The minor pilin PilV provides a conserved adhesion site throughout the antigenically variable meningococcal type IV pilus.

Neisseria meningitidis utilizes type IV pili (T4P) to adhere to and colonize host endothelial cells, a process at the heart of meningococcal invasive diseases leading to meningitis and sepsis. T4P are polymers of an antigenically variable major pilin building block, PilE, plus several core minor pilins that initiate pilus assembly and are thought to be located at the pilus tip. Adhesion of N. meningitidis to human endothelial cells requires both PilE and a conserved noncore minor pilin PilV, but the localization of PilV and its precise role in this process remains to be clarified. Here, we show that both PilE and PilV promote adhesion to endothelial vessels in vivo. The substantial adhesion defect observed for pilV mutants suggests it is the main adhesin. Consistent with this observation, superresolution microscopy showed the abundant distribution of PilV throughout the pilus. We determined the crystal structure of PilV and modeled it within the pilus filament. The small size of PilV causes it to be recessed relative to adjacent PilE subunits, which are dominated by a prominent hypervariable loop. Nonetheless, we identified a conserved surface-exposed adhesive loop on PilV by alanine scanning mutagenesis. Critically, antibodies directed against PilV inhibit N. meningitidis colonization of human skin grafts. These findings explain how N. meningitidis T4P undergo antigenic variation to evade the humoral immune response while maintaining their adhesive function and establish the potential of this highly conserved minor pilin as a vaccine and therapeutic target for the prevention and treatment of N. meningitidis infections.

Colonization of dermal arterioles by Neisseria meningitidis provides a safe haven from neutrophils.

The human pathogen Neisseria meningitidis can cause meningitis and fatal systemic disease. The bacteria colonize blood vessels and rapidly cause vascular damage, despite a neutrophil-rich inflammatory infiltrate. Here, we use a humanized mouse model to show that vascular colonization leads to the recruitment of neutrophils, which partially reduce bacterial burden and vascular damage. This partial effect is due to the ability of bacteria to colonize capillaries, venules and arterioles, as observed in human samples. In venules, potent neutrophil recruitment allows efficient bacterial phagocytosis. In contrast, in infected capillaries and arterioles, adhesion molecules such as E-Selectin are not expressed on the endothelium, and intravascular neutrophil recruitment is minimal. Our results indicate that the colonization of capillaries and arterioles by N. meningitidis creates an intravascular niche that precludes the action of neutrophils, resulting in immune escape and progression of the infection.

Type IV pilus retraction enables sustained bacteremia and plays a key role in the outcome of meningococcal sepsis in a humanized mouse model.

Neisseria meningitidis (the meningococcus) remains a major cause of bacterial meningitis and fatal sepsis. This commensal bacterium of the human nasopharynx can cause invasive diseases when it leaves its niche and reaches the bloodstream. Blood-borne meningococci have the ability to adhere to human endothelial cells and rapidly colonize microvessels. This crucial step enables dissemination into tissues and promotes deregulated inflammation and coagulation, leading to extensive necrotic purpura in the most severe cases. Adhesion to blood vessels relies on type IV pili (TFP). These long filamentous structures are highly dynamic as they can rapidly elongate and retract by the antagonistic action of two ATPases, PilF and PilT. However, the consequences of TFP dynamics on the pathophysiology and the outcome of meningococcal sepsis in vivo have been poorly studied. Here, we show that human graft microvessels are replicative niches for meningococci, that seed the bloodstream and promote sustained bacteremia and lethality in a humanized mouse model. Intriguingly, although pilus-retraction deficient N. meningitidis strain (ΔpilT) efficiently colonizes human graft tissue, this mutant did not promote sustained bacteremia nor induce mouse lethality. This effect was not due to a decreased inflammatory response, nor defects in bacterial clearance by the innate immune system. Rather, TFP-retraction was necessary to promote the release of TFP-dependent contacts between bacteria and, in turn, the detachment from colonized microvessels. The resulting sustained bacteremia was directly correlated with lethality. Altogether, these results demonstrate that pilus retraction plays a key role in the occurrence and outcome of meningococcal sepsis by supporting sustained bacteremia. These findings open new perspectives on the role of circulating bacteria in the pathological alterations leading to lethal sepsis.

Lower Body Lift in the Massive Weight Loss Patient: A New Classification and Algorithm for Gluteal Augmentation.

BACKGROUND: An often-neglected part of the lower body lift procedure is the gluteal region. The objective of this study was to classify massive weight loss patients undergoing a body lift procedure and provide a safe, standardized approach for gluteal augmentation. METHODS: A retrospective review of all body lift procedures performed between January of 2012 and January of 2017 was conducted. Patients undergoing a lower body lift with or without gluteal augmentation were included for analysis. Patients were classified as follows: type I, minimal lower and upper back fat and deflated buttock; type II, substantial lower back fat, minimal upper back fat, and deflated buttock; type III, substantial lower and upper back fat and deflated buttock; and type IV, good buttock projection. Type I patients had gluteal implants, type II patients had autologous flap augmentation, type III patients had gluteal lipofilling, and type IV patients did not have any gluteal augmentation. RESULTS: Two hundred eighty patients were included for analysis. Two hundred thirty-eight underwent concomitant gluteal augmentation (85 percent): 213 had autologous flaps (76 percent), 13 had gluteal implants (5 percent), and 12 had large-volume lipofilling (4 percent). Forty-two patients underwent a body lift with no gluteal augmentation (15 percent). Gluteal augmentation did not increase the rate of complications. In both groups, no skin necrosis, venous thrombosis, or pulmonary embolism was reported. Patients who had a sleeve gastrectomy had significantly lower odds of complications compared with gastric bypass (OR, 0.45; p = 0.017). CONCLUSION: A standardized algorithmic approach for gluteal augmentation may optimize the result without increasing the complication rate. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.

Augmentation of tendon healing with an injectable tendon hydrogel in a rat Achilles tendon model.

BACKGROUND: Many unsolved problems in plastic and hand surgery are related to poor healing of acute and chronic tendon injuries. The authors hypothesized that tendon healing could be augmented by the addition of a tendon-derived, extracellular matrix hydrogel that would guide tissue regeneration. METHODS: Both Achilles tendons of 36 Wistar rats were given full-thickness injuries approximately 5 mm long and 0.5 mm wide from the tendon insertion at the calcaneus to the midsubstance. The hydrogel was injected into the injury site of one leg and compared with control saline in the other. The ultimate failure load, ultimate tensile stress, and stiffness were evaluated at 2, 4, and 8 weeks. Tendon cross-sections underwent histologic analysis (hematoxylin and eosin and picrosirius red) after the animals were killed. Statistical analysis of biomechanical data was performed using a paired t test. RESULTS: There was no significant difference in strength between gel and saline injections in ultimate failure load (p = 0.15), ultimate tensile stress (p = 0.42), or stiffness (p = 0.76) at 2 weeks. However, there was a significant difference in ultimate failure load (74.8 ± 11.6 N versus 58.4 ± 14.2 N; p = 0.02) at 4 weeks. The difference in ultimate tensile stress (p = 0.63) and stiffness (p = 0.08) remained insignificant. By 8 weeks, there was no significant difference in strength in ultimate failure load (p = 0.15), ultimate tensile stress (p = 0.39), or stiffness (p = 0.75). CONCLUSIONS: Treatment with the tendon hydrogel significantly increases the ultimate failure load of tendons at the critical 4-week time point, and is a promising method for augmentation of tendon healing.

Design and characterization of an injectable tendon hydrogel: a novel scaffold for guided tissue regeneration in the musculoskeletal system.

A biocompatible hydrogel consisting of extracellular matrix (ECM) from human tendons is described as a potential scaffold for guided tissue regeneration and tissue engineering purposes. Lyophilized decellularized tendons were milled and enzymatically digested to form an ECM solution. The ECM solution properties are assessed by proteome analysis with mass spectrometry, and the material's rheological properties are determined as a function of frequency, temperature, and time. In vivo application of the gel in a rat model is assessed for remodeling and host cell repopulation. Histology for macrophage invasion, fibroblast repopulation, and nanoscale properties of the gel is assessed. Gel interaction with multipotent adipoderived stem cells (ASCs) is also addressed in vitro to assess possible cytotoxicity and its ability to act as a delivery vehicle for cells. Proteome analysis of the ECM-solution and gel mass spectroscopy identified the most abundant 150 proteins, of which two isoforms of collagen I represented more than 55% of the sample. Rheology showed that storage (G') and loss (G″) of the ECM solution were stable at room temperature but displayed sigmoidal increases after ∼15 min at 37°C, matching macroscopic observations of its thermo responsiveness. G' and G″ of the gel at 1 rad/s were 213.1±19.9 and 27.1±2.4 Pa, respectively. Electron microscopy revealed fiber alignment and good structural porosity in the gel, as well as invasion of cells in vivo. Histology also showed early CD68(+) macrophage invasion throughout the gel, followed by increasing numbers of fibroblast cells. ASCs mixed with the gel in vitro proliferated, indicating good biocompatibility. This ECM solution can be delivered percutaneously into a zone of tendon injury. After injection, the thermoresponsive behavior of the ECM solution allows it to polymerize and form a porous gel at body temperature. A supportive nanostructure of collagen fibers is established that conforms to the three-dimensional space of the defect. This hydrogel holds the distinctive composition specific for tendon ECM, where tissue-specific cues facilitate host cell infiltration and remodeling. The results presented indicate that injectable ECM materials from tendon may offer a promising alternative in the treatment of tendinopathies and acute tendon injuries.

Decellularized tendon-bone composite grafts for extremity reconstruction: an experimental study.

BACKGROUND: Restoration of biomechanical strength following surgical reconstruction of tendon or ligament insertion tears is challenging because these injuries typically heal as fibrous scars. The authors hypothesize that injuries at the tendon-bone interface would benefit from reconstruction with decellularized composite tendon-bone grafts. METHODS: Tendon-bone grafts were harvested from Sprague-Dawley rats. Grafts subjected to decellularization were compared histologically and biomechanically with untreated grafts ex vivo and in a new in vivo model. Wistar rats underwent Sprague-Dawley allograft reconstruction using a pair-matched design. The rats were killed at 2 or 4 weeks. B-cell and macrophage infiltration was determined using immunohistochemistry, and explants were tested biomechanically. RESULTS: Decellularization resulted in a decrease in cells from 164 ± 61 (untreated graft) to 13 ± 7 cells per high-power field cells (p < 0.005) and a corresponding significant decrease in DNA content, and preserved scaffold architecture of the tendon-bone interface. Biomechanical comparison revealed no difference in failure load (p = 0.32), ultimate tensile stress (p = 0.76), or stiffness (p = 0.22) between decellularized grafts and untreated controls. Following in vivo reconstruction with tendon-bone interface grafts, decellularized grafts were stronger than untreated grafts at 2 weeks (p = 0.047) and at 4 weeks (p < 0.005). A persistent increase in B-cell and macrophage infiltration was observed in both the capsule surrounding the tendon-bone interface and the tendon substance in untreated controls. CONCLUSION: Decellularized tendon-bone grafts display better biomechanical properties at early healing time points and a decreased immune response compared with untreated grafts in vivo.

Human flexor tendon tissue engineering: in vivo effects of stem cell reseeding.

BACKGROUND: Tissue-engineered human flexor tendons may be an option to aid in reconstruction of complex upper extremity injuries with significant tendon loss. The authors hypothesize that human adipose-derived stem cells remain viable following reseeding on human tendon scaffolds in vivo and aid in graft integration. METHODS: Decellularized human flexor tendons harvested from fresh-frozen cadavers and reseeded with green fluorescent protein-labeled pooled human adipose-derived stem cells were examined with bioluminescent imaging and immunohistochemistry. Reseeded repaired tendons were compared biomechanically with unseeded controls following implantation in athymic rats at 2 and 4 weeks. The ratio of collagen I to collagen III at the repair site was examined using Sirius red staining. To confirm cell migration, reseeded and unseeded tendons were placed either in contact or with a 1-mm gap for 12 days. Green fluorescent protein signal was then detected. RESULTS: Following reseeding, viable cells were visualized at 12 days in vitro and 4 weeks in vivo. Biomechanical testing revealed no significant difference in ultimate load to failure and 2-mm gap force. Histologic evaluation showed host cell invasion and proliferation of the repair sites. No increase in collagen III was noted in reseeded constructs. Cell migration was confirmed from reseeded constructs to unseeded tendon scaffolds with tendon contact. CONCLUSIONS: Human adipose-derived stem cells reseeded onto decellularized allograft scaffolds are viable over 4 weeks in vivo. The movement of host cells into the scaffold and movement of adipose-derived stem cells along and into the scaffold suggests biointegration of the allograft.

Decellularized human tendon-bone grafts for composite flexor tendon reconstruction: a cadaveric model of initial mechanical properties.

PURPOSE: After complex hand trauma, restoration of tendon strength is challenging. Tendon insertion tears typically heal as fibrous scars after surgical reconstruction and create a weak point at the tendon-bone interface. In addition, major tendon loss may overwhelm the amount of available autograft for reconstruction. An off-the-shelf product may help address these challenges. We hypothesized that decellularized human flexor digitorum profundus and distal phalanx tendon-bone composite grafts were a feasible option for flexor tendon reconstruction after complex hand trauma. By replacing the entire injured composite segment, the need for tendon repair within the tendon sheath, reconstruction of the tendon-bone interface, and use of limited autograft could be eliminated. METHODS: Paired human cadaver forearms were dissected to obtain the flexor digitorum profundus tendon with an attached block of distal phalanx. Tendon-bone grafts were pair-matched and divided into 2 groups: decellularized grafts (n = 12) and untreated (control) grafts (n = 11). Grafts in the decellularized group were subjected to physiochemical decellularization. Pair-matched tendon-bone grafts (decellularized and untreated) were placed back into the flexor tendon sheath and secured distally using a tie-over button and proximally by weaving the graft into the flexor digitorum superficialis tendon in the distal forearm. The ultimate load, location of failure, and excursion were determined. RESULTS: Decellularized tendon-bone composite grafts demonstrated no significant difference in ultimate failure load or stiffness compared with untreated grafts. Both groups eventually failed in varied locations along the repair. The most common site of failure in both groups was the tie-over button. The decellularized group failed at the tendon-bone insertion in 3 specimens (25%) compared with none in the untreated group. Both groups demonstrated an average tendon excursion of approximately 82 mm before failure. CONCLUSIONS: Decellularization of human flexor tendon-distal phalanx tendon-bone constructs did not compromise initial strength despite chemical and mechanical decellularization in a cadaveric model. At the time of repair, decellularized flexor tendon-bone grafts can exceed the strength and excursion needed for hand therapy immediately after reconstruction. CLINICAL RELEVANCE: These tendon-bone grafts may become an option for complex hand reconstruction at or near tendon-bone insertions and throughout the tendon sheath. Further work is required to assess the role of reseeding in an in vivo model.

Physicochemical decellularization of composite flexor tendon-bone interface grafts.

BACKGROUND: Extremity injuries involving tendon attachment to bone are difficult to address. Clinically, tendon-bone interface allografts must be decellularized to reduce immunogenicity. Composite grafts are difficult to decellularize because chemical agents cannot reach cells between tissues. In this study, the authors attempted to optimize tendon-bone interface graft decellularization. METHODS: Human flexor digitorum profundus tendons with attached distal phalanx were harvested from cadavers and divided into four groups. Group 1 (control) was untreated. Group 2 (chemical) was chemically treated with 5% peracetic acid, 0.1% ethylenediaminetetraacetic acid, and 0.1% sodium dodecyl sulfate. Group 3 (low-power) underwent targeted ultrasonication for 3 minutes (22,274 J, 126W) followed by chemical decellularization. Group 4 (high-power) underwent targeted ultrasonication for 10 minutes (88,490 J, 155W) followed by chemical decellularization. Decellularization was assessed histologically with hematoxylin and eosin stain and stains for major histocompatibility complex I stains. Cell counts were performed. The ultimate tensile load of decellularized grafts (group 4) were compared with pair-matched untreated grafts (group 1). RESULTS: Average cell counts were 100 ± 41, 27 ± 10, 12 ± 11, and 6 ± 11 per high-power field for groups 1, 2, 3, and 4, respectively (p < 0.001). Decellularization using physical and chemical treatments (groups 3 and 4) resulted in substantial reduction of cells and major histocompatibility complex I molecules. There was no difference in ultimate tensile load between treated (group4) and untreated (group 1) samples (p > 0.5). CONCLUSIONS: Physicochemical decellularization of tendon-bone interface grafts using targeted ultrasonication and chemical treatment resulted in near-complete reduction in cellularity and maintenance of tensile strength. In the future, these decellularized composite scaffolds may be used for reconstruction of tendon-bone injuries.

New concepts and technologies in reconstructive hand surgery.

Complex traumatic injuries and degenerative conditions of the hand continue to lead to significant impairment and disability. From technical innovations to regenerative concepts, this article presents the latest advances in the dynamic field of hand surgery in which worldwide efforts are made around the globe to repair, regenerate, or restore each composite tissue forming the hand. The systematic method by which finger replantation is performed, from bony fixation to skin closure, provides a platform for discussion of the newest innovations available to reconstructive hand surgeons.

Human flexor tendon tissue engineering: revitalization of biostatic allograft scaffolds.

Cadaveric tendon allografts form a readily available and underutilized source of graft material. Because of their material properties, allografts are biomechanically and biologically superior to synthetic scaffolds. However, before clinical use, allografts must undergo decellularization to reduce immunogenicity and oxidation to increase porosity, leaving a nonvital biostatic scaffold. Ex vivo seeding, or revitalization, is thought to hasten graft incorporation and stimulate intrinsic tendon healing, permitting early mobilization and return to function. In this study, we examined physical and biochemical augmentation methods, including scaffold surface scoring (physical) and rehydration of lyophilized scaffolds in serum (biochemical). Scaffolds were divided into four groups: (1) scored scaffolds, (2) lyophilized scaffolds rehydrated in fetal calf serum (FCS), (3) scaffolds both scored and rehydrated in FCS, and (4) control scaffolds. Scaffolds were reseeded with adipose-derived stem cells (ADSCs). Reseeding efficacy was quantified by a live cell and total cell assays and qualified histologically with hematoxylin and eosin, live/dead and SYTO green nucleic acid stains, TUNEL apoptosis stains, procollagen stains, and transmission electron microscopy. Scaffold-seeded cell viability at up to 2 weeks in vitro and up to 4 weeks in vivo was demonstrated with bioluminescent imaging of scaffolds seeded with luciferase-positive ADSCs. The effect of seeding on scaffold biomechanical properties was demonstrated with evaluation of ultimate tensile stress (UTS) and an elastic modulus (EM). We found that scaffold surface scoring led to an increase in live and total cell attachment and penetration (MTS assay, p<0.001 and DNA assay, p=0.003, respectively). Histology confirmed greater total cell number in both construct core and surface in scored compared with unscored constructs. Cells reseeded on scored constructs displayed reduced apoptosis, persistent procollagen production, and had a similar ultrastructural relationship to the surrounding matrix as native tenocytes on transmission electron microscopy. Rehydration of lyophilized scaffolds in serum did not improve reseeding. Seeded constructs demonstrated greater UTS and EM than unseeded constructs. Scaffolds seeded with ADSC-luc2-eGFP demonstrated persistent viability for at least 2 weeks in vitro. In conclusion, tendon surface scoring increases surface and core reseeding in vitro and may be incorporated as a final step in allograft processing before clinical implantation.