Development of a lethal, closed-abdomen grade V hepato-portal injury model in non-coagulopathic swine.

BACKGROUND: Hemorrhage within an intact abdominal cavity remains a leading cause of preventable death on the battlefield. Despite this need, there is no existing closed-cavity animal model to assess new hemostatic agents for the preoperative control of intra-abdominal hemorrhage. METHODS: We developed a novel, lethal liver injury model in non-coagulopathic swine by strategic placement of two wire loops in the medial liver lobes including the hepatic and portal veins. Distraction resulted in grade V liver laceration with hepato-portal injury, massive bleeding, and severe hypotension. Crystalloid resuscitation was started once mean arterial pressure (MAP) fell below 65 mm Hg. Monitoring continued for up to 180 min. RESULTS: We demonstrated 90% lethality (9/10) in swine receiving injury and fluid resuscitation, with a mean survival time of 43 min. Previous efforts in our laboratory to develop a consistently lethal swine model of abdominal solid organs, including preemptive anticoagulation, a two-hit injury with controlled hemorrhage prior to liver trauma, and the injury described above without resuscitation, consistently failed to result in lethal injury. CONCLUSION: This model can be used to screen other interventions for pre hospital control of noncompressible.

Intracoronary delivery of mesenchymal stem cells at high flow rates after myocardial infarction improves distal coronary blood flow and decreases mortality in pigs.

OBJECTIVES: Evaluate the effects of pressure and duration of intracoronary (IC) infusion of mesenchymal stem cells (MSCs) on delivery efficiency and safety after myocardial infarction (MI). BACKGROUND: Standard IC delivery of MSCs can lead to intravascular plugging and reduced coronary blood flow. The optimal delivery pressure and duration is unknown. METHODS: Immediately after MI pigs were randomized to 1 of 3 delivery protocols of 5 x 10(7) iron-fluorescent microspheres labeled MSCs, control received 2 ml infusions at 1 ml/min (five times), very high flow rate (VHFR) a single 10 ml infusion at 60 ml/min and the high flow rate (HFR) group a single 10 ml infusion at 20 ml/min. TIMI grade flow was assessed throughout the procedure and at sacrifice (day 14). MSCs distribution was analyzed in isolated hearts by 4.7T MRI. Delivery efficiency was quantified via fluorescent microsphere recovery using a magnetic separation technique and by light microscopy. RESULTS: TIMI grade flow did not change following MI (all groups TIMI 3). However, following MSCs delivery only 18% (2/11) of control animals had TIMI 3 blood flow vs. 56% (5/9) in VHFR and 67% (4/6) in HFR (P = 0.03). As a consequence, 63% of control animals died within 24 hr, 33% in VHFR and none in HFR (P = 0.02). MSCs delivery in the infarct tissue did not differ between the groups (P = 0.06). CONCLUSIONS: A single MSCs infusion at 20 ml/min resulted in improved coronary blood flow and decreased mortality, without sacrificing delivery efficiency.

Slit-surface electrospinning: a novel process developed for high-throughput fabrication of core-sheath fibers.

In this work, we report on the development of slit-surface electrospinning--a process that co-localizes two solutions along a slit surface to spontaneously emit multiple core-sheath cone-jets at rates of up to 1 L/h. To the best of our knowledge, this is the first time that production of electrospun core-sheath fibers has been scaled to this magnitude. Fibers produced in this study were defect-free (i.e. non-beaded) and core-sheath geometry was visually confirmed under scanning electron microscopy. The versatility of our system was demonstrated by fabrication of (1) fibers encapsulating a drug, (2) bicomponent fibers, (3) hollow fibers, and (4) fibers from a polymer that is not normally electrospinnable. Additionally, we demonstrate control of the process by modulating parameters such as flow rate, solution viscosity, and fixture design. The technological achievements demonstrated in this work significantly advance core-sheath electrospinning towards commercial and manufacturing viability.

Contractile forces generated by articular chondrocytes in collagen-glycosaminoglycan matrices.

The objective of the study was to directly measure the force of contraction of adult articular chondrocytes and to examine their contractile behavior in collagen-glycosaminoglycan analogs of extracellular matrix by live cell imaging in vitro. The contractile forces generated by passages 2 and 3 adult canine articular chondrocytes were measured using a cell force monitor. The contractile behavior of the cells was also directly imaged as they were cultured in collagen-glycosaminoglycan scaffolds. Passage 2 cells seeded in a collagen-glycosaminoglycan scaffold were capable of generating a force of 0.3 nN/cell. Chondrocytes subcultured through a third passage generated a force twice that level, paralleling the increase in the alpha-smooth muscle actin (SMA) content of the cells as demonstrated by Western blot analysis. Treatment of passage 3 cells with staurosporine reduced the force of contraction by approximately one-half, reflecting the effects of this agent in reducing the SMA content of the cells and disrupting the microfilaments. These values compare with 1 nN previously reported for lapine dermal fibroblasts of passage 5-7, using the same apparatus. Direct live cell imaging documented the contractile behavior of the articular chondrocytes in the collagen-glycosaminoglycan matrix in the time frame in which the force was directly measured in the cell force monitor. This imaging demonstrated how the cells acted individually and in unison to buckle the collagen struts making up the matrix. Adult articular chondrocytes are capable of generating a SMA-enabled force of contraction sufficient to model extracellular matrix molecules.

Self-expanding foam for prehospital treatment of severe intra-abdominal hemorrhage: dose finding study.

BACKGROUND: Noncompressible abdominal bleeding is a significant cause of preventable death on the battlefield and in the civilian trauma environment, with no effective therapies available at point of injury. We previously described the development of a percutaneously administered, self-expanding, poly(urea)urethane foam that improved survival in a lethal Grade V hepatic and portal vein injury model in swine. In this study, we hypothesized that survival with foam treatment is dose dependent. METHODS: A high-grade hepatoportal injury was created in a closed abdominal cavity, resulting in massive noncompressible hemorrhage. After injury, the animals were divided into five groups. The control group (n = 12) was treated only with fluid resuscitation, and four polymer groups received different dose volumes (Group 1, n = 6, 64 mL; Group 2, n = 6, 85 mL; Group 3, n = 18, 100 mL; and Group 4, n = 10, 120 mL) in addition to fluids. Ten minutes after injury, the foam was percutaneously administered, and animals were monitored for 3 hours. RESULTS: Survival with hepatoportal injury was highest in Group 4 (90%) and decreased in a dose-dependent fashion (Group 3, 72%; Group 2, 33%; Group 1, 17%). All polymer groups survived significantly longer than the controls (8.3%). Hemorrhage rate was reduced in all groups but lowest in Group 4 versus the control group (0.34 [0.052] vs. 3.0 [1.3] mL/kg/min, p < 0.001). Increasing foam dose volume was associated with increased peak intra-abdominal pressure (88.2 [38.9] in Group 4 vs. 9.5 [3.2] in the controls, p < 0.0001) and increased incidence of focal bowel injuries. CONCLUSION: The self-expanding foam significantly improves survival in a dose-dependent fashion in an otherwise lethal injury. Higher doses are associated with better survival but resulted in the need for bowel resection.

Self-expanding polyurethane polymer improves survival in a model of noncompressible massive abdominal hemorrhage.

BACKGROUND: Intracavitary noncompressible hemorrhage remains a significant cause of preventable death on the battlefield. Two dynamically mixed and percutaneously injected liquids were engineered to create an in situ self-expanding polymer foam to facilitate hemostasis in massive bleeding. We hypothesized that intraperitoneal injection of the polymer could achieve conformal contact with sites of injury and improve survival in swine with lethal hepatoportal injury. METHODS: High grade hepatoportal injury was created in a closed abdominal cavity, resulting in massive noncoagulopathic, noncompressible hemorrhage. Animals received either standard battlefield fluid resuscitation (control, n = 12) or fluid resuscitation plus intraperitoneal injection of hemostatic foam (polymer, n = 15) and were monitored for 3 hours. Blood loss was quantified, and all hepatoportal injuries were inspected for consistency. RESULTS: Before intervention, all animals initially experienced severe, profound hypotension and near-arrest (mean arterial pressure at 10 minutes, 21 [5.3] mm Hg). Overall survival at 3 hours was 73% in the polymer group and 8% in the control group (p = 0.001). Median survival time was more than 150 minutes in the polymer group versus 23 minutes (19-41.5 minutes) in the control group (p < 0.001), and normalized blood loss in the polymer group was 0.47 (0.30) g/kg per minute versus 3.0 (1.3) g/kg per minute in the controls (p = < 0.001). All hepatoportal injuries were anatomically similar, and the polymer had conformal contact with injured tissues. CONCLUSION: Intraperitoneal polymer injection during massive noncompressible hemorrhage reduces blood loss and improves survival in a lethal, closed-cavity, hepatoportal injury model. Chronic safety and additional efficacy studies in other models are needed.

A new technique for calculating individual dermal fibroblast contractile forces generated within collagen-GAG scaffolds.

Cell-mediated contraction plays a critical role in many physiological and pathological processes, notably organized contraction during wound healing. Implantation of an appropriately formulated (i.e., mean pore size, chemical composition, degradation rate) three-dimensional scaffold into an in vivo wound site effectively blocks the majority of organized wound contraction and results in induced regeneration rather than scar formation. Improved understanding of cell contraction within three-dimensional constructs therefore represents an important area of study in tissue engineering. Studies of cell contraction within three-dimensional constructs typically calculate an average contractile force from the gross deformation of a macroscopic substrate by a large cell population. In this study, cellular solids theory has been applied to conventional column buckling relationships to quantify the magnitude of individual cell contraction events within a three-dimensional, collagen-glycosaminoglycan scaffold. This new technique can be used for studying cell mechanics with a wide variety of porous scaffolds that resemble low-density, open-cell foams. It extends previous methods for analyzing cell buckling of two-dimensional substrates to three-dimensional constructs. From data available in the literature, the mean contractile force (Fc) generated by individual dermal fibroblasts within the collagen-glycosaminoglycan scaffold was calculated to range between 11 and 41 nN (Fc=26+/-13 nN, mean+/-SD), with an upper bound of cell contractility estimated at 450 nN.

Tensional forces influence gene expression and sutural state of rat calvariae in vitro.

BACKGROUND: Theories regarding the cause of craniosynostosis that are more than 15 years old cite the role that tensional forces play in the normal and abnormal development of the cranial suture. These theories highlight the effect of stress bands originating from the skull base to the vertex, guiding sutural development. METHODS: In this study, the normally fusing posterior intrafrontal suture of the rat was subjected to 3 mN of tensional force for 30 minutes per day. The suture was then assessed for patency, proliferation, apoptosis, and transforming growth factor (TGF)-beta signaling components. RESULTS: Sutures that were subjected to tensional force were histologically patent at the end of 14 days. This was in contrast to sutures that were maintained without force. Proliferative and apoptotic activity was increased also in sutures maintained open artificially. Interestingly, levels of active TGF-beta-signaling components were also increased in force-maintained sutures. CONCLUSIONS: Sutural maintenance by mechanical force is concurrent with modulation of cellular activity and protein expression reminiscent of the open suture. This study demonstrates the dynamic reciprocity existing between biochemical activity and morphologic state. Although it is known that changes in TGF-betas and fibroblast growth factors can cause sutural fusion, this is the first study to demonstrate that abrogation of sutural closure is responsible for growth factor signaling modulation.

Repeated direct endomyocardial transplantation of allogeneic mesenchymal stem cells: safety of a high dose, "off-the-shelf", cellular cardiomyoplasty strategy.

BACKGROUND: Efficacy of cellular cardiomyoplasty seems to occur in a dose-related manner. However, the safety of multiple transendomyocardial transplantation procedures to administer high cell dosages has not been previously reported. The aims of this study were to assess the short- and intermediate-term results of a repeated cell administration strategy and evaluate the safety of an "off-the-shelf" allogeneic mesenchymal stem cell (MSC) source. METHODS: Porcine bone marrow-derived MSCs were culture-expanded through three cycles in vitro before transplantation. Yorkshire swine weighing 30-40 kg were allocated to receive the total dose during 1 injection procedure or divided over 2 procedures separated by 14 days, as follows: (i) 400x10(6) allogeneic MSC (n=5), (ii) 800x10(6) allogeneic MSC divided in 2 doses (n=5), (iii) cryopreservant vehicle containing 10% DMSO, 5% porcine serum and 85% PlasmaLyte A, 14 days apart (n=2), or (iv) sterile saline 14 days apart (n=2). During each procedure, twenty 0.5 ml aliquots of the assigned injectant were administered using the Stiletto Endocardial Direct Injection Catheter System, targeting at the left ventricular anterior, septal and lateral walls under fluoroscopy. Vital signs and electrocardiograms were recorded during the procedure and at 24 h. The animals were examined daily and cardiac enzymes were measured immediately post-procedure, and on days 1, 15 and 90. Necropsy and histopathology were performed at day 90. RESULTS: Mean transendocardial injection procedure time was 40+/-10 min. All ventricular target areas were accessed by the Stiletto system. Ventricular ectopic beats and/or non-sustained ventricular tachycardia associated with catheter contact or endomyocardial injections were observed in all cases. However, no sustained ventricular arrhythmia, anaphylaxis, or significant cardiac enzyme release was seen. One mortality resulted from air embolism during the procedure. All other swine survived from the time of recovery until planned sacrifice at day 90 and had normal physical examination findings. The 3-month histopathology showed no evidence of rejection, calcification, teratoma or myocardial infarction. CONCLUSION: Repeated endomyocardial transplantation of high dose, bone marrow-derived allogeneic cells appeared safe in a large animal, human surrogate model. Such cellular cardiomyoplasty strategy warrants further investigation.

A quantitative, randomized study evaluating three methods of mesenchymal stem cell delivery following myocardial infarction.

AIMS: Mesenchymal stem cells (MSCs), rare bone marrow-derived stem cell precursors of non-haematopoietic tissues, have shown promise in potentially repairing infarcted myocardium. These and similar cell types are being tested clinically, but understanding of delivery and subsequent biodistribution is lacking. This study was designed to quantitatively compare MSC engraftment rates after intravenous (IV), intracoronary (IC), or endocardial (EC) delivery in a porcine myocardial infarction (MI) model. METHODS AND RESULTS: Allogeneic, male MSCs were cultured from porcine bone marrow aspirates. Iridium nanoparticles were added during culturing and internalized by the MSCs. An MI was induced in female swine (27-40 kg in size) by prolonged balloon occlusion of the mid-left anterior descending artery. Animals (n = 6 per group) were randomized to one of three delivery methods. Cellular engraftment was determined 14+/-3 days post-delivery by measuring ex-vivo the iridium nanoparticle concentration in the infarct. Confirmation of cellular engraftment utilized both DiI and fluorescence in situ hybridization (FISH) labelling techniques. During MSC infusion, no adverse events were noted. However, following IC infusion, half of the pigs exhibited decreased blood flow distal to the infusion site. At 14 days, the mean number of engrafted cells within the infarct zone was significantly greater (P< or =0.01) following IC infusion than either EC injection or IV infusion and EC engraftment was greater than IV engraftment (P< or =0.01). There was less systemic delivery to the lungs following [EC vs. IV (P = 0.02), EC vs. IC (P = 0.06)]. Both DiI and FISH labelling demonstrated the presence of engrafted male MSCs within the female infarcted tissue. CONCLUSION: IC and EC injection of MSCs post-MI resulted in increased engraftment within infarcted tissue when compared with IV infusion, and IC was more efficient than EC. However, IC delivery was also associated with a higher incidence of decreased coronary blood flow. EC delivery into acutely infarcted myocardial tissue was safe and well tolerated and was associated with decreased remote organ engraftment with compared with IC and IV deliveries.