Characterization of a full-thickness decellularized and lyophilized human placental membrane for clinical applications.

Allografts derived from live-birth tissue obtained with donor consent have emerged as an important treatment option for wound and soft tissue repairs. Placental membrane derived from the amniotic sac consists of the amnion and chorion, the latter of which contains the trophoblast layer. For ease of cleaning and processing, these layers are often separated with or without re-lamination and the trophoblast layer is typically discarded, both of which can negatively affect the abundance of native biological factors and make the grafts difficult to handle. Thus, a full-thickness placental membrane that includes a fully-intact decellularized trophoblast layer was developed for homologous clinical use as a protective barrier and scaffold in soft tissue repairs. Here, we demonstrate that this full-thickness placental membrane is effectively decellularized while retaining native extracellular matrix (ECM) scaffold and biological factors, including the full trophoblast layer. Following processing, it is porous, biocompatible, supports cell proliferation in vitro, and retains its biomechanical strength and the ability to pass through a cannula without visible evidence of movement or damage. Finally, it was accepted as a natural scaffold in vivo with evidence of host-cell infiltration, angiogenesis, tissue remodelling, and structural layer retention for up to 10 weeks in a murine subcutaneous implant model.

iTClamp-Mediated Wound Closure Speeds Control of Arterial Hemorrhage With or Without Additional Hemostatic Agents.

BACKGROUND: Exsanguination is the leading cause of preventable posttraumatic death, especially in the prehospital arena. Traditional hemorrhage control methods involve packing the wound with hemostatic agents, providing manual pressure, and then applying a pressure dressing to stabilize the treatment. This is a lengthy process that frequently destabilizes upon patient transport. Conversely, the iTClamp, a compact wound closure device, is designed to rapidly seal wound edges mechanically, expediting clot formation at the site of injury. OBJECTIVES: To determine the efficacy of the iTClamp with and without wound packing in the control of a lethal junction hemorrhage. METHODS: Given the limited available information regarding the efficacy of the iTClamp in conjunction with traditional hemostatic agents, this study used a swine model of severe junctional hemorrhage. The goal was to compare a multiagent strategy using the iTClamp in conjunction with XSTAT to the traditional method of Combat Gauze packing with pressure dressing application. Readouts include application time, blood loss, and rebleed occurrence. RESULTS: Mean application times of the iTClamp treatment alone or in conjunction with other hemostatic agents were at least 75% faster than the application time of Combat Gauze with pressure dressing. Percent blood loss was not significantly different between groups but trended the highest for Combat Gauze treated swine, followed by iTClamp plus XSTAT, iTClamp alone and finally iTClamp plus Combat Gauze. CONCLUSION: The results from this study demonstrate that the iTClamp can be effectively utilized in conjunction with hemostatic packing to control junctional hemorrhages.

A 24-month retrospective update: follow-up hospitalization charges and readmissions in US lumbar fusion surgeries using a cellular bone allograft (CBA) versus recombinant human bone morphogenetic protein-2 (rhBMP-2).

BACKGROUND: The objectives of this study were to build upon previously-reported 12-month findings by retrospectively comparing 24-month follow-up hospitalization charges and potentially-relevant readmissions in US lumbar fusion surgeries that employed either recombinant human bone morphogenetic protein-2 (rhBMP-2) or a cellular bone allograft comprised of viable lineage-committed bone cells (V-CBA) via a nationwide healthcare system database. METHODS: A total of 16,172 patients underwent lumbar fusion surgery using V-CBA or rhBMP-2 in the original study, of whom 3,792 patients (23.4%) were identified in the current study with all-cause readmissions during the 24-month follow-up period. Confounding baseline patient, procedure, and hospital characteristics found in the original study were used to adjust multivariate regression models comparing differences in 24-month follow-up hospitalization charges (in 2020 US dollars) and lengths of stay (LOS; in days) between the groups. Differences in potentially-relevant follow-up readmissions were also compared, and all analyses were repeated in the subset of patients who only received treatment at a single level of the spine. RESULTS: The adjusted cumulative mean 24-month follow-up hospitalization charges in the full cohort were significantly lower in the V-CBA group ($99,087) versus the rhBMP-2 group ($124,389; P < 0.0001), and this pattern remained in the single-level cohort (V-CBA = $104,906 vs rhBMP-2 = $125,311; P = 0.0006). There were no differences between groups in adjusted cumulative mean LOS in either cohort. Differences in the rates of follow-up readmissions aligned with baseline comorbidities originally reported for the initial procedure. Subsequent lumbar fusion rates were significantly lower for V-CBA patients in the full cohort (10.12% vs 12.00%; P = 0.0002) and similar between groups in the single-level cohort, in spite of V-CBA patients having significantly higher rates of baseline comorbidities that could negatively impact clinical outcomes, including bony fusion. CONCLUSIONS: The results of this study suggest that use of V-CBA for lumbar fusion surgeries performed in the US is associated with substantially lower 24-month follow-up hospitalization charges versus rhBMP-2, with both exhibiting similar rates of subsequent lumbar fusion procedures and potentially-relevant readmissions.

Lumbar spine fusion outcomes using a cellular bone allograft with lineage-committed bone-forming cells in 96 patients.

BACKGROUND: Instrumented posterior lumbar fusion (IPLF) with and without transforaminal interbody fusion (TLIF) is a common treatment for low back pain when conservative interventions have failed. Certain patient comorbidities and lifestyle risk factors, such as obesity and smoking, are known to negatively affect these procedures. An advanced cellular bone allograft (CBA) with viable osteogenic cells (V-CBA) has demonstrated high fusion rates, but the rates for patients with severe and/or multiple comorbidities remain understudied. The purpose of this study was to assess fusion outcomes in patients undergoing IPLF/TLIF using V-CBA with baseline comorbidities and lifestyle risk factors known to negatively affect bone fusion. METHODS: This was a retrospective study of de-identified data from consecutive patients at an academic medical center who underwent IPLF procedures with or without TLIF, and with V-CBA. Baseline patient and procedure characteristics were assessed. Radiological outcomes included fusion rates per the Lenke scale. Patient-reported clinical outcomes were evaluated via the Oswestry Disability Index (ODI) and Visual Analog Scale (VAS) for back and leg pain. Operating room (OR) times and intraoperative blood loss rates were also assessed. RESULTS: Data from 96 patients were assessed with a total of 222 levels treated overall (mean: 2.3 levels) and a median follow-up time of 16 months (range: 6 to 45 months). Successful fusion (Lenke A or B) was reported for 88 of 96 patients (91.7%) overall, including in all IPLF-only patients. Of 22 patients with diabetes in the IPLF+TLIF group, fusion was reported in 20 patients (90.9%). In IPLF+TLIF patients currently using tobacco (n = 19), fusion was reported in 16 patients (84.3%), while in those with a history of tobacco use (n = 53), fusion was observed in 48 patients (90.6%). Successful fusion was reported in all 6 patients overall with previous pseudarthrosis at the same level. Mean postoperative ODI and VAS scores were significantly reduced versus preoperative ratings. CONCLUSION: The results of this study suggest that V-CBA consistently yields successful fusion and significant decreases in patient-reported ODI and VAS, despite patient comorbidities and lifestyle risk factors that are known to negatively affect such bony healing.

Red-Green Tactical Lighting Is Preferred for Suturing Wounds in a Simulated Night Environment.

BACKGROUND: Delivering medical care in nighttime conditions is challenging, as 25% of Special Operations medical Operators have reported that problems with lighting contributed to poor casualty outcomes. Red light is often used in nighttime operations but makes blood detection difficult and diminishes depth perception and visual acuity. Red-green combination lighting may be superior for differentiating blood from tissue and other fluids but had not been tested versus red-only or green-only lighting for combat-related medical procedures, such as wound suturing. METHODS: Dark-adapted medical resident physicians (N = 24) sutured 6cm long, 3cm deep, full-thickness lacerations in deceased swine under red-only, green-only, and red-green lighting provided by a tactical flashlight using a randomized within-subjects design. Time to suture completion, suture quality, user ratings, and user preference data were contrasted at p < .05. This study was approved by Naval Medical Center Portsmouth IRB. RESULTS: Suture completion time and suture quality were similar across all lighting conditions. Participants rated red-green lighting as significantly easier for identifying blood, identifying instruments, and performing suturing (p < .01). Red-green lighting was preferred by 83% of participants compared to 8% each for red-only and green-only (p < .001). CONCLUSIONS: Pending further study under tactical conditions, red-green lighting is tentatively recommended for treating battlefield wounds in low-light environments.

Histologic case series of human acellular dermal matrix in superior capsule reconstruction.

BACKGROUND: Acellular dermal matrix (ADM) allografts are commonly used in the surgical treatment of complex and irreparable rotator cuff tears. Multiple studies report that superior capsule reconstruction (SCR) using ADM has resulted in short-term clinical success as assessed via radiographic and patient-reported outcomes. However, limited information is available regarding the biologic fate of these grafts in human subjects. This case series describes histologic results from 8 patients who had reoperations, during which the previously implanted ADMs were removed. These explanted ADMs were subjected to histologic analysis with the hypothesis that they would have evidence of recellularization, revascularization, and active remodeling. METHODS: Eight patients, 38-82 years old, underwent reoperation 6-38 months after undergoing SCR. ADM explants were voluntarily shipped to the manufacturer for histologic analysis. Each graft's structure and composition were qualitatively evaluated by 1 or more of the following histologic stains: hematoxylin and eosin, safranin O, and Russell-Movat pentachrome. Pan-muscle actin staining also assessed the level of neovascularization, potential myoblast or myocyte infiltration, and muscle tissue development in the graft, and was analyzed to determine the proportion of graft that had been recellularized in situ. RESULTS: Grafts showed varying levels of gross and microscopic incorporation with the host. An uneven, but high, overall degree of recellularization, revascularization, and active remodeling was observed. The degree of remodeling correlated with implant duration. These results are consistent with successful biologic reconstruction of the superior shoulder capsule. CONCLUSIONS: The present histologic analysis suggests that ADMs used in SCR undergo active recellularization, revascularization, and remodeling as early as 6 months after implantation, and that graft recellularization positively correlates with duration of implantation. These results represent a significant advancement in our knowledge regarding biologic incorporation of ADMs used in SCR.

A large database study of hospitalization charges and follow-up re-admissions in US lumbar fusion surgeries using a cellular bone allograft (CBA) versus recombinant human bone morphogenetic protein-2 (rhBMP-2).

BACKGROUND: The objective of this study was to retrospectively compare initial procedure and 12-month follow-up hospitalization charges and resource utilization (lengths of stay; LOS) for lumbar fusion surgeries using either recombinant human bone morphogenetic protein-2 (rhBMP-2) or a cellular bone allograft comprised of viable lineage-committed bone cells (V-CBA) via a large US healthcare system database. Potentially relevant re-admissions during the follow-up period were also assessed. METHODS: A total of 16,172 patients underwent lumbar fusion surgery using V-CBA or rhBMP-2, of whom 3503 (21.66%) patients had follow-up re-admission data. Initial patient, procedure, and hospital characteristics were assessed to determine confounding factors. Multivariate regression modeling compared differences in hospitalization charges (in 2018 US dollars) and LOS (in days) between the groups, as well as incidences of potentially relevant re-admissions during the 12-month follow-up period. RESULTS: The adjusted mean initial procedure and 12-month follow-up hospital charges were significantly lower in the V-CBA group versus the rhBMP-2 group ($109,061 and $108,315 versus $160,191 and $130,406, respectively; P < 0.0001 for both comparisons). This disparity remained in an ad hoc comparison of charges for initial single-level treatments only (V-CBA = $103,064, rhBMP-2 = $149,620; P < 0.0001). The adjusted mean initial LOS were significantly lower in the V-CBA group (3.77 days) versus the rhBMP-2 group (3.88 days; P < 0.0001), but significantly higher for the cumulative follow-up hospitalizations in the 12-month follow-up period (7.87 versus 7.46 days, respectively; P < 0.0001). Differences in rates of follow-up re-admissions aligned with comorbidities at the initial procedure. Subsequent lumbar fusion rates were comparable, but significantly lower for V-CBA patients who had undergone single-level treatments only, in spite of V-CBA patients having significantly higher rates of initial comorbidities that could negatively impact clinical outcomes. CONCLUSIONS: The results of this study indicate that use of V-CBA for lumbar fusion surgeries performed in the US may result in substantially lower overall hospitalization charges versus rhBMP-2, with both exhibiting similar rates of 12-month re-admissions and subsequent lumbar fusion procedures.

Multilevel instrumented posterolateral lumbar spine fusion with an allogeneic cellular bone graft.

BACKGROUND: Low back pain (LBP) is the leading cause of absence from work, disability, and impaired quality of life. Fusion surgery may be indicated when non-operative treatments have failed to provide relief. Surgery may include the use of fusion-enhancing implants, such as cellular bone allografts (CBAs). The purpose of this retrospective study was to evaluate efficacy and safety of one CBA (V-CBA) in patients who underwent instrumented posterolateral fusion (IPLF). METHODS: Retrospective data were collected from 150 consecutive patients who had undergone IPLF surgery between January 1, 2015, and March 31, 2018, in which V-CBA was used. All surgeries were performed by one surgeon. V-CBA was mixed with local autograft bone. Patient diagnoses included degenerative disc disease, spondylosis, spondylolisthesis, or spondylolysis with or without stenosis. Standing anteroposterior (AP) and lateral images were collected prior to surgery and again at the terminal visit, which took place between 6 and 33 months post-operatively. De-identified images were assessed radiologically. Adverse events were documented. The primary composite endpoint of fusion status was dependent upon two main criteria: bridging bone per the Lenke scale (classified as "A" definitely solid or "B" possibly solid) and posterior hardware status (intact). Lenke scale C or D were categorized as pseudarthrosis. RESULTS: Eighty-seven male and 63 female patients (613 levels total) underwent IPLF in which V-CBA was implanted. An average of 4.1 levels was treated, with 59.3% of patients having undergone treatment for more than 3 levels. Twenty-nine percent of patients had diabetes. Fifty-two percent of patients had previously used nicotine products, and 12% were current smokers. Sixteen serious adverse events were recorded and included lumbar seroma, cerebrospinal fluid leak, wound dehiscence, pneumonia, urinary tract infection, and myocardial infarction. Successful fusion (Lenke scale "A" or "B") was recorded in 148 out of 150 patients (98.7%), or 608 out of 613 levels. The total pseudarthrosis rate was 0.8%. CONCLUSIONS: The use of V-CBA combined with local autograft in multilevel IPLF resulted in successful fusions in 98.7% of patients. These results are particularly robust given the complex nature of many of these cases: 89 patients had 4 or more surgical levels, and many patients had multiple comorbidities. LEVEL OF EVIDENCE: IV.

Randomized Comparative Assessment of Three Surgical Cricothyrotomy Devices on Airway Mannequins.

BACKGROUND: Airway obstruction is the second leading cause of preventable battlefield death, at least in part because surgical cricothyrotomy (SC) failure rates remain unacceptably high. Ideally, SC should be a rapid, simple, easily-learned, and reliably-performed procedure. Currently, 3 SC devices meet Tactical Combat Casualty Care (TCCC) standards: The Tactical CricKit® (TCK), Control-CricTM(CC), and Bougie-assisted Technique (BAT). However, no previous studies have compared these devices in application time, application success, user ratings, and user preference. METHODS: United States Navy Corpsmen (N = 25) were provided 15 minutes of standardized instruction, followed by hands-on practice with each device on airway mannequins. Participants then performed SC with each of the 3 devices in a randomly assigned sequence. In this within-subjects design, application time, application success, participant ratings, and participant preference data were analyzed using repeated-measures ANOVA, regression, and non-parametric statistics at p < 0.05. RESULTS: Application time for CC (M = 184 sec, 95% CI 144-225 sec) was significantly slower than for BAT (M = 135 sec, 95% CI 113-158 sec, p < 0.03) and TCK (M = 117 sec, 95% CI 93-142 sec, p < 0.005). Success was significantly greater for BAT (76%) than for TCK (40%, p < 0.02) and trended greater than CC (48%, p = 0.07). CC was rated significantly lower than TCK and BAT in ease of application, effectiveness, and reliability (each p < 0.01). User preference was significantly (p < 0.01) higher for TCK (58%) and BAT (42%) than for CC (0%). Improved CC blade design was the most common user suggestion. CONCLUSION: While this study was limited by the use of mannequins in a laboratory environment, present results indicate that none of these devices was ideal for performing SC. Based on slow application times, low success rates, and user feedback, the Control-CricTM cannot be recommended until improvements are made to the blade design.

A Pilot Study of Four Intraosseous Blood Transfusion Strategies.

BACKGROUND: Intraosseous (IO) access is used by military first responders administering fluids, blood, and medications. Current IO transfusion strategies include gravity, pressure bags, rapid transfusion devices, and manual push-pull through a three-way stopcock. In a swine model of hemorrhagic shock, we compared flow rates among four different IO blood transfusion strategies. METHODS: Nine Yorkshire swine were placed under general anesthesia. We removed 20 to 25mL/kg of each animal's estimated blood volume using flow of gravity. IO access was obtained in the proximal humerus. We then autologously infused 10 to 15mL/kg of the animal's estimated blood volume through one of four randomly assigned treatment arms. RESULTS: The average weight of the swine was 77.3kg (interquartile range, 72.7kg-88.8kg). Infusion rates were as follows: gravity, 5mL/min; Belmont rapid infuser, 31mL/min; single-site pressure bag, 78mL/min; double-site pressure bag, 103mL/min; and push-pull technique, 109mL/min. No pulmonary arterial fat emboli were noted. CONCLUSION: The optimal IO transfusion strategy for injured Servicemembers appears to be single-site transfusion with a 10mL to 20mL flush of normal saline, followed immediately by transfusion under a pressure bag. Further study, powered to detect differences in flow rate and clinical complications. is required.

Proteomic analysis of media from lung cancer cells reveals role of 14-3-3 proteins in cachexia.

AIMS: At the time of diagnosis, 60% of lung cancer patients present with cachexia, a severe wasting syndrome that increases morbidity and mortality. Tumors secrete multiple factors that contribute to cachectic muscle wasting, and not all of these factors have been identified. We used Orbitrap electrospray ionization mass spectrometry to identify novel cachexia-inducing candidates in media conditioned with Lewis lung carcinoma cells (LCM). RESULTS: One-hundred and 58 proteins were confirmed in three biological replicates. Thirty-three were identified as secreted proteins, including 14-3-3 proteins, which are highly conserved adaptor proteins known to have over 200 binding partners. We confirmed the presence of extracellular 14-3-3 proteins in LCM via western blot and discovered that LCM contained less 14-3-3 content than media conditioned with C2C12 myotubes. Using a neutralizing antibody, we depleted extracellular 14-3-3 proteins in myotube culture medium, which resulted in diminished myosin content. We identified the proposed receptor for 14-3-3 proteins, CD13, in differentiated C2C12 myotubes and found that inhibiting CD13 via Bestatin also resulted in diminished myosin content. CONCLUSIONS: Our novel findings show that extracellular 14-3-3 proteins may act as previously unidentified myokines and may signal via CD13 to help maintain muscle mass.

Neutral sphingomyelinase-3 mediates TNF-stimulated oxidant activity in skeletal muscle.

AIMS: Sphingolipid and oxidant signaling affect glucose uptake, atrophy, and force production of skeletal muscle similarly and both are stimulated by tumor necrosis factor (TNF), suggesting a connection between systems. Sphingolipid signaling is initiated by neutral sphingomyelinase (nSMase), a family of agonist-activated effector enzymes. Northern blot analyses suggest that nSMase3 may be a striated muscle-specific nSMase. The present study tested the hypothesis that nSMase3 protein is expressed in skeletal muscle and functions to regulate TNF-stimulated oxidant production. RESULTS: We demonstrate constitutive nSMase activity in skeletal muscles of healthy mice and humans and in differentiated C2C12 myotubes. nSMase3 (Smpd4 gene) mRNA is highly expressed in muscle. An nSMase3 protein doublet (88 and 85 kD) is derived from alternative mRNA splicing of exon 11. The proteins partition differently. The full-length 88 kD isoform (nSMase3a) fractionates with membrane proteins that are resistant to detergent extraction; the 85 kD isoform lacking exon 11 (nSMase3b) is more readily extracted and fractionates with detergent soluble membrane proteins; neither variant is detected in the cytosol. By immunofluorescence microscopy, nSMase3 resides in both internal and sarcolemmal membranes. Finally, myotube nSMase activity and cytosolic oxidant activity are stimulated by TNF. Both if these responses are inhibited by nSMase3 knockdown. INNOVATION: These findings identify nSMase3 as an intermediate that links TNF receptor activation, sphingolipid signaling, and skeletal muscle oxidant production. CONCLUSION: Our data show that nSMase3 acts as a signaling nSMase in skeletal muscle that is essential for TNF-stimulated oxidant activity.

Mitochondria dysfunction in lung cancer-induced muscle wasting in C2C12 myotubes.

AIMS: Cancer cachexia is a syndrome which results in severe loss of muscle mass and marked fatigue. Conditioned media from cachexia-inducing cancer cells triggers metabolic dysfunction in skeletal muscle, including decreased mitochondrial respiration, which may contribute to fatigue. We hypothesized that Lewis lung carcinoma conditioned medium (LCM) would impair the mitochondrial electron transport chain (ETC) and increase production of reactive oxygen species, ultimately leading to decreased mitochondrial respiration. We incubated C2C12 myotubes with LCM for 30 min, 2, 4, 24 or 48 h. We measured protein content by western blot; oxidant production by 2',7'-dichlorofluorescin diacetate (DCF), 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF), and MitoSox; cytochrome c oxidase activity by oxidation of cytochrome c substrate; and oxygen consumption rate (OCR) of intact myotubes by Seahorse XF Analyzer. RESULTS: LCM treatment for 2 or 24 h decreased basal OCR and ATP-related OCR, but did not alter the content of mitochondrial complexes I, III, IV and V. LCM treatment caused a transient rise in reactive oxygen species (ROS). In particular, mitochondrial superoxide (MitoSOX) was elevated at 2 h. 4-Hydroxynonenal, a marker of oxidative stress, was elevated in both cytosolic and mitochondrial fractions of cell lysates after LCM treatment. CONCLUSION: These data show that lung cancer-conditioned media alters electron flow in the ETC and increases mitochondrial ROS production, both of which may ultimately impair aerobic metabolism and decrease muscle endurance.