4.13 Screening Waitlisted Transplant Patients for Swine MHC Class II Antibodies

Posted on January 18, 2018

J. M. Ladowski1, G. Martens1, L. Reyes1, Z. Wang1, M. Tector1, A. J. Tector1  1University Of Alabama at Birmingham,Birmingham, Alabama, USA

Introduction: Xenotransplantation, using genetically-modified swine, represents a solution to the organ shortage and is rapidly approaching clinical trials. Avoiding recipients with donor-specific major histocompatibility complex (MHC) antibodies greatly extends graft life. There exist many tools to screen allotransplant recipients for the presence, specificity, and consequence of antibodies but few reagents to screen potential xenotransplant recipients. Therefore, the prevalence, specificity, and immunogenic potential of anti-swine MHC class II antibodies remains unknown. 

Methods: Broadly positive swine MHC class II cells were generated by transfection with a human class II transactivator transgene and single antigen cells were made by expression of swine class II heavy chains. Serum from 237 waitlisted patients were screened using a flow cytometry crossmatch. Positive samples were further assessed for complement activation with a complement dependent cytotoxicity assay. 

Results: The screening flow cytometry crossmatch revealed 30/237 (12.6%) samples with potential anti-pig MHC class II antibodies. The 30 sera were tested on single allele swine cells and showed binding predominately to swine DQ alleles. Antibodies were able to activate complement and showed an increased killing of MHC-DQ relative to MHC-DR positive cells.      

Conclusion: Reagents to screen potential xenotransplant recipients for the presence, specificity, and consequence of antibodies were produced. The methods detected many patients with haplotype specific anti-swine MHC class II antibodies capable of activating complement, especially against swine-DQ alleles. Lessons from allotransplantation show importance of screening and avoiding patients with these donor-specific antibodies.  

 

4.10 Characterization of Porcine Xenoantibody Response In Vitro

Posted on January 18, 2018

K. Pham1, N. Wilson-Schlei1, R. Redfield1  1University Of Wisconsin,Department Of Surgery,Madison, WI, USA

Introduction:  One possible solution to the current shortage of donor organs is the use of xenografts. A potential model for viable xenotransplantation has been porcine organs but the associated immune barriers has delayed its transition to clinical practice. A key component to this barrier is the difference in carbohydrate structures expressed on porcine cells, which serve as antigens to humans. Even with the use of carbohydrate pathway knock-out pigs in combination with immunosuppression, there are still issues with long term graft survival and function in non-human primates. The goal of our experiment is to characterize the pre-existing immune response of porcine xenotransplantation in vitro. 

Methods:  Using six samples of naïve rhesus macaque serum, we performed a flow crossmatch assay with porcine peripheral blood mononuclear cells (PBMC). Porcine PBMC’s were incubated with three different groups: media alone (used as a negative control), porcine plasma (self), and rhesus macaque plasma (the experimental group). We analyzed IgG and IgM binding between lymphocytes and macrophages using flow cytometry. Results were reported as median fluorescence intensity (MFI) and unpaired two-tailed T test was used for statistical analysis.

Results: It was found that naturally occurring anti-pig IgG binding had significantly increased MFI in the six rhesus macaque plasma samples when incubated with porcine PBMC (P value < 0.01). Anti-pig IgG binding was also found to be most prevalent in cells outside the lymphocyte gate.  In terms of IgG binding, MFI did not change for lymphocytes when compared to negative control (self and no plasma). IgG binding to non-lymphoid cells resulted in an average Δ MFI of 100 when compared to negative controls. Interestingly, there appeared to be a heterogeneous response when comparing individual amounts of anti-pig IgG and IgM. Ranges for IgG were of 737 to 1046 MFI. As for IgM, MFI ranged from 291 to 430.

Conclusion: With this experiment, we confirm previous reports that naturally occurring antibodies do bind to xenoantigens (Burlak, 2014, Ezzelarab, 2006, Rood, 2006). There appears to be an increased presence of IgG binding, particularly among cells that may be macrophages. This is consistent with previous results that implicated cells which appeared to be macrophages and xenoantibody of the IgG isotype. However, we need to analyze the data to determine whether the cells in this gate, in which most binding occurred, bind CD68 to show they are indeed macrophages. Future considerations will involve a more in-depth investigation of the role of macrophages in this flow crossmatch assay and also if PBMC’s represent a valid model for xenograft failure. 

 

4.11 Assessing Pollution Samples for Autoimmune Promoting Activity In Vitro

Posted on January 18, 2018

H. Tran1, C. O’Driscoll1, E. Hoffmann1, J. Fechner1, J. Mezrich1  1University Of Wisconsin,Transplant/Surgery/SMPH,Madison, WI, USA

Introduction: Autoimmune diseases have been dramatically increasing in incidence worldwide with over 80 recognized diseases. Particulate matter (PM) from airborne pollution has been strongly associated with aggravating autoimmunity in humans, but it has been difficult to characterize mechanisms for aggravation of disease and identify which exposures are most pathological. Our group studies the role the aryl hydrocarbon receptor (AHR) on T-cell differentiation and effector function. Our previous studies suggest that inhalation of polycyclic aromatic hydrocarbons (PAHs) found in PMs may impact autoimmune disease through the AHR. We hypothesized that different pollution samples will induce varying, yet predictable effects on autoimmune disease depending on the balance of the fractions of chemicals in the sample. To characterize this, mice were exposed to inhaled pollution samples (urban dust particle (UDP) and two diesel emission particles (DEP1 and DEP2)) and control two weeks prior to induction of experimental autoimmune encephalomyelitis (EAE). Inhalation of both diesel samples significantly aggravated EAE, while exposure to UDP trended towards ameliorating disease. In this study we explored in vitro assays that could characterize the pathologic effects of different exposures.

Methods:  Splenocytes of 2D2 mice, transgenic for a T-cell receptor specific to MOG (the antigen that mice respond to in EAE), were stimulated with MOG peptide and LPS (to mature DCs). Dilutions of the three samples were added to the cells. The cells were cultured for 4-5 days and the supernatants of the cultures were harvested. ELISA was used to measure IFNg and IL-17 levels, as measures of immune activation. Cytokine bead arrays were also used to measure additional cytokines. We cultured bone-marrow derived dendritic cells (BMDC) in another assay, exposed them in culture for 24 hours with pollutants or control, and assayed them for cytokine production.

Results: In the MOG assay, all three PMs led to decreased levels of IL-6. Diesel exposure increased IFNg, but decreased IL-10 expression, a cytokine known to suppress immune responses. In contrast, UDP had no effect on IFNg expression, but increased IL-17 and IL-10 expression. In the BMDC assay, exposure to UDP increased levels of IL-10 production while the diesel samples did not. UDP also increased IDO expression in DCs, an enzyme involved in the production of regulatory T cells.

Conclusion: The current data suggests that the in vitro assays may have potential for screening the autoimmune promoting activity in samples of pollution. UDP, which is made up of multiple different sources of pollution, is known to include a complex mixture of chemicals, which may affect many different cell-types, whereas diesel samples, derived from single sources of pollution, may have more predictable and specific consequences on T-cell differentiation and disease. Further characterization of this will allow improved strategies for avoidance and remediation of polluted environments.

 

4.09 Regional Differences in Elastic Strain Correlate with Altered Mechanotransduction and Keloid Progression

Posted on January 18, 2018

T. Dohi1, J. Padmanabhan1, P. Than1, S. Akaishi3, M. Terashima2, N. Matsumoto3, R. Ogawa3, G. C. Gurtner1  1Stanford University,Department Of Surgery,Palo Alto, CA, USA 2Stanford University,Department Of Civil & Environmental Engineering,Palo Alto, CA, USA 3Nippon Medical School,Department Of Plastic, Reconstructive And Aesthetic Surgery,Bunkyo, TOKYO, Japan

Introduction: There is high morbidity, mortality, and cost related to fibroproliferative disorders. Of these, keloids are unique in that they progress beyond the original site of injury and form large, linear scars often of significantly different shape from the initial wound. The unknown pathophysiology, high rate of recurrence, and lack of effective treatment modalities makes keloid disease a challenging clinical problem. Progression beyond the original injury site and extension into normal peripheral skin highlights the zone between the keloid and surrounding skin as a target for investigation.

Methods:  We analyzed posture-related changes in local strain in various human anatomic locations correlated with keloid formation (n=10). Additionally, we developed a finite element method (FEM) analytic model to study the effect of von Mises stress and true strain in keloid-containing regions of skin. We also performed targeted-molecular analysis of human keloid-containing skin samples to identify the key biochemical pathways involved in keloid progression (n=5). 

Results: Anatomic regions prone to keloid formation are subject to high-levels of multidirectional posture-related strain. Focusing on these areas we created a model using finite element analysis and demonstrated that multidirectional loading leads to high true strain in the peripheral tissue surrounding the keloid. Furthermore, comparative immunohistochemical staining of human keloid tissue, peripheral tissue and control skin revealed that the peripheral tissue exhibits high levels of proliferation and altered mechanotransduction components including HSP27 and NFkB.

Conclusion: In summary, we describe a novel mechanism through which keloid disease may progress beyond the original wound site. Specifically, we show that changes in human posture result in elevated true strain in tissues immediately surrounding keloids in anatomic regions that are prone to keloid formation. We found that key mechanotransduction signaling pathways are altered in these regions, which may underlie the unique spread of keloid disease beyond the original wound site. These findings identify several molecular targets for therapy with potential for rapid clinical translation.

 

4.08 Notch Activator Jagged1 Results in Increased Closure Rates in an Ex Vivo Murine Skin Wound Model 1

Posted on January 18, 2018

Z. Aburjania1, T. W. King1  1University Of Alabama at Birmingham,Plastic Surgery,Birmingham, Alabama, USA

Introduction:
Decreased rates of wound healing affect millions of patients annually. We are interested in discovering novel strategies to enhance the wound healing process in diabetic patients.  We have previously shown that inhibiting Notch inhibits wound healing. Based upon our previous work, we propose that upregulation of Notch would increase rates of wound healing.  JAG1 is a known activator of Notch. Therefore, we hypothesized that applying topical JAG1 to ex vivo excisional wounds on the backs of mice would result in increased Notch activity, and thus an increased wound healing rate as compared to untreated wounds.

Methods:
Skin biopsies from 12-week old, healthy mice, (1-cm2 full-thickness) were cultured ex vivo. A 4-mm wound was created in the center of the skin biopsy. A topical application onto the open wound bed of JAG1 (10 nM) or vehicle (PBS) was applied daily for 14 days. Digital photographs were taken daily and the skin was processed for histological and protein analysis on days 3, 7, 10, and 14. The wounds were analyzed using ImageJ software. Wound area was calculated as a percent area of the original wound size. Statistical significance was defined as p<0.05 using the students’ t-test.

Results:
Partial to complete re-epithelialization was seen in the wounded tissues over the experimental period in both the control & JAG1 treated groups. The mouse skin treated with topical JAG1 had an increased rate of wound closure when compared to wounds treated with PBS.

Conclusion:
JAG1 increases the rate of re-epithelialization of cutaneous wounds in an ex vivo murine wound-healing model, indicating that Notch signaling plays a crucial role in wound healing in mice. Based upon our findings, further study of Notch in wound healing should be conducted which may then lead to better therapeutics for the wound healing process in patients. 

1

4.07 A Novel Human Xenograft Model to study Strategies in Articular Cartilage Regeneration.

Posted on January 18, 2018

M. P. Murphy1, M. Lopez1, R. C. Ransom1, O. Marecic1, R. E. Brewer1, L. S. Koepke1, S. Mascharak1, C. F. Chan1, M. T. Longaker1  1Stanford University,Surgery,Palo Alto, CA, USA

Introduction:  Currently there are no effective strategies for regenerating articular cartilage in diseases such as osteoarthritis (OA). Our group has utilized the mouse model to study the effects of surgical, chemical and cellular manipulation in articular cartilage regeneration. We aim to understand the effects of surgical, chemical and cellular manipulation in articular cartilage regeneration in a novel xenograft human model.

Methods: We transplanted the phalanges of 18-week old fetal specimens subcutaneously in the dorsum of immuno-compromised NSG P3 mice. After we confirmed viabiliy of the human xenograft using MRI, microCT and on gross inspection we performed microfracture (MF) surgery on the articular joints. Histological composition was assessed using Movat’s Pentachrome stain and Safranin O/Fast green stain. Immunohistochemistry (IHC) was performed assessing levels of Col 1, 2, 10 and MMP13. Proliferation was assessed with EdU labelling in histology and intracellular FACS. 

Results: We successfully developed a model of investigating the effects of surgical manipulation on human articular cartilage regeneration. (Figure 1) Following 6 weeks of implantation the xenograft had grown in size. The microCT images show calcification and MRI show viable articular cartilage. We have found that similar to our mouse model, fibrocartilage forms after MF. On histology and IHC we have determined the difference between normal articular cartilage and MF tissue. Cellular proliferation increases following injury.

Conclusion: We believe that our surgical technique and topical factors including BMP2 and Avastin will provide regenerative surgeons with a new approach to treating OA. We will further augment these factors with induced SSC from human Adipose-derived Stromal Cells. Our findings provide us with a "preclinical" human model that can be utilised to effectively investigate strategies for articular cartilage regeneration. 

 

4.05 Human Small Intestine Transplantation: Jejunum is more Susceptible to Ischemia than Ileum

Posted on January 18, 2018

T. Lysyy1, M. Finotti1,2, R. Morotti1, A. S. Munoz-Abraham1, A. Bertacco2, R. Agarwal1, C. Ibarra1, R. Patron1, A. Alkukhun1, F. D’Amico1,2, M. Rodriguez-Davalos1, D. Mulligan1, J. Geibel1  1Yale University School Of Medicine,Surgery,New Haven, CT, USA 2University Of Padua,Transplantation And Heptobiliary/Surgery,Padua, PADUA, Italy

Introduction: The human small intestine is the most ischemia-sensitive organ among all transplanted abdominal organs. Intestinal transplantation (IT) is an effective alternative for total parenteral nutrition in patients with short-bowel syndrome or intestinal failure. Postoperative complications of IT (including graft failure, acute rejection, and necrosis) are correlated to poor preservation and prolonged ischemia time. In this study, we evaluated the sensitivity of human small intestinal grafts to ischemic injury in two different hypothermic preservation conditions, and compared the extent of ischemic damage in both proximal (jejunum) and distal (ileum) segments.

Methods: Eighteen human small intestines were procured: 14 grafts were obtained on site with cold ischemia time (CIT) range of 1-4 hours (avg. 2.03 hrs), and 4 were shipped to our institution with CIT range of 5-9 hours (avg. 7.56 hrs). Histological samples were collected from jejunum and ileum intestinal segments at different time points (2 samples per time point from each segment, n=208 samples). The first set of samples was obtained at the time of procurement/organ delivery (T0) (baseline histological assessment). A second set was obtained at T0 with samples stored at 4oC for mean 8.6 hours (T8). A third set was obtained after mean 8.6 hours of continuous hypothermic perfusion using an intestinal perfusion unit (IPU) (PT8). All samples were fixed with 10% formalin prior to a blinded histological evaluation. Samples were scored using the Park/Chiu system. Scores were analyzed with Graph Pad Prism 6 for each group.

Results:In the group with CIT 1-4 hrs at T0, there was no histological score difference between the jejunum and ileum (mean  score in proximal samples 0.928 vs distal 0.961).  However, at T8 (mean score in proximal was 2.10 vs distal 1.39, (p<0.0005)) and PT8 (mean proximal 1.89 vs distal 1.25, (p<0.0005)), there is a statistically significant difference in the ischemic scores of the jejunum and ileum. In the second group with CIT 5-9 hrs the mean ischemic scores at T0 were 2.38 in jejunum and 1 in ileum (p<0.0232). At T8, the mean scores were 3 for jejunum and 3.75 for ileum, with no statistical difference (p>0.05). Similarly, at PT8 (mean ischemic scores: proximal 3.12 vs distal 3.87), there was no difference in the degree of ischemia between the two segments, (p>0.05).

Conclusion:The proximal segment (jejunum) of the human intestine is more susceptible to ischemia than the distal segment (ileum). Prolonged CIT (5-9 hrs) increases the degree of ischemic damage, affecting the jejunum more than ileum. The early use of the intestinal perfusion unit after procurement (<5 hours) appeared to reduce ischemic injury in the jejunum compared to static cold storage. Furthermore, selective transplantation of the more distal segment of the small intestine (ileum) could reduce ischemia-related postoperative complications and improve clinical outcomes and patient survival.

4.06 Cellular and Mechanical Mechanisms Underlying Regeneration in Mandibular Distraction Osteogenesis

Posted on January 18, 2018

R. C. Ransom1,2, A. C. Carter3, A. Salhotra1,2, T. Leavitt1, O. C. Marecic1,2, M. Lopez1,2, M. Murphy1,2, C. K. Chan1,2, D. C. Wan1, H. Y. Chang3, M. T. Longaker1,2  1Hagey Laboratory For Pediatric Regenerative Medicine,Department Of Surgery, Division Of Plastic And Reconstructive Surgery, Stanford University School Of Medicine,Stanford, CA, USA 2Institute For Stem Cell Biology And Regenerative Medicine,Stanford University,Stanford, CA, USA 3Center For Personal Dynamic Regulomes,Stanford University,Stanford, CA, USA

Introduction:  Accumulated evidence indicates that mechanical cues, which include physical forces, alterations in extracellular matrix mechanics and changes in cell shape, are transmitted to the nucleus directly or indirectly to orchestrate transcriptional activities that are crucial for tissue regeneration. Although mechanotransduction is thought to occur via integration of multiple signaling pathways, the precise mechanism leading to downstream cellular responses is not well understood. We have developed a mouse model of mandibular distraction osteogenesis (DO) which allows for tracing of cell fate and genetic dissection of mechanotransduction during bone formation.

Methods:  We examined cell-type-specific responses to mechanical force within distinct subpopulations of the mouse skeletal stem cell (mSSC) hierarchy. After determining that bone, cartilage, and stromal tissue are clonally derived in mice from lineage-restricted stem and progenitor cells in vivo, we employed this strategy to purify specific skeletogenic populations during mandibular distraction osteogenesis by prospective isolation using FACS. We employed the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) to profile open chromatin landscapes in these cell populations to understand the epigenetic changes in response to distraction. To investigate the role of mechanotransduction via focal adhesion kinase (FAK) in distraction osteogenesis, we inhibited FAK using the small molecule PF57223, a specific and potent inhibitor of FAK signaling. Three-dimensional reconstruction of μCT images of gradually distracted specimens revealed disrupted bone formation under conditions of FAK inhibition. ATAC-seq was employed for determination of FAK-responsive regions of the epigenome within each subpopulation of the skeletal stem cell hierarchy.

Results: We show that mechanical force augments the numbers and function of multiple cell populations across the skeletal hierarchy, including mouse skeletal stem and progenitor cells and their differentiated subsets. Mechanistically, distraction induces robust cell-matrix interactions that are coupled to cell-specific transcriptional responses via epigenomic pathways and pharmacological inactivation of this pathway disrupts bone formation. 

Conclusion: Here we employ a rigorous mandibular DO model in mice that is genetically dissectable, allowing for detailed examination of the fundamental principles regulating de novo bone formation. The identification of the cellular source of regeneration, the timeline for progenitor cell response, and determination of how these cells transduce physical stimuli to enact a regenerative response may provide new and effective strategies for reconstruction of the craniofacial skeleton.

 

4.03 Scaffold-free 3D-Bioprinting (3DBP) of A Porcine Liver Model

Posted on January 18, 2018

C. A. Vega1, L. J. Smith2,3, K. A. Altman4, P. Li4, B. Ekser4  1Indiana University School Of Medicine,Indianapolis, IN, USA 2Indiana University – Purdue University Indianapolis,3D-Bioprinting Core,Indianapolis, IN, USA 3Indiana University School Of Medicine,Department Of Radiology And Imaging Sciences,Indianapolis, IN, USA 4Indiana University School Of Medicine,Division Of Transplant Surgery, Department Of Surgery,Indianapolis, IN, USA

Introduction: Xenotransplantation (XTx) could be the solution to the lack of transplantable organs. Recently, great progress has been made in XTx research and there are currently >26 different genetically-engineered (GE) pigs. The most complex GE pig contains six different gene knock-outs or -ins. However, no researcher knows what would be the best genetic combination for XTx. The production of GE pigs is very expensive and would be quite time consuming for each genetic combination. Capitalizing on a new 3D bioprinting technology, we propose to use GE cells to generate a scaffold-free 3D pig liver tissue constructs, which can be used to study human immunological and coagulative responses in a time and cost effective way.                                                   

Methods: A step-wise model for 3DBP was developed which included (i) determination of optimal size of spheroids, (ii) determination of optimal time of spheroid formation and stability, (iii) designing and printing of the most suitable 3D structure, and (iv) determination of viability of the 3D-bioprinted structure. The optimization of 3DBP was also based on (i) porcine hepatocyte isolation; (ii) cell aggregate (spheroid) diameter, roundness, smoothness, durability, stability, and viability; (iii) the ratio and combination of different cell types; (iv) and the assembly (printing) of tissue constructs using spheroids.  

 

Results: Using a combination of porcine fetal fibroblasts and liver derived cells (LDCs, CD31+) (10:1 ratio), 450-500µm diameter spheroids were generated after 2-3 days of plating and successfully used to print 3 preliminary constructs (Fig.1). These 2 cell-type 3D-bioprinted constructs were later tested for their viability/structure after 1, 2, and 3 weeks of printing (Fig.1). Next, a combination of fibroblasts, hepatocytes, and LDCs (2:1:0.1 ratio) formed spheroids 5 days after plating and were then used to print our first 3 cell-type construct. Most of the spheroids remained intact in this 3 cell-type constructs. After incubation for an additional 5 days, this 3-cell construct fused and formed a stable 3D structure. Histology (H&E staining) shows promising results.

Conclusion: We have successfully printed a scaffold-free 3D Bioprinted Porcine Liver Model containing hepatocytes, liver derived endothelial cells, and fibroblasts. Further cell ratio optimization is required to produce functional spheroids capable of forming a more stable construct in the future. Using CRISPR-Cas9 technology, desired GE pig cells will be printed for future constructs which will allow us to explore the immunogenic and coagulative properties of pig tissues in the human body. 

4.04 Discordant Impact on Coagulation Between HTK and UW

Posted on January 18, 2018

A. Kam1, H. B. Moore1, C. B. Walker1, J. L. Dexter-Meldrum1, M. Chapman1, T. Johnson1, M. A. Adams1, T. Pshak1, I. Kam1, E. Pomfret1, T. L. Nydam1  1University Of Colorado Denver,Surgery,Aurora, CO, USA

Introduction:  Less than optimal outcomes following donation after cardiac death (DCD) donation are in part attributed to the development of microthrombi within the microvasculature of the graft. Using thromboelastography (TEG) during DCD recovery we have found numerous donors retain full clotting strength despite heparinization. We therefore sought to determine the effects of two commonly used preservation solutions, University of Wisconsin (UW) solution and Histidine-Tryptophan-Ketoglutarte (HTK), on clot formation and fibrinolysis. We hypothesize HTK will have a greater effect on promoting hypocoagulability due to its lower viscosity.

Methods:  Coagulation was assessed by TEG using whole blood diluted to 25%, 50%, and 75% with each preservative solution including HTK, UW and normal saline (NS). NS was used as a control to adjust for dilutional effects. The following TEG parameters were recorded: clot initiation (R time), fibrin polymerization (angle), and clot strength [maximum amplitude (MA) and clot lysis 30 minutes after reaching MA (LY30)].  For each solution, the dilution concentration was correlated to TEG indices with Spearman’s Rho test.  Solutions were contrasted between each other at set concentrations using a Friedman test.  Data was described as the median.

Results: Eight healthy volunteers donated blood. The serial dilution of whole blood with NS correlated with a prolonged R time (0.527 p=0.008), shallow angle (-0.693 p<0.001), lower MA (-0.811 p<0.001), and lower LY30 (-0.482 p=0.017).  This was similar to HTK (R time 0.564 p=0.004; angle -0.752 p<0.001; MA -0.818 P<0.001 and LY30 -0.643 p=0.001).  UW dilution had similar correlations with the exception that LY30 had a positive correlation with dilution (0.410 p=0.047). When contrasting TEG indices at different dilutions, no significant differences were seen until the dilution reached 75%.  Whole blood diluted with 75% HTK had significantly reduced fibrin polymerization compared to NS and UW (17° vs 32° vs 28° p=0.005 Figure). Whereas UW had significantly increased LY30 compared to NS and HTK (1.9% vs 0% vs 0% p<0.001).

Conclusion: Different preservation solutions have similar effects on coagulation as normal saline until 75% of whole blood has been replaced.  However, at 75% dilution, these solutions have different contributions to disturbances in TEG indices promoting hypocoagulability. HTK promotes a weaker clot from impaired fibrin polymerization, while UW promotes fibrinolysis.  These data support tailoring preservation solution to the pre-existing hypercoagulabilty seen in donors in order to optimize organ recovery.
 

4.01 Conserved FcRN-albumin interaction: implications for porcine-to-human renal xenotransplantation.

Posted on January 18, 2018

J. R. Butler1,2, M. R. Greg2, J. Ladowski2, Z. Wang2, J. L. Estrada2, M. Tector2, A. Tector2  1Indiana University School Of Medicine,Indianapolis, IN, USA 2University Of Alabama at Birmingham,Birmingham, Alabama, USA

Introduction: Historically, animal models of renal xenotransplantation have been challenged by acute humoral rejection. When studied in pig-to-primate models, there have been inconsistent reports of postoperative proteinuria; because proteinuria is a hallmark of complement-mediated graft damage, it remains unclear whether renal handling of serum macromolecules represents a physiologic barrier to clinical application in the absence of immune-mediated injury. Recently, serum albumin has been identified as a renal filtrate; serum proteostasis is dependent on post-glomerulus albumin absorption. In particular, the neonatal Fc receptor (FcRn) has been identified as a primary driver of renal proximal tubule albumin absorption in humans. The object of this study was to query the potential for porcine FcRn to promote absorption of human albumin in the absence of immunologic injury.

Methods: Intravital 2-photon microcopy has been instrumental in the study of renal albumin handling. Though this process has identified albumin as a renal filtrate in rat, mouse, and human, this is the first reported use of dual photon microcopy to assess the porcine kidney. After mapping the path of albumin across the filtration barrier in the porcine kidney, we established a fluorescence-based in vitro model of cellular uptake to query the ability of porcine proximal tubule cells to effectively absorb human albumin. With an established model to asses cross-species albumin-epithelial interactions at the cellular level, we sought to better understand the relationship at the molecular level. To this end we used a Biacore sensorgram to establish kenetics of the FcRn-albumin interaction. Finally, at the organismal level, we utilized a rejection free pig-to-primate model of life-sustaining renal transplant to analyze the potential loss of serum protein macromolues.

Results: Intravital 2-photon microcopy successfully followed labeled human albumin through the porcine kidney. Similar to results from other animal models, albumin is filtered at the porcine glomerulus. An in vitro cellular model of albumin uptake supports the ability of porcine renal proximal tubal cells to absorb human albumin. At the molecular level, porcine FcRn binds human albumin with the same affinity it does autologous porcine albumin; KD values of 7.22e-7 vs 3.92e-7 respectively (p= 0.21). Finally, when studied in vivo in a porcine to primate model free of immune-mediated graft damage the porcine kidney does not produce significant proteinuria.

Conclusion: Despite historical concern that renal loss of albumin would cause clinically significant proteinuria in porcine-to-primate xenotransplantation, this is not observed in the absence of immune-mediated graft injury. At the organ, cellular, and molecular level the porcine kidney process human albumin in a physiologic manner. Proteinuria does not represent an independent barrier to the clinical application of porcine-to-human xenotransplantation.

4.02 Automated Quantification of Soft Endpoints in Wound Healing Analysis

Posted on January 18, 2018

S. Mascharak1, A. L. Moore1,2, B. Duoto1, D. S. Foster1, R. E. Jones1,3, G. Wernig4, M. T. Longaker1  1Stanford University,Dept. Surgery,Palo Alto, CA, USA 2Brigham And Women’s Hospital,Dept. Surgery,Boston, MA, USA 3University Of Texas Southwestern Medical Center,Dept. Surgery,Dallas, TX, USA 4Stanford University,Dept. Pathology,Palo Alto, CA, USA

Introduction:  Studies on dermal wound healing and scarring commonly rely on qualitative assessments of morphological and histological characteristics. These can be time consuming and prone to individual observer bias. Furthermore, it is not always feasible to visually ascertain metrics of scar organization on the micron scale. We have developed a package of image processing methods to automate quantification of morphological and histological dermal scar features. Using this tool, we elucidate the effects of an anti-scarring treatment on collagen content, fiber organization, and hair follicle neogenesis after dermal wounding. 

Methods:  Two stented dermal wounds were made on the dorsa of C57BL/6 mice and injected with various concentrations (PBS, 0.1, 2, 20 mg/mL) of a potential anti-scarring drug (Doxycycline). Histology (Masson’s Trichrome, Picrosirius Red) and hair follicle neogenesis were assessed at 14 and 28 days, respectively, and analyzed with Matlab 2017a. Histological features were measured using a combination of color deconvolution, adaptive filtering, and skeletonization of individual collagen fibers. Hair follicles were segmented using top-hat and shape filters.

Results: Automated quantification of color-deconvoluted histology images showed a significant decrease in collagen deposition for wounds treated with anti-scarring drug (*p = 0.036, 2 mg/mL vs PBS, Fig. 1A-C). Color deconvolution also segmented mature (red) and immature (green) collagen fibers in birefringent Picrosirius Red-stained samples, revealing a concomitant increase in immature collagen deposition (*p = 0.018, 2 mg/mL vs PBS, Fig. 1D Picro Green). Next, images of collagen fibers were skeletonized to trace individual tracks, revealing significant decreases in mature fiber length (*p = 0.042, 2 mg/mL vs PBS), number (*p = 0.036), and branching (*p = 0.038) after treatment (Fig. 1E-F). Strikingly, alignment of mature collagen fibers decreased after treatment (*p = 0.048, 2 mg/mL vs PBS), indicating basket-weave organization reminiscent of unwounded skin. Shape filtration segmented hair follicles, dramatically speeding up the counting process (Fig. 1G). A significant decrease in follicles was observed after treatment (*p = 0.025, 2 mg/mL vs PBS), though no change in morphology was observed (Fig. 1H). 

Conclusion: Automated image processing facilitates histological and morphological study of scars. Additionally, analysis of collagen fibers reveals differences in scar organization after treatment with Doxycycline, that would not be apparent to the naked eye. Based on these findings, we will expand the use of image processing tools to models of hypertrophic scarring and fetal wound healing, towards the goal of new quantitative benchmarks for scar analysis. 

3.20 Large-field of View Laparoscopic Visualization Utilizing Multiple Miniaturized Cameras

Posted on January 18, 2018

J. Kim1, A. Watras1, H. Liu1, Z. Zeng1, J. A. Greenberg2, C. P. Heise2, Y. Hu1, H. Jiang1  1University Of Wisconsin-Madison,Electrical And Computer Engineering,Madison, WI, USA 2University Of Wisconsin School Of Medicine And Public Health,Department Of Surgery,Madison, WI, USA

Introduction: Laparoscopic surgery has numerous clinical benefits for patients including decreased postoperative pain, decreased wound morbidity, earlier recovery, and improved cosmesis. The quality and extent of intra-abdominal visualization is crucial during laparoscopic procedures. However, the current system that uses a single camera has many drawbacks in terms of the quality and extent of visualization as well as operative efficiency. We created a multi-camera visualization system with a larger field of view (FoV) compared to standard laparoscopic cameras for improved operative efficiency.

Methods: Our multi-camera visualization system utilizes four cameras with a specially designed trocar and a real-time video stitching program. We performed a bean drop task in a commercially available simulator box to test our large-FoV visualization system. Our video stitching program used the video data from four individual cameras and combined these four videos in real-time to provide large FoV. A projective transformation was calculated using matched feature points for mapping each image from different cameras into a single coordinate system. One of the four cameras was considered to provide the main view and then the required transformations were computed to map the images from four different cameras to the coordinate system of the main view.

Results: The four cameras are deployed and retrieved by the mechanical system of the trocar. The working port is not occupied by the cameras during operation and allows insertion of surgical instruments. Therefore, our system can reduce the number of ports or free up a surgical port, a potential advantage of our system. As a demonstration, we successfully performed a bean drop task in the trainer box by using our large-FoV visualization system. The figure below shows that a bean was picked up by a grasper and dropped into the hole of an inverted cup. The red dotted circles are traces of a moving bean picked by a grasper. The real-time stitched video can track the complete motion of the bean without any physical camera maneuver (Figure (a, b)), which is another clear advantage over current laparoscopic cameras that need to be operated separately by an assistant. In contrast, an image from a single camera cannot track the whole trajectory of the bean and cover the beans and the cup simultaneously (Figure (c, d)).

Conclusion: Our multi-camera visualization system provides a large FoV, frees up a surgical port, and eliminates the needs of physical maneuvering of the laparoscopic camera (thus the camera operating assistant). This system can provide larger extent of intra-abdominal visualization and may lead to higher efficiency of operation.

 

3.19 Effects of Exogenous Del1 Protein on Human Skeletal Stem Cell Proliferation

Posted on January 18, 2018

T. Boyko1,2, O. Marecic1, M. Lopez1, C. Chan1, M. T. Longaker1, G. P. Yang1  1Stanford University School Of Medicine,Surgery,Stanford, CALIFORNIA, USA 2University At Buffalo,Surgery,Buffalo, NY, USA

Introduction:  Millions of people fracture their bones yearly, 5% of which fail to heal properly. Risk factors for this include old age, osteoporosis and diabetes. Failure of bony fusion also complicates thousands of orthopedic, spinal and maxillofacial surgeries including joint replacements, spinal fusion and dental implants resulting in significant morbidity and increased costs. Skeletal progenitor cells have recently been described. They give rise to three main tissues of the skeletal system: bone, cartilage and stroma, and are responsible for bone healing. We have previously shown that the extracellular matrix protein Del1 acts to promote proliferation and prevents apoptosis of the subpopulation of mouse skeletal progenitor cells involved in fracture healing – theBone Cartilage Stroma Progenitor cells (BCSPs). Del1 knockout mice heal fractures with less bone, and the healed bone is weaker than that of wild type control fractures. We hypothesized that addition of exogenous Del1 protein could promote proliferation of human skeletal stem cell (HSSC).

Methods:  Femur heads of three patients undergoing hip replacement surgery, age 70, 71 and 75, were obtained. Following mechanical and enzymatic digestion, HSSCs were isolated via FACS, using previously identified cell surface markers. HSCCs were plated in vitro with or without the addition of human Del1 protein. Proliferation was measured daily over 5 days.

Results: The addition of exogenous human Del1 protein to cultured HSSCs led to increased proliferation rates compared to untreated HSSCs. Cell numbers reached statistical significance on the third day of treatment (p<0.05) and remained statistically significant over the remaining days cells were counted in all three human samples.

Conclusion: Del1 knockout mice do not have a significant phenotype without injury. Following fracture, knockout mice are able to heal, but with significantly less bone. We have shown that the underlying mechanism consists of Del1 acting as a pro-proliferative and anti-apoptotic factor for mouse skeletal progenitor cells. In this study, we demonstrate that exogenous human Del1 can promote proliferation of human skeletal progenitor cells. This suggests that Del1 has the potential for therapeutic use to reduce morbidity from failure of bone fracture healing or inadequate bone fixation of an implant.

 

3.18 Diversity of Enterococcus faecalis in the Early Microbiota of Rats

Posted on January 18, 2018

P. T. Delaplain1, J. Wang1, B. Bell1, A. Grishin1, H. Ford1  1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA

Introduction:
Although necrotizing enterocolitis (NEC) is believed to be associated with bacterial colonization of the neonatal gut, the exact nature of the relationship between NEC and specific groups of bacteria or characteristics of bacterial populations remains largely elusive. Analysis of bacterial populations using high throughput sequencing of 16S RNA brought only limited success, as the same bacterial species were found in sick and healthy individuals. Our previous studies implicated a clinically relevant strain of Cronobacter muytjensii, but not several other strains of this species, as a causative agent of NEC. This lead us to hypothesize that NEC is caused by a plethora of opportunistic pathogens, which may belong to a variety of bacterial species, whereas colonization with non-pathogenic bacteria may be innocuous or even protective. In the framework of this hypothesis, we are characterizing multiple isolates of one of the most common first colonizers, Enterococcus faecalis, and their role in the pathogenesis of experimental NEC.

Methods:
Neonates obtained from timed pregnant rats sourced from Charles River or Harlan were either breast fed or formula fed. E. faecalis bacteria were isolated from the intestines of 4-day-old rats based on their thermotolerance, ability to grow in the presence of azide, characteristic appearance upon Gram staining, and 16S RNA sequencing. The isolates were further characterized for colony morphology, hemolysis, gelatin liquefaction, sugar utilization, antibiotic resistance, and spectra of DNA HindIII restriction fragments. Isolates that possessed different combinations of the corresponding phenotypic traits were considered different strains. The strains identified were examined for their ability to activate the pro-inflammatory transcription factor NF-κ B in IEC-6 enterocytes using Western blotting with anti-Iκ Bα  antibodies. 

Results:
21 different strains were identified among the 146 independent isolates of E. faecalis. DNA restriction enzyme digestion revealed two unique genomic patterns. Some, but not all strains activated NF-κ B in IEC-6 cells. In addition, small colony size on standard media seemed to correlate with a preference for anaerobic conditions. 

Conclusion:
There is a considerable diversity of E. faecalis even in animals coming from the same specific pathogen-free environment. Some bacterial phenotypes, specifically hemolysis, proteolysis, antibiotic resistance, and ability to trigger inflammatory response in enterocytes, may be causatively associated with pathogenicity in NEC. Further characterization of these bacteria may identify both effective first colonizers as well as potentially pathogenic or protective strains.
 

3.16 Acta2, Tnc, and Col24a1 Expression are Associated with Fibroblast Formation of Abdominal Adhesions

Posted on January 18, 2018

D. Foster1,2, C. D. Marshall1,2, R. C. Ransom2, A. Manjunath3, G. Gulati3, M. S. Hu2, C. C. Chan3, W. T. Leavitt2, A. L. Moore2, L. A. Barnes2, M. Murphy2, M. T. Longaker1,2,3  1Stanford University,Department Of Surgery,Stanford, CA, USA 2Stanford University,Hagey Laboratory For Pediatric Regenerative Medicine,Stanford, CA, USA 3Stanford University,Institute For Stem Cell Biology And Regenerative Medicine,Stanford, CA, USA

Introduction:  After surgical interventions or secondary to fibrotic disease, intra-abdominal adhesions can form. The presence of adhesions makes further surgery challenging and can have other negative consequences such as infertility, bowel obstructions and chronic pain. Adhesions are thought to form by fibroblast collagen deposition, similar to cutaneous scar formation. The mechanisms underlying adhesion formation, including origin of the fibroblasts involved or signaling process governing this phenomenon, however, remain poorly characterized. Currently, there is no effective therapy to prevent or treat adhesive disease.  

Methods:  Intra-abdominal adhesions were established between the bowel and the peritoneal lining of the abdominal sidewall in PDGFRα-GFP mice, in which PDGFRα (platelet derived growth factor receptor alpha), a pan-fibroblast marker, is tagged with green fluorescent protein (GFP) [Fig. 1A]. Sham-surgery PDGFRα-GFP mice were used for comparison. Once formed, the adhesion, abdominal wall and bowel wall tissues were extracted and digested with collagenase. FACS sorting and quantitative PCR confirmed fibroblast identity via expression of known fibroblast genes. Bulk RNA sequencing was conducted on sorted fibroblasts and gene expression was compared between the adhesion and sham-surgery cohorts. For the next aim of this project, human abdominal adhesion tissue is being harvested from ileostomy takedown patients. All experiments were approved by Stanford University’s IRB or APLAC, as applicable. 

Results: FACS sorting of mouse adhesion tissue showed consistent expression of fibroblast genes including PDGFRα, Vim (vimentin), and Col1a2 (encodes for collagen 1). This was confirmed with quantitative PCR. RNA sequencing showed significantly higher expression of relevant fibrosis-associated genes in both male and female mice from the adhesion cohort including Acta2, Tnc, and Col24a1, compared to sham [Fig. 1B]. Acta2 encodes for smooth muscle α-2-actin (αSMA) and is expressed by myofibroblasts and smooth muscle cells. Tnc encodes for tenascin C, an extracellular matrix protein, and Col24a1 is a member of the collagen gene family involved in type 1 collagen regulation. Human adhesion tissue will be sorted and sequenced, and gene expression will be analyzed and compared with mouse results. The results of our human adhesion tissue experiments are forthcoming. 

Conclusion: Identification of this gene expression pattern in adhesions presents the opportunity for possible therapeutic targets. Comparison of our results from mouse experiments with cellular activity and gene expression patterns in human tissue will bring us closer to developing a potential therapy to combat adhesive disease. 

3.17 Anti-fibrosis Effect of Novel Oridonin Analog CYD0618 via Suppression of NF-κB Pathway

Posted on January 18, 2018

C. B. Cummins1, X. Wang1, J. Xu2, Y. Ding2, H. Chen2, J. Zhou2, R. Radhakrishnan1  1University Of Texas Medical Branch,Department Of Surgery,Galveston, TX, USA 2University Of Texas Medical Branch,Department Of Pharmacology And Toxicology,Galveston, TX, USA

Introduction:
Liver fibrosis is characterized as excessive deposition of the extracellular matrix (ECM) proteins, especially collagen type I. Activated hepatic stellate cells (HSCs) are the primary cell type responsible for ECM deposition, and NF-κB signal has been reported as one of major mediators of HSC activation. Previously, our team reported oridonin, a bioactive diterpenoid isolated from Rabdosia rubescens, exhibited anti-hepatic fibrogenetic activity In vitro. In the present study, we examined the effects of its novel derivative CYD0618 on HSC viability, apoptosis and NF-κB signaling.  

Methods:
The proliferation effects of CYD0618 treatment on activated human and rat HSC cell lines LX-2 and HSC-T6 were measured by Alamar Blue Assay. Apoptosis was measured by Cell Death ELISA. Cellular proteins were determined by Western blots and immunofluorescence.

Results:
CYD0618 significantly inhibited LX-2 cells proliferation in a dose-dependent manner with an IC50 value of ~0.45 μM after 48 hours treatment, this was ~15 fold more potent than the parent compound oridonin. Similar effects were seen in HSC-T6 cells with an IC50 of ~0.75 μM. Cell apoptosis was induced by CYD0618 in both cell lines. CYD0618 treatment increased cell cycle inhibitory protein p21, p27, and induced apoptosis marker cleaved poly (ADP-ribose) polymerase (c-PARP), while significantly suppressing the expression of collagen type I. Notably, CYD0618 blocked lipopolysaccharides(LPS)-induced NF-κB p65 nuclear translocation and DNA binding activity, in addition to preventing LPS-induced NF-κB inhibitory protein IκBα phosphorylation and degradation. LPS-stimulated NF-κB downstream target cytokines IL-6, MCP-1 were also attenuated by CYD0618. It has been reported that phosphorylation of the NF-κB p65 on the serine 536 residue affects its nuclear translocation and transcription of target genes. Our data showed that endogenous NF-κB p65 S536 phosphorylation was inhibited by CYD0618 treatment in a time-dependent fashion. Importantly, NF-κB specific chemical inhibitor Bay-11-0781 was found to inhibit the proliferation, and promote apoptosis in both LX-2 and HSC-T6 cells.

Conclusion:
The potent anti-hepatic fibrogenetic effect of CYD0618 may be mediated via suppression of the NF-κB pathway.
 

3.15 Investigating the Integrin α3β1–dependent Secretome in Keratinocytes: Implications for Wound Healing

Posted on January 18, 2018

L. A. DeFreest1, L. Van De Water2, C. DiPersio2  1Albany Medical College,Department Of Surgery, Division Of General Surgery, Section Of Trauma And Critical Care,Albany, NY, USA 2Albany Medical College,Department Of Surgery And Department Of Regenerative & Cancer Cell Biology,Albany, NY, USA

Introduction: Integrin receptors expressed in epidermal keratinocytes have been shown to have important roles in cutaneous wound healing including paracrine stimulation of angiogenesis, regulation of keratinocyte proliferation and migration, and production and remodeling of the extracellular matrix (ECM) to which they adhere. Our laboratory has investigated the roles of the laminin-binding integrin α3β1 in keratinocytes using both in vivo and in vitro models of wound healing. Our previous data supports a role for α3β1 in modulation of these processes by regulating the secretion of proteins into the extracellular milieu.  Our long term goal is to elucidate the α3β1-dependent paracrine and autocrine signaling mechanisms that modulate the wound microenvironment to promote efficient wound healing.

Methods: Using a cell culture model of activated mouse keratinocytes (MK cells), we have prepared medium conditioned by MK cells that are homozygous for a null mutation in the gene that encodes the α3 integrin subunit (i.e. lacking α3β1 integrin), or MK cells stably transfected with human α3 (i.e. expressing α3β1 integrin). These conditioned media were analyzed by mass spectrometry to identify proteins secreted by MK cells in an α3β1–dependent manner, with a focus on those proteins that were upregulated at least four-fold in the presence of α3β1. Selected proteins in both media and lysates from MK cells were then evaluated by western blot to confirm and quantitate the secreted proteins.

Results: Of the >7000 proteins identified by mass spectrometry, nineteen were upregulated more than four-fold in medium from α3β1–expressing mouse keratinocytes. These proteins corresponded to a wide variety of keratinocyte functions including ECM, pro-angiogenic, and immunological species.  Interestingly, lactotransferrin (LTfn) secretion was up-regulated 18-fold in α3β1-expressing cells.  Western blot confirmed a significant upregulation of LTfn secretion as well as production of the protein in cell lysates.

Conclusion: Proteomic analysis of conditioned media from MK cell lines identified numerous secreted proteins that are upregulated by α3β1 indicating an important role for this integrin in controlling the keratinocyte secretome. Lactotransferrin has previously been shown to have a role in wound healing and has anti-microbial and immunomodulatory properties making it an interesting candidate for further study. Interestingly, LTfn is known to be expressed in response to estrogen and under the control of the estrogen receptor, suggesting an intersection of signaling pathways downstream of integrins and the estrogen receptor which has not been previously studied in detail. Elucidation of the signaling mechanisms that result in α3β1–dependent production of LTfn may lead to novel therapeutics to promote closure of surgical wounds.
 

3.12 FXR May Be Necessary for Experimental Murine LPS Induced Peritonitis Intestinal Damage

Posted on January 18, 2018

M. U. Mallicote1, O. Escobar1, C. Gayer1  1Children’s Hospital Los Angeles,Los Angeles, CA, USA

Introduction: Bile acids are known to stimulate the Farnesoid X receptor (FXR) pathway in the intestines; however, there are discrepancies in the literature about the effects of FXR activation on the intestinal barrier. FXR activation has been reported to decrease gut permeability in chronic injury models, although FXR activation is known to decrease intestinal cell proliferation. Given these inconsistencies, we hypothesize that FXR activation is detrimental to the intestinal barrier in an acute injury model.

 

Methods: In vivo, wild-type (WT) and global FXR knock-out (FXR-KO) mice were injected with lipopolysaccharide (LPS) or saline via intra-peritoneal route to induce intestinal injury and gavage-fed FITC-dextran. Animals were sacrificed at 16 hours. Barrier function was assessed by serum FITC levels and mRNA expression of IL-6, IL-10, TNF, and IL1b was assessed by RT-PCR.

 

Results: Barrier permeability in WT mice was significantly increased in LPS versus saline controls. This effect, however, was significantly attenuated in FXR-KO mice. LPS increased FXR mRNA expression in WT but not in FXR-KO. Of the cytokines tested, only TNF was elevated in both WT and FXR-KO mice injected with LPS versus saline controls. FXR-KO mice injected with LPS also showed highly elevated levels of IL-6 and IL1b whereas the WT mice did not. Interestingly, the FXR-KO mice also exhibited high levels of IL-10 an anti-inflammatory cytokine.

 

Conclusions: In WT mice, LPS-induced inflammation mainly leads to elevated levels of TNF. In FXR-KO mice there is elevation of IL-6, TNF, and IL1b, however intestinal barrier damage appears to be attenuated. Of note, in FXR-KO mice there was elevation of the anti-inflammatory cytokine IL-10 which may be the cause of the attenuated barrier damage we are seeing. Determining why the absence of FXR leads to IL-10 induction may lead to the development of novel therapies for regulating the intestinal epithelium during acute injury and may help prevent gut-origin sepsis.

3.14 C-Jun Induction Leads to Increased Scar Formation and Fibrosis in Mice

Posted on January 18, 2018

B. Duoto1,2, A. Moore1,3, D. Foster1, R. E. Jones1,4, S. Mascharak1, G. Wernig5, M. Longaker1  1Stanford Univserity,Surgery,Stanford, CALIFORNIA, USA 2San Jose State University,Biology,San Jose, CALIFORNIA, USA 3Brigham And Women’s HospitalBrigham And Women’s Hospital,Surgery,Boston, MASSACHUSSETTS, USA 4University Of Texas Southwestern Medical Center,Surgery,Dallas, TX, USA 5Stanford University,Pathology,Stanford, CALIFORNIA, USA

Introduction:

Fibrosis and scar formation are major clinical issues which result in disfigurement and permanent functional loss. In both adults and children, excessive fibrosis after surgery or injury can result in complications that are difficult to treat, often recur, and have few effective therapeutic options. Additionally, the only animal models that exist to simulate these processes in humans include the red Duroc pig and rabbit ear, which are expensive, difficult to use, and do not provide transgenic modeling. Recently, a paper describing a transgenic mouse strain that utilizes over-expression of c-Jun, an AP-1 transcription factor, to induce global tissue fibrosis was published. We hypothesize that local induction of c-Jun in the same transgenic mouse would result in increased scarring and fibrosis.

Methods:
            Stented excisional dorsal wounding was performed on c-JuntetO; R26-M2rtTA mice along with injections of phosphate buffered saline (PBS), or with c-Jun inducing agent doxycycline at 0.1mg/mL, and 2mg/mL concentrations. Induction and dressing changes were performed every other day until wounds were completely healed. Wounds were then harvested and stained with hematoxylin and eosin for scar thickness and trichrome for collagen deposition. These assays quantify stain-specific wound characteristics.

Results:
            Preliminary histological staining data uncovered a significantly increased scar thickness in the c-JuntetO R26-M2rtTA mice as compared to the C57BL/6J PBS control mice (*p<0.0001) and the C57BL/6J 2mg/mL doxycycline control mice (*p<0.0001). Comparatively, in regards to the amount of scar collagen deposition, no significance was found between the c-JuntetO R26-M2rtTA mice and the C57BL/6J PBS control mice (p=0.0832) as well as the C57BL/6J 2mg/mL doxycycline control mice (p=0.1692).

Conclusion:

Histological stains of c-JuntetO R26-M2rtTA dorsal wounds support that there are distinct differences in scar formation in the c-Jun transgenic model as compared to controls. These data support that this novel mouse model can be developed to study the molecular pathways which lead to, and inhibit, fibrosis. In future studies we will investigate novel inhibitors of fibrosis in this animal model, study the scar forming fibroblasts in greater detail, and compare our results to human specimens. 

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