Drug suspending during wound healing effectively weakens immunosuppression-related complications by preserving CD8+ T cell function.

Immunosuppressive medications, which interfere with the activation and proliferation of T and B cells, increase the risk of wound healing complications. To address it, this study aimed to validate the feasibility of drug suspending during wound healing, whilst exploring the mechanisms exerted by T cells, which are important in the wound healing process. For this, a mouse skin wound model was set up. Tacrolimus (FK506) and fingolimod (FTY720) were both administered intraperitoneally prior to wounding to inhibit the T cell activation and migration, respectively. Flow-cytometric analysis subsequently revealed the functional T cell subtypes detected during the healing process. A CD8a antibody was also administered to deplete CD8+ T cells in vivo to verify their specific function. It was found that FK506 or FTY720 administration delayed the early phase of wound healing by reducing collagen production, which was also supported by the downregulation of col1a1, col3a1 and tgfb1. However, there was no significant difference in the total healing period. Both spleen- and skin-derived CD8+ T cells were proliferated and activated after injury without intervention, whereas CD4+ T cells showed no significant changes. Furthermore, selectively depleting CD8+ T cells retarded the healing process by downregulating collagen production-associated genes (col1a1, col3a1, tgfß1 and en1) and proteins (collagen type 1 and 3). In addition, the CD8a antibody decreased the expression of genes lta, tnfa, il13 and il13ra, and protein interleukin-13Rα. In conclusion, suspending immunosuppressive drugs during wound healing was shown to be feasible through restraining the migration of activated T cells. CD8+ T cells represented the primary functional subtype positively associated with wound healing.

An Updated Surgical Approach of Using Flap and Cutaneous Nerve in the Lateral Arm to Reconstruct Complex Injuries of the Forearm and Hand: A Case Series Study.

BACKGROUND: Complex injuries involving the nerves and other soft tissues in the forearm and hand lead to functional and aesthetic defects. In such situations, multiple types of nerve autografts and flap donor sites are available. However, multiple donor sites cause donor morbidity in different locations and may lead to awkward operational positions. Therefore, based on the anatomical characterization, we aimed to modify the utilization of the lateral arm donor site for reconstruction, which restricts donor morbidity in the affected upper extremity. METHODS: We report a case series (N = 6) using a lateral arm flap (LAF) to reconstruct complex soft tissue defects in the forearm, palm, and finger. The posterior antebrachial cutaneous nerve (PACN) is the primary option for nerve bridging, whereas the LAF can carry the lower lateral brachial cutaneous nerve (LBCN) as a sensory flap. Once the PACN was insufficient, the LBCN was harvested simultaneously. All the cases included in this study were performed between January 2012 and August 2021. Demographic information, flap and nerve characteristics, complications, and hand function were analyzed. RESULTS: The LAF plus PACN or plus LBCN as nerve autograft, both successfully repaired 6 complex injuries: 2 cases in the forearm side, 1 in the hand palm, and 3 in the finger defects. Posterior antebrachial cutaneous nerve was the most used (8-15 cm), and LBCN plus PACN was used to bridge nerve defects when necessary (in total, 20 and 21 cm). The average follow-up time was 19.7 months. The disabilities of the arm, shoulder and hand score ranged between 6 and 12, and the mean 2-point discrimination values ranged between 6 and 12. The Semmes-Weinstein monofilament test result was under 5.46. In addition, 2 patients underwent a secondary debulking surgery. The average length of hospital stay was 10.4 days. Hematoma occurred in 2 cases, and all patients reported numbness in the donor nerve innervated areas. CONCLUSIONS: This surgical refinement can reconstruct complex injuries in the forearm and hand. In addition, this approach restricts donor morbidity in the affected limb, comforts the operational position, and is achieved under brachial plexus anesthesia.

Prediction of pedicled flap survival preoperatively by operating indocyanine green angiography at 1,450 nm wavelength: an animal model study.

Background: Predicting flap viability benefits patients by reducing complications and guides flap design by reducing donor areas. Due to varying anatomy, obtaining individual vascular information preoperatively is fundamental for designing safe flaps. Although indocyanine green angiography (ICGA) is a conventional tool in intraoperative assessment and postoperative monitoring, it is rare in preoperative prediction. Methods: ICGA was performed on 20 male BALB/c mice under five wavelengths (900/1,000/1,100, /1,250/1,450 nm) to assess vascular resolution after ICG perfusion. A "mirrored-L" flap model with three angiosomes was established on another 20 male BALB/c mice, randomly divided into two equal groups. In Group A, a midline between angiosomes II and III was used as a border. In Group B, the points of the minimized choke vessel caliber marked according to the ICG signal at 1,450 nm wavelength (ICG1450) were connected. Necrotic area calculations, pathohistological testing, and statistical analysis were performed. Results: The vascular structure was clearly observed at 1,450 nm wavelength, while the 900 to 1,100 nm failed to depict vessel morphology. Necrosis was beyond the borderline in 60% of Group A. Conversely, 100% of Group B had necrosis distal to the borderline. The number of choke vessels between angiosomes II and III was positively correlated with the necrotic area (%). The pathohistological findings supported the gross observation and analysis. Conclusion: ICG1450 can delineate the vessel structure in vivo and predict the viability of pedicled skin flaps using the choke vessel as the border between angiosomes.

Chemokine (C-C motif) ligand 2-enhanced adipogenesis and angiogenesis of human adipose-derived stem cell and human umbilical vein endothelial cell co-culture system in adipose tissue engineering.

Human adipose-derived stem cells (hADSCs) and human umbilical vein endothelial cells (HUVECs) co-cultured in vitro are widely used in adipose tissue engineering but exhibit various limitations. Chemokine (C-C motif) ligand 2 (CCL2) has been proved essential during adipogenesis and angiogenesis in vivo. We examined whether adipogenesis and angiogenesis could also be directly promoted by CCL2 in vitro. Cells were cultured with 0, 10, 50, and 100 ng/ml CCL2. The effects of CCL2 on adipogenesis of hADSCs, and lipid accumulation in the positive control group (hADSCs), blank control group (hADSCs + HUVECs), and experimental group (hADSCs + HUVECs + CCL2) in the hADSC and HUVEC direct co-culture system were evaluated by Oil Red O staining. Angiogenesis in the presence of CCL2 was evaluated by Matrigel tube formation assay. Angiogenic- and adipogenic-associated gene and protein expression in the co-culture system were measured by Quantitative Real-time Polymerase Chain Reaction and western blotting, respectively. All concentrations of CCL2 promoted hADSC adipogenic differentiation and HUVEC tube formation (P < 0.05). Following direct co-culture, the experimental group accumulated more lipid droplets than the positive control (P < 0.0001), whereas the latter showed better adipogenesis than the blank control group. 50 ng/ml CCL2 exhibited stronger adipogenic and angiogenic potential than other concentrations. After 72 h of direct co-culture, the mRNA expression of adipogenic differentiation (peroxisome proliferators-activated receptorsγ, CCAAT/enhancer binding protein-α, Leptin, and lipoprotein lipase) and angiogenic genes (vascular endothelial growth factor-A, vascular endothelial growth factor receptor 2, matrix metalloprotein (MMP) 9, and 14) in the experimental group was much higher than in the control (P < 0.05). The addition of 50 ng/ml CCL2 in the system resulted in elevated phosphorylated Protein kinase B/AKT expression. In summary, CCL2 directly promoted adipogenesis of hADSCs and angiogenesis of HUVECs under both mono-culture and co-culture condition in vitro possibly by enhancing AKT phosphorylation. An optimal concentration of 50 ng/ml CCL2 could improve the adipogenesis and angiogenesis of hADSC and HUVEC co-culture system.

Construction of adipose tissue using a silica expander capsule and cell sheet-assembled of decellularized adipose tissue.

Delayed neovascularization and unstable adipose formation are major confounding factors in adipose tissue engineering. A system using decellularized adipose tissue (DAT), adipose-derived stem cells (ADSCs), and human umbilical vein endothelial cells (HUVECs) has been preliminarily studied, but it requires optimization, as adipogenic and angiogenic capabilities for maintaining a stable construct shape are limited. The current study aimed to address these limitations. Our initial modification involved the addition of exogenous chemokine (C-C motif) ligand 2 (CCL2), which resulted in enhanced adipogenesis and angiogenesis. However, further improvement was required due to delayed blood recanalization. To further optimize the system, a vascularized fibrous capsule derived from an implanted silica expander was utilized as a second modification. We hypothesized this would function as both a microbioreactor to fix the seed cells and exogenous CCL2 locally and as a vascular bed to promote neovascularization. Compared with that of the CCL2 loaded ADSC-HUVECs cell sheet assembled DAT system, adding the silica expander capsule resulted in significantly increased construct stability, new vessel intensity, a greater number of Oil Red O-positive lipid droplets, more enhanced tissue remodeling, and upregulated peroxisome proliferator-activated receptor gamma (PPARγ) & leptin expression. Thus, these two modifications helped optimize the currently available ADSC-HUVEC cell sheet assembled DAT system, providing an adipose tissue construction strategy with enhanced adipogenesis and angiogenesis to reconstruct soft tissue defects. Moreover, close-to-normal leptin expression provided the engineered adipose tissue with a glucometabolic function, in addition to remodeling capabilities. STATEMENT OF SIGNIFICANCE: Delayed neovascularization and unstable adipose formation are the two major problems in tissue engineering adipose. Here, we introduced an adipose tissue engineering construction strategy using a silica expander capsule along with hADSCs-HUVECs cell sheet-assembled DAT in a CCL2-rich microenvironment. Our data suggested that CCL2 could improve angiogenesis and adipogenesis in vitro and in vivo. The addition of tissue expander capsule could further improve the stability of construction and fabricated adipose tissue with increased new vessel intensity, greater numbers of Oil Red O-positive lipid droplets, more enhanced tissue remodeling, and upregulated leptin expression. CCL2 and expander capsule can have clinical utility for soft tissue defects repair, and these two factors can be useful in other tissue engineering.