HLA Awareness in tissue decellularization: A paradigm shift for enhanced biocompatibility, studied on the model of the human fascia lata graft.

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.

Multimodal analyses of immune cells during bone repair identify macrophages as a therapeutic target in musculoskeletal trauma.

Musculoskeletal traumatic injuries (MTI) involve soft tissue lesions adjacent to a bone fracture leading to fibrous nonunion. The impact of MTI on the inflammatory response to fracture and on the immunomodulation of skeletal stem/progenitor cells (SSPCs) remains unknown. Here, we used single-nucleus transcriptomic analyses to describe the immune cell dynamics after bone fracture and identified distinct macrophage subsets with successive pro-inflammatory, pro-repair and anti-inflammatory profiles. Concurrently, SSPCs transition via a pro- and anti-inflammatory fibrogenic phase of differentiation prior to osteochondrogenic differentiation. In a preclinical MTI mouse model, the injury response of immune cells and SSPCs is disrupted leading to a prolonged pro-inflammatory phase and delayed resolution of inflammation. Macrophage depletion improves bone regeneration in MTI demonstrating macrophage involvement in fibrous nonunion. Finally, pharmacological inhibition of macrophages using the CSF1R inhibitor Pexidartinib ameliorates healing. These findings reveal the coordinated immune response of macrophages and skeletal stem/progenitor cells as a driver of bone healing and as a primary target for the treatment of trauma-associated fibrosis.

Multimodal analyses of immune cells during bone repair identify macrophages as a therapeutic target in musculoskeletal trauma.

Musculoskeletal traumatic injuries (MTI) involve soft tissue lesions adjacent to a bone fracture leading to fibrous nonunion. The impact of MTI on the inflammatory response to fracture and on the immunomodulation of skeletal stem/progenitor cells (SSPCs) remains unknown. Here, we used single cell transcriptomic analyses to describe the immune cell dynamics after bone fracture and identified distinct macrophage subsets with successive pro-inflammatory, pro-repair and anti-inflammatory profiles. Concurrently, SSPCs transition via a pro- and anti-inflammatory fibrogenic phase of differentiation prior to osteochondrogenic differentiation. In a preclinical MTI mouse model, the injury response of immune cells and SSPCs is disrupted leading to a prolonged pro-inflammatory phase and delayed resolution of inflammation. Macrophage depletion improves bone regeneration in MTI demonstrating macrophage involvement in fibrous nonunion. Finally, pharmacological inhibition of macrophages using the CSF1R inhibitor Pexidartinib ameliorates healing. These findings reveal the coordinated immune response of macrophages and skeletal stem/progenitor cells as driver of bone healing and as a primary target for the treatment of trauma-associated fibrosis. Summary: Hachemi et al. report the immune cell atlas of bone repair revealing macrophages as pro-fibrotic regulators and a therapeutic target for musculoskeletal regeneration. Genetic depletion or pharmacological inhibition of macrophages improves bone healing in musculoskeletal trauma.

Collagen molecular organization preservation in human fascia lata and periosteum after tissue engineering.

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.