of the thoracic SC was significantly different between 3 groups of short, intermediate and long SCs, while no significant difference was observed in the lumbar and sacral SC regions. Highly significant correlations were observed between the thoracic SC length, on the one hand, and the total SC length, the thoracic VC length and the total VC length, on the other hand. The very constant length of the lumbar and sacral SC regions was not correlated with any regional SC or VC lengths. CONCLUSION:This study confirms the constant length of the lumbar and sacral SC regions, independently of any other regional SC and VC measurements, while the thoracic region of the SC appears to be the main contributor in the variation of the total SC length.

Massive bone allograft decellularized by perfusion is a solution for large bone defect reconstructions. Perfusion-based decellularization offers a solution by removing cellular components while preserving the non-immunogenic matrix. This study evaluates the in vivo integration and mechanical properties of perfusion-decellularized bone grafts compared to "fresh-frozen" grafts, both before implantation and after explantation. Standardized porcine femoral grafts were categorized into non-irradiated, irradiated, and explanted groups, with half undergoing perfusion decellularization. Biomechanical tests, including screw pull-out test, compression, and 3-point bending test, were performed. Results indicate that while decellularization increases graft brittleness, Vickers indentation and compression tests showed no significant differences between groups. In our study, decellularization reduced the mechanical strength of allografts both before and after implantation. However, since the risk of rupture occurred only under mechanical loads exceeding the physiological range, perfusion-decellularized bone grafts remain a valid strategy for bone repair.

Last twenties, tissue engineering has rapidly advanced to address the shortage of organ donors. Decellularization techniques have been developed to mitigate immune rejection and alloresponse in transplantation. However, a clear definition of effective decellularization remains elusive. This study compares various decellularization protocols using the human fascia lata model. Morphological, structural and cytotoxicity/viability analyses indicated that all the five tested protocols were equivalent and met Crapo's criteria for successful decellularization. Interestingly, only the in vivo immunization test on rats revealed differences. Only one protocol exhibited Human Leucocyte Antigen (HLA) content below 1% residual threshold, the only criterion preventing rat immunization with an absence of rat anti-human IgG switch after one month (N=4 donors for each of the 7 groups, added by negative and positive controls, n=28). By respecting a refined set of criteria, i.e. lack of visible nuclear material, <50ng DNA/mg dry weight of extracellular matrix, and <1% residual HLA content, the potential for adverse host reactions can be drastically reduced. In conclusion, this study emphasizes the importance of considering not only nuclear components but also major histocompatibility complex in decellularization protocols and proposes new guidelines to promote safer clinical development and use of bioengineered scaffolds.

BACKGROUND:Numerous protocols exist concerning the decellularization of the esophagus, a potential alternative to the classical surgical approach for the reconstruction of the digestive tract after esophagectomy. This systematic literature review (SLR) aimed to provide an overview of the effectiveness of the current protocols. METHODS:This SLR was conducted in PubMed, EMBASE, and Scopus until September 2025. Study selection, data extraction, and quality assessment were performed by two independent reviewers according to the inclusion/exclusion criteria. RESULTS:A total of 2494 references were screened after removing duplicates. Among these references, 26 articles were included. The large majority of studies (24/26) used Sodium Dodecyl Sulfate (SDS) or Sodium DeoxyCholate (SDC), and the most common physical method was the cannulation of the esophagus (17/26). The animal model was very heterogenous. All protocols except one showed no residual cell nuclei, with only 5/19 papers confirming a satisfactory residual amount of DNA. The assessment of the extracellular matrix (ECM)-mostly qualitative-revealed global preservation but with a systematic loss of glycosaminoglycans (GAGs). CONCLUSIONS:The decellularization of the esophagus is feasible, but the definition of the optimal protocol to achieve this goal remains difficult because of the important heterogeneity among the different studies.

Introduction: The fascia lata (FL), a dense connective tissue enveloping the deep structures of the thigh, has served as an avascular scaffold for decades in reconstructive surgeries, but never as a standalone vascularized flap, because its vasculature is only described by perforating vessels coming from the lateral circumflex femoral artery without specific intrinsic vasculature. However, the demand for vascularized tissue replacements requires a thorough understanding of this inherent vascularization. Based on the anterolateral thigh (ALT) flap knowledge, this study explores the detailed quantitative vascular architecture of the FL, from the perspective of using it as a standalone vascularized flap. Material & methods: High-resolution microfocus X-ray computed tomography (microCT) of Angiofil-latex injected flaps allowed us to study the specific fascial distribution of perforators. Dissections of 11 fresh-frozen lower limbs identified the main perforating arterioles. The intrinsic vascular network within the FL tissue was then analyzed using microCT. Results: The study confirmed an average of 2.5 f 0.5 main perforating trunks giving rise to 5.6 f 2.9 perforating arterioles per thigh. Notably, microCT revealed a distinct intrinsic vascular network within the FL tissue, with tiny arterioles of 55.39 f 28.47 mu m radius and a volumetric density of 33,347,960 f 23,243,879 mu m3 /mm2. This intrinsic vasculature exhibited a high density of bifurcations (branching nodes=1.36 f 2.26/mm2), demonstrating its potential use as a vascularized flap. Discussion: This study unveils the intrinsic vascularization of the FL, supporting its utility as a mega-thin vascularized flap in reconstructive surgery. Based on this vascular carrier, these findings also open new applications for tissue engineering and personalized medicine, toward an improvement of surgical outcomes and patient recovery.

BACKGROUND:Total esophageal reconstruction after failure of conventional techniques is a significant surgical challenge, particularly in fragile patients who have undergone multiple surgical procedures. Traditional options, including gastroplasties, colon transfers, and free jejunal flaps, are associated with high morbidity rates, failure rates, or surgical complexity. METHODS:The authors report an innovative 4-step strategy for esophageal reconstruction using a presternal skin tube created with tissue expanders and perforator flap techniques. Six patients with previous definitive esophageal exclusion underwent reconstruction between 2009 and 2023. The procedure involved patients with complex medical history and multiple reconstruction failures, including gastroplasties, coloplasties, or ileocoloplasties, and consisted of chest skin expansion with tissue expanders, tubularization of a perforator flap based on internal mammary vessels, anastomosis of the distal skin tube with a jejunal Y loop, and proximal anastomosis to the native cervical esophagus. RESULTS:The entire reconstructive process was completed over 17.5 ± 7.3 months. All patients achieved restoration of digestive tract continuity. Minor complications, including wound dehiscence and fistulas, were observed in all patients, but resolved without major intervention. Functional outcomes were favorable, with a maximum follow-up of 15 years and patients tolerating liquids, semisolids, and solid foods after 2 to 3 months. Imaging and endoscopy confirmed lumen integrity, absence of strictures, and static skin-like epithelium without ulceration. CONCLUSIONS:This staged reconstruction offers a viable and safe alternative for total esophageal replacement in complex, high-risk cases, avoiding additional extensive intrathoracic procedures. The presternal neoesophagus provides acceptable function with manageable complications. CLINICAL QUESTION/LEVEL OF EVIDENCE:Therapeutic, IV.

Introduction: Massive bone allografts enable the reconstruction of critical bone defects in numerous conditions (e.g. tumoral, infection or trauma). Unfortunately, their biological integration remains insufficient and the reconstruction may suffer from several postoperative complications. Perfusion-decellularization emerges as a tissue engineering potential solution to enhance osseointegration. Therefore, an intrinsic vascular study of this novel tissue engineering tool becomes essential to understand its efficacy and applicability. Material and methods: 32 porcine long bones (humeri and femurs) were used to assess the quality of their vascular network prior and after undergoing a perfusion-decellularization protocol. 12 paired bones were used to assess the vascular matrix prior (N = 6) and after our protocol (N = 6) by immunohistochemistry. Collagen IV, Von Willebrand factor and CD31 were targeted then quantified. The medullary macroscopic vascular network was evaluated with 12 bones: 6 were decellularized and the other 6 were, as control, not treated. All 12 underwent a contrast-agent injection through the nutrient artery prior an angio CT-scan acquisition. The images were processed and the length of medullary vessels filled with contrast agent were measured on angiographic cT images obtained in control and decellularized bones by 4 independent observers to evaluate the vascular network preservation. The microscopic cortical vascular network was evaluated on 8 bones: 4 control and 4 decellularized. After injection of gelatinous fluorochrome mixture (calcein green), non-decalcified fluoroscopic microscopy was performed in order to assess the perfusion quality of cortical vascular lacunae. Results: The continuity of the microscopic vascular network was assessed with Collagen IV immunohistochemistry (p-value = 0.805) while the decellularization quality was observed through CD31 and Von Willebrand factor immunohistochemistry (p-values <0.001). The macroscopic vascular network was severely impaired after perfusion-decellularization; nutrient arteries were still patent but the amount of medullary vascular channels measured was significantly higher in the control group compared to the decellularized group (p-value <0.001). On average, the observers show good agreement on these results, except in the decellularized group where more inter-observer discrepancies were observed. The microscopic vascular network was observed with green fluoroscopic signal in almost every canals and lacunae of the bone cortices, in three different bone locations (proximal metaphysis, diaphysis and distal metaphysis). Conclusion: Despite the aggressiveness of the decellularization protocol on medullary vessels, total porcine long bones decellularized by perfusion retain an acellular cortical microvascular network. By injection through the intact nutrient arteries, this latter vascular network can still be used as a total bone infusion access for bone tissue engineering in order to enhance massive bone allografts prior implantation.

Introduction: Nipple–areolar complex (NAC) reconstruction after breast cancer surgery is challenging and does not always provide optimal long-term esthetic results. Therefore, generating a NAC using tissue engineering techniques, such as a decellularization–recellularization process, is an alternative option to recreate a specific 3D NAC morphological unit, which is then covered with an in vitro regenerated epidermis and, thereafter, skin-grafted on the reconstructed breast.Materials and methods: Human NACs were harvested from cadaveric donors and decellularized using sequential detergent baths. Cellular clearance and extracellular matrix (ECM) preservation were analyzed by histology, as well as by DNA, ECM proteins, growth factors, and residual sodium dodecyl sulfate (SDS) quantification. In vivo biocompatibility was evaluated 30 days after the subcutaneous implantation of native and decellularized human NACs in rats. In vitro scaffold cytocompatibility was assessed by static seeding of human fibroblasts on their hypodermal side for 7 days, while human keratinocytes were seeded on the scaffold epidermal side for 10 days by using the reconstructed human epidermis (RHE) technique to investigate the regeneration of a new epidermis.Results: The decellularized NAC showed a preserved 3D morphology and appeared white. After decellularization, a DNA reduction of 98.3% and the absence of nuclear and HLA staining in histological sections confirmed complete cellular clearance. The ECM architecture and main ECM proteins were preserved, associated with the detection and decrease in growth factors, while a very low amount of residual SDS was detected after decellularization. The decellularized scaffolds were in vivo biocompatible, fully revascularized, and did not induce the production of rat anti-human antibodies after 30 days of subcutaneous implantation. Scaffold in vitro cytocompatibility was confirmed by the increasing proliferation of seeded human fibroblasts during 7 days of culture, associated with a high number of living cells and a similar viability compared to the control cells after 7 days of static culture. Moreover, the RHE technique allowed us to recreate a keratinized pluristratified epithelium after 10 days of culture.Conclusion: Tissue engineering allowed us to create an acellular and biocompatible NAC with a preserved morphology, microarchitecture, and matrix proteins while maintaining their cell growth potential and ability to regenerate the skin epidermis. Thus, tissue engineering could provide a novel alternative to personalized and natural NAC reconstruction.

IntroductionTo mitigate the post-operative complication rates associated with massive bone allografts, tissue engineering techniques have been employed to decellularize entire bones through perfusion with a sequence of solvents. Mechanical assessment was performed in order to compare conventional massive bone allografts and perfusion/decellularized massive bone allografts.Material and methodsTen porcine femurs were included. Five were decellularized by perfusion. The remaining 5 were left untreated as the "control" group. Biomechanical testing was conducted on each bone, encompassing five different assessments: screw pull-out, 3-points bending, torsion, compression and Vickers indentation.ResultsUnder the experimental conditions of this study, all five destructive tested variables (maximum force until screw pull-out, maximum elongation until screw pull-out, energy to pull out the screw, fracture resistance in flexion and maximum constrain of compression) were statistically significantly superior in the control group. All seven nondestructive variables (Young's modulus in flexion, Young's modulus in shear stress, Young's modulus in compression, Elastic conventional limit in compression, lengthening to rupture in compression, resilience in compression and Vickers Hardness) showed no significant difference.DiscussionDescriptive statistical results suggest a tendency for the biomechanical characteristics of decellularized bone to decrease compared with the control group. However, statistical inferences demonstrated a slight significant superiority of the control group with destructive mechanical stresses. Nondestructive mechanical tests (within the elastic phase of Young's modulus) were not significantly different.

Large bone defect regeneration remains a major challenge for orthopedic surgeons. Tissue engineering approaches are therefore emerging in order to overcome this limitation. However, these processes can alter some of essential native tissue properties such as intermolecular crosslinks of collagen triple helices, which are known for their essential role in tissue structure and function. We assessed the persistence of extracellular matrix (ECM) properties in human fascia lata (HFL) and periosteum (HP) after tissue engineering processes such as decellularization and sterilization. Harvested from cadaveric donors (N = 3), samples from each HFL and HP were decellularized following five different chemical protocols with and without detergents (D1-D4 and D5, respectively). D1 to D4 consisted of different combinations of Triton, Sodium dodecyl sulfate and Deoxyribonuclease, while D5 is routinely used in the institutional tissue bank. Decellularized HFL tissues were further gamma-irradiated (minimum 25 kGy) in order to study the impact of sterilization on the ECM. Polarized light microscopy (PLM) was used to estimate the thickness and density of collagen fibers. Tissue hydration and content of hydroxyproline, enzymatic crosslinks, and non-enzymatic crosslinks (pentosidine) were semi-quantified with Raman spectroscopy. ELISA was also used to analyze the maintenance of the decorin (DCN), an important small leucine rich proteoglycan for fibrillogenesis. Among the decellularization protocols, detergent-free treatments tended to further disorganize HFL samples, as more thin fibers (+53.7%) and less thick ones (−32.6%) were recorded, as well as less collagen enzymatic crosslinks (−25.2%, p = 0.19) and a significant decrease of DCN (p = 0.036). GAG content was significantly reduced in both tissue types after all decellularization protocols. On the other hand, HP samples were more sensitive to the D1 detergent-based treatments, with more disrupted collagen organization and greater, though not significant loss of enzymatic crosslinks (−37.4%, p = 0.137). Irradiation of D5 HFL samples, led to a further and significant loss in the content of enzymatic crosslinks (−29.4%, p = 0.037) than what was observed with the decellularization process. Overall, the results suggest that the decellularization processes did not significantly alter the matrix. However, the addition of a gamma-irradiation is deleterious to the collagen structural integrity of the tissue.

Critical bone loss can have several origins: infections, tumors or trauma. Therefore, massive bone allograft can be a solution for limb salvage. Such a biological reconstruction should have the ideal biomechanical qualities. However, their complication rate remains too high. Perfusion-decellularization of massive allografts could promote the vitality of these grafts, thereby improving their integration and bone remodeling.Three perfusion-decellularized massive bone allografts were compared to 3 fresh frozen massive bone allografts in a preclinical in vivo porcine study using an orthopedic surgery model. Three pigs each underwent a critical diaphyseal femoral defects followed by an allogeneic intercalary femoral graft on their both femurs (one decellularized and one conventional fresh frozen as “native”) to reconstruct the defect. Clinical imaging was performed over 3 months of follow-up. The grafts were then explanted and examined by non-decalcified histology, fluoroscopic microscopy and immunohistochemistry.Bone consolidation was achieved in both groups at the same time. However, the volume of bone callus appeared to be greater in the decellularized group. Histology demonstrated a superior bone remodeling in the decellularized group, with a higher number of osteoclasts (p < 0.001) and larger areas of osteoid matrix and newly formed bone as compared to the “native” group. Immunohistochemistry showed a superior vitality and remodeling in both the cortical and medullary cavities for osteocalcin (p < 0.001), Ki67 (p < 0.001), CD3 (p < 0.001) and α-SMA (p < 0.001) as compared the “native” group. Three months after implantation, the decellularized grafts were proven to be biologically more active compared to native grafts. Fluoroscopic microscopy revealed more ossification fronts in the depth of the decellularized grafts (p = 0.021).This pilot study provides the first in vivo demonstration on the enhanced biological capacities of massive bone allograft decellularized by perfusion as compared to conventional massive bone allografts.

The fascial system has gained recognition for its integral role in connecting skin, superficial and deep fasciae, and underlying muscles. However, consensus on its microstructure depending on its topography remains elusive as well as its implications in clinical practices, such as reconstructive surgery and physiotherapy techniques. This study focuses on the iliotibial tract (ITT) implicated in the iliotibial band syndrome. The goal is to describe microstructural characteristics using classical 2D histology and cryogenic contrast-enhanced microcomputed tomography (cryo-CECT) such as the total thickness, number of layers, layer thickness, fibre orientation and tortuosity, according to the specific topography. The total thickness of the ITT varied across topographic regions, with the superior part being on average thicker but non-significantly different from the other regions. The inferior part showed heterogeneity, with the anterior region (AI) being the thinnest and the posterior one (PI) the thickest. The ITT exhibited 1-3 layers, with no significant differences among regions. Most commonly, it consisted of two layers, except for the antero-superior (AS) and antero-middle (AM) regions, which sometimes had only one layer. The posterior regions frequently had 2 or 3 layers, with the PI region having the highest mean (2.7 layers). The intermediate layer was the thickest one, varying from the AI region (0.368 mm ± 0.114) to the PI region (0.640 mm ± 0.305). The superficial layer showed regional variability, with the AS region being the thinnest. The deep layer appeared thinner than the superficial one. Fibre orientation analysis indicated that the intermediate layer mainly consisted of oblique longitudinal fibres, orientated downward and forward, while the superficial and deep layers had transversal or oblique transversal fibres. Cryo-CECT 3D observations confirmed these findings, revealing distinct orientations for different layers. Fibre tortuosity exhibited differences based on orientation. Transversal fibres (>65°) were significantly less tortuous than longitudinal fibres (<25°) and oblique intermediate fibres (25°-65°), aligning with 3D plot observations. This quantitative study highlights various microstructural characteristics of the ITT, offering insights into its regional variations. The analysis accuracy is increased due to the novel technology of cryo-CECT which emerges as a valuable tool for precise assessment of 3D fibre orientation and tortuosity. These findings contribute to a deeper understanding of the ITT structure, useful in clinical practices, such as reconstructive surgery and physiotherapy, and future research endeavours.

INTRODUCTION:Postmortem evaluation of the human vascular system has a long history, with advancements ranging from dissections to modern imaging techniques like computed tomography (CT scan). This study designs a novel combination of Angiofil, a liquid radiopaque polymer, and latex, a flexible cast material, for cadaveric vascular analysis. MATERIAL & METHODS:The aim was to synergize the advantages of both components, providing accurate radiological images and optimal dissection conditions. Three arterial territories (lateral circumflex femoral artery, profunda brachii artery, and radial artery) were injected and assessed through CT scans and dissections. RESULTS:The Angiofil-latex mixture allowed successful visualization of the vascular networks, offering a simple, reproducible, and non-toxic approach. Quantitative assessments of the three territories, including diameters and lengths, showed comparable results between CT scan and dissection. DISCUSSION:The technique precision and versatility make it an accessible and valuable tool for anatomical studies, potentially extending its application to MRI analyses. Overall, the Angiofil-latex combination presents a cost-effective solution for researchers, offering enhanced visibility and detailed anatomical insights for various applications, including anatomical variation studies.

IntroductionIn tissue engineering, the establishment of sufficient vascularization is essential for tissue viability and functionality. Inadequate vascularization disrupts nutrients and oxygen supply. Nonetheless, regenerating intricate vascular networks represents a significant challenge. Consequently, research efforts devoted to preserving and regenerating functional vascular networks in engineered tissues are of paramount importance. The present work aims to validate a decellularisation process with preservation of the vascular network and extracellular matrix (ECM) components in fasciocutaneous flaps.MethodFive vascularized fasciocutaneous flaps from cadaveric donors were carefully harvested from the anterolateral thigh (ALT), preserving the main perforator of the fascia lata. The entire ALT flap underwent decellularization by perfusion using a clinically validated chemical protocol. Fluoroscopy and computed tomography (CT) were used to analyze the persistence of the vascular network within the flap, pre- and post-decellularization. Histological analysis, including hematoxylin and eosin staining, and quantitative DNA assessment evaluated decellularization efficacy. Further qualitative (immunohistochemistry, IHC) and quantitative analyses were conducted to assess the preservation of ECM components, such as collagen, glycosaminoglycans, and elastin.ResultOn average, the ALT flap maintains 82% of the perfusion area (p = 0.094) post-treatment. Histological analysis confirmed decellularization efficacy and revealed structural rearrangement. Paired analysis revealed a significant decrease in DNA levels (<14.8 ng/mg of dry weight, p****< 0.0001) and well-maintained ECM. IHC indicated the persistence of elastine, collagen IV and laminin. Quantitative analysis confirmed elastin (p = 0.44) and collagen persistence (+74%, p*** = 0.001, albeit with a decrease in matrix glycosaminoglycans (-41%, p*** = 0.01)ConclusionDecellularization effectively removed cells, while preserving the ECM overall and maintaining some vascular network integrity. Yet, further study is needed to validate these findings, involving microCT examination of the vascular network and its ability to support cell colonization and viability.

AbstractWe report a rare life‐threatening presentation of postsurgical pyoderma gangrenosum (PG) after augmentation mammoplasty in a 32‐year‐old woman. Six days after surgery, the patient presented with fever and erythema at surgical wounds. In view of a suspected postsurgical infection, the patient was first treated with antibiotics and removal of breast protheses. In spite of this treatment, in a few days the patient developed a state of shock with multiorgan failure. The antibiotic coverage was broadened several times and multiple extensive surgical debridement with resection of the mammary glands were performed but did not improve the clinical situation. Twelve days after admission, the diagnosis of PG with systemic features was suspected and clinical improvement was observed within 24 h of methylprednisolone administration. PG can mimic the cutaneous and systemic features of necrotizing wound infection of surgical site. Biopsy and culture are critical to differentiating necrotizing neutrophilic dermatosis from necrotizing infection. The diagnosis of this less common form of PG is challenging and this case highlights how the frequent misdiagnosis with infection may result in ineffective antibiotic treatment and how the unnecessary surgical debridement may prolong and exacerbate the condition.

Decellularization techniques have advanced to reduce the risk of immune rejection in transplantation. Validation of these protocols typically relies on Crapo's criteria1, which include the absence of visible nuclei and low DNA content. In our study, five decellularization protocols were compared to determine the optimal approach for human fascia lata (HFL) samples. However, our findings raised questions as to why recipients can still develop immunity despite meeting validation criteria.HFL samples were decellularized using four protocols with SDS-Triton X100-DNase (D1 to D4-HFL) and one protocol using solvent-detergent-based baths (D5-HFL). The decellularized samples (D-HFL) were compared to native samples (N-HFL) using histology, and DNA content was measured. The human leukocyte antigen (HLA) content within the matrix was assessed using western blot analysis. Both D-HFL and N-HFL samples, along with negative control patches, were implanted in the backs of 28 Wistar rats. Anti-human IgG serum levels were evaluated after one month.H&E and Hoechst staining revealed the absence of residual cells in all decellularization protocols. DNA content was consistently below the critical threshold (p<0.05). All implanted D-HFL samples resulted in significantly lower anti-human IgG levels compared to N-HFL (p<0.01). However, 2.5 out of 4 rats developed immunity after being implanted with D1 to D4-HFL, with varying levels of anti-human IgG. Only rats implanted with D5-HFL showed undetectable levels of IgG and were considered non-immunized. Western blot analysis indicated that only D5-HFL had a residual HLA content below 1%.The literature on decellularization has primarily relied on Crapo's criteria, which do not consider the role of HLA mismatch in acute immune rejection. Our results suggest that a residual HLA content below 1% should also be considered to prevent immunization, even if other validation criteria are met. Further research is needed to evaluate the impact of residual HLA levels on human allotransplantation outcomes.

Background:Calcific aortic stenosis (AS) is the most prevalent heart valve disease in developed countries. The aortic valve cusps progressively thicken and the valve does not open fully due to the presence of calcifications. In vivo imaging, usually used for diagnosis, does not allow the visualization of the microstructural changes associated with AS.Methods:Ex vivo high-resolution microfocus computed tomography (microCT) was used to quantitatively describe the microstructure of calcified aortic valve cusps in full 3D. As case study in our work, this quantitative analysis was applied to normal-flow low-gradient severe AS (NF-LG-SAS), for which the medical prognostic is still highly debated in the current literature, and high-gradient severe AS (HG-SAS).Results:The volume proportion of calcification, the size and number of calcified particles and their density composition was quantified. A new size-based classification considering small-sized particles that are not detected with in vivo imaging was defined for macro-, meso- and microscale calcifications. Volume and thickness of aortic valve cusps, including the complete thickness distribution, were also determined. Moreover, changes in the cusp soft tissues were also visualized with microCT and confirmed by scanning electron microscopy images of the same sample. NF-LG-SAS cusps contained lower relative amount of calcifications than HG-SAS. Moreover, the number and size of calcified objects and the volume and thickness of the cusps were also lower in NF-LG-SAS cusps than in HG-SAS.Conclusions:The application of high-resolution ex vivo microCT to stenotic aortic valve cusps provided a quantitative description of the general structure of the cusps and of the calcifications present in the cusp soft tissues. This detailed description could help in the future to better understand the mechanisms of AS.

The purpose of this study is to enhance massive bone allografts osseointegration used to reconstruct large bone defects. These allografts show >50% complication rate requiring surgical revision in 20% cases. A new protocol for total bone decellularisation exploiting the vasculature can offer a reduction of postoperative complication by annihilating immune response and improving cellular colonization/ osseointegration. The nutrient artery of 18 porcine bones - humerus/femur/radius/ulna - was cannulated. The decellularization process involved immersion and sequential perfusion with specific solvents over a course of one week. Perfusion was realized by a peristaltic pump (mean flow rate: 6ml/min). The benefit of arterial perfusion was compared to a control group kept in immersion baths without perfusion. Bone samples were processed for histology (HE, Masson's trichrome and DAPI for cell detection), immunohistochemistry (IHC : Collagen IV/elastin for intraosseous vascular system evaluation, Swine Leukocyte Antigen – SLA for immunogenicity in addition to cellular clearance) and DNA quantification. Sterility and solvent residues in the graft were also evaluated with thioglycolate test and pH test respectively. Compared to native bones, no cells could be detected and residual DNA was <50ng/mg dry weight. Intramedullary spaces were completely cleaned. IHC showed the preservation of intracortical vasculature with channels bounded by Collagen IV and elastin within Haversian systems. IHC also showed a significant decrease in SLA signaling. All grafts were sterile at the last decellularization step and showed no solvent residue. The control group kept in immersion baths, paired with 6 perfused radii/ulnae, showed that the perfusion is mandatory to ensure complete decellularisation. Our results prove the effectiveness of a new concept of total bone decellularisation by perfusion. These promising results could lead to a new technique of Vascularized Composite Allograft transposable to pre-clinical and clinical models.