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Objectives The potential for beneficial effects of adipose-derived stem cells (ASCs) on myocardial perfusion and left ventricular dysfunction in myocardial ischemia (MI) has not been tested following intravenous delivery. Methods Surviving pigs following induction of MI were randomly assigned to 1 of 3 different groups: the placebo group (n = 7), the single bolus group (SB) (n = 7, 15 × 107 ASCs), or the divided dose group (DD) (n = 7, 5 × 107 ASCs/day for three consecutive days). Myocardial perfusion defect area and coronary flow reserve (CFR) were compared during the 28-day follow-up. Also, serial changes in the absolute number of circulating CD4+T and CD8+T cells were measured. Results The increases in ejection fraction were significantly greater in both the SB and the DD groups compared to the placebo group (5.4 ± 0.9%, 3.7 ± 0.7%, and −0.4 ± 0.6%, respectively), and the decrease in the perfusion defect area was significantly greater in the SB group than the placebo group (−36.3 ± 1.8 and −11.5 ± 2.8). CFR increased to a greater degree in the SB and the DD groups than in the placebo group (0.9 ± 0.2, 0.8 ± 0.1, and 0.2 ± 0.2, respectively). The circulating number of CD8+T cells was significantly greater in the SB and DD groups than the placebo group at day 7 (3,687 ± 317/µL, 3,454 ± 787/µL, and 1,928 ± 457/µL, respectively). The numbers of small vessels were significantly greater in the SB and the DD groups than the placebo group in the peri-infarct area. Conclusions Both intravenous SB and DD delivery of ASCs are effective modalities for the treatment of MI in swine. Intravenous delivery of ASCs, with its immunomodulatory and angiogenic effects, is an attractive noninvasive approach for myocardial rescue. © 2014 Wiley Periodicals, Inc.

Objectives To examine the comparative fate of adipose-derived stem cells (ASCs) as well as their impact on coronary microcirculation following either retrograde coronary venous or arterial delivery.

Purpose A delayed full-thickness wound-healing model was developed and used for examining the capacity of adipose-derived stem cells (ASCs), either alone or in platelet-rich fibrin gels, to promote healing. Methods and Materials Four pigs received electron beam radiation to the dorsal skin surface. Five weeks after radiation, subcutaneous fat was harvested from nonirradiated areas and processed to yield ASCs. Two weeks later, 28 to 30 full-thickness 1.5-cm 2 wounds were made in irradiated and nonirradiated skin. Wounds were treated with either saline solution, ASCs in saline solution, platelet-rich plasma (PRP) fibrin gel, ASCs in PRP, or non-autologous green fluorescence protein–labeled ASCs. Results The single radiation dose produced a significant loss of dermal microvasculature density (75%) by 7 weeks. There was a significant difference in the rate of healing between irradiated and nonirradiated skin treated with saline solution. The ASCs in PRP-treated wounds exhibited a significant 11.2% improvement in wound healing compared with saline solution. Enhancement was dependent on the combination of ASCs and PRP, because neither ASCs nor PRP alone had an effect. Conclusions We have created a model that simulates the clinically relevant late radiation effects of delayed wound healing. Using this model, we showed that a combination of ASCs and PRP improves the healing rates of perfusion-depleted tissues, possibly through enhancing local levels of growth factors.

The authors examined the efficacy of adipose stem cells, when supplied either alone or in platelet-rich fibrin gels, to improve wound healing.A porcine full-thickness wound model was used to compare six topical treatments: platelet-poor plasma; platelet-rich plasma; autologous adipose stem cells plus platelet-poor plasma; autologous adipose stem cells plus platelet-rich plasma; allogeneic adipose stem cells containing green fluorescent protein plus platelet-poor plasma; and saline (control). One week after isolation, adipose stem cells were applied to full-thickness wounds on the paraspinal and thoracic regions of three pigs (44 wounds per pig; each treatment was applied to eight separate wounds). Each wound was monitored over 21 days for closure, cosmesis, and histopathology.There was no significant difference in the reepithelialization rate, but treatments containing adipose stem cells demonstrated increased microvessel densities (31.75 +/- 5.73 vessels/cm2 versus 7.93 +/- 3.61 vessels/cm2) compared with groups without adipose stem cells. Wound cosmesis was improved in the adipose stem cell plus platelet-rich plasma group compared with other treatment groups (p0.05). Vascular endothelial growth factor levels detected in matrices containing adipose stem cells were approximately 7-fold higher compared with platelet-rich plasma or platelet-poor plasma (p0.05). Localization of transgenic green fluorescent protein plus adipose stem cells indicated incorporation near neovasculature.In normal healing wounds, adipose stem cells appear to enhance the healing process only when provided in a fibrin gel vehicle containing a number of complementary wound-healing trophic factors. Perivascular adipose stem cell localization suggests a function in enhancing blood supply through providing physical and paracrine support to newly forming vessels.

This study quantifies myocardial transfection following percutaneous retrograde coronary venous delivery (RCVD) of a plasmid encoding human Del-1. RCVD of Del-1, GFP plasmid, or marker dye was conducted in 14 pigs. After selective cannulation of a coronary vein, a delivery site was confirmed by contrast injection and myocardial blush. Ten milliliters of plasmid hDel-1 or GFP was administered. Animals were euthanized 3 and 7 days post-RCVD. hDel-1 gene expression was evaluated by quantitative RT-PCR. An average myocardial expression of 4.5 x 10(5) copies hDel-1/microg total RNA was observed within the approximately 5 x 5 cm(2) target tissue of the left ventricle. GFP expression was detected by fluorescent microscopy. hDel-1 protein expression was confirmed by immunohistochemistry. Regionalized myocardial expression was found in all pigs. hDel-1 RNA was not found in distant tissues except in the three pigs with prominent venovenous washout (PVW). These levels were 3 to 4 log unites lower than those found in myocardium. Single retrograde coronary venous administration resulted in efficient regional myocyte transfection of hDel-1 and GFP. This method may be useful and clinically feasible for myocardial angiogenesis therapy.

Background —Catheter-based intrapericardial (IPC) delivery of therapeutic agents has recently been demonstrated. Paclitaxel is known to inhibit vascular smooth muscle cell proliferation. This study examined the effect of IPC instillation of paclitaxel on neointimal proliferation after balloon overstretch of porcine coronary arteries. Methods and Results —Overstretch injury of coronary arteries was followed by IPC administration of micellar paclitaxel at low dose (LD, 10 mg; n=6) or high dose (HD, 50 mg; n=7) or of control micelles (50 mg, n=5). Animals were euthanized 28 days after balloon dilation. Arterial injury indices were no different among the groups. The neointimal area, maximal intimal thickness, and adventitial thickness were significantly reduced in both LD (0.47±0.04 mm 2 , 0.43±0.03 mm, and 0.35±0.02 mm, respectively) and HD (0.51±0.06 mm 2 , 0.42±0.03 mm, and 0.38±0.03 mm, respectively) paclitaxel groups compared with the control group (0.79±0.07 mm 2 , 0.56±0.02 mm, and 0.47±0.02 mm, respectively; P P Conclusions —IPC space delivery of a single dose of paclitaxel significantly reduces vessel narrowing in this balloon-overstretch model. This effect is mediated by reduction of neointimal mass as well as positive vascular remodeling.

Objective and Design We characterized the mean peak vertical forces (MFz) in five groups of dogs which underwent transection of the left anterior cruciate ligament (ACLT) or sham ACLT and ipsilateral dorsal root ganglionectomy or sham-ganglionectomy, and the relationship of these forces to the severity of osteoarthritis (previously reported) 72 weeks after arthrotomy. Group I ( N =7) underwent ACLT; Group II ( N =8) underwent ACLT followed 52 weeks later by ganglionectomy; Group III ( N =7) underwent ganglionectomy followed 2 weeks later by ACLT; Group IV ( N =7) underwent sham-ganglionectomy followed 2 weeks later by ACLT; Group V ( N =8) underwent ganglionectomy followed 2 weeks later by sham-ACLT. The dogs were evaluated 2, 6, 12, 24, 52 and 72 weeks after arthrotomy. Results From 6 weeks after arthrotomy until death, the left hindlimb MFz in Group V was significantly greater ( P 0.05) than that in the other four groups. The MFz of all groups which underwent ACLT decreased after arthrotomy. While the MFz of Group III (very severe OA) was about 10–20% greater than that of Groups I, II and IV (mild OA) 6 and 12 weeks after ACLT, and generally about 5–10% greater subsequently, this difference was not statistically significant. The MFz of Group II returned to pre-ganglionectomy levels, rather than to baseline levels, following ganglionectomy. Conclusions (1) since the ipsilateral limb of dogs with ganglionectomy+sham ACLT bore normal amounts of weight throughout most of the postsurgical period, and its knee did not develop OA, one cannot argue that the knee was protected from OA because the limb was not used; (2) the fact that the MFz of dogs which underwent ACLT+ganglionectomy returned to pre-ganglionectomy levels, rather than baseline, is consistent with the hypothesis that the unstable joint was protected from accelerated breakdown by a central nervous system that was reprogramed by sensation from the unstable limb; (3) the slightly—but consistently—greater MFz of dogs which underwent ganglionectomy+ ACLT may contribute to the acceleration of OA in this model.

Summary Objective and Design: transection of the anterior cruciate ligament 2 weeks after ipsilateral hindlimb deafferentation leads to osteoarthritis of the knee joint within 3 weeks. We analyzed the gait of six dogs that underwent this procedure in order to identify kinematic changes that could account for this rapid joint degeneration. All animals were video taped 1, 3, 6, 9 and 13 weeks after surgery while they trotted on a treadmill. Results: In each dog, extension of the hip, knee and ankle joints of the unstable limb was increased, and the yield phase of the unstable knee was delayed or attenuated. When killed, five of six dogs showed a large full-thickness cartilage ulcer on the distal and/or anterior surface of the medial femoral condyle of the unstable knee; in the sixth dog, a smaller ulcer was observed. However, the severity of pathology in each individual was not obviously related to differences among the dogs in postoperative joint kinematics. Conclusions: These data, and results of prior studies in humans and dogs, suggest that knee hyperextension resulting from limb deafferentation, and knee instability resulting from anterior cruciate ligament transection, operate in concert to create a mechanical environment (i.e., increased tibiofemoral separation and changes in the loading of articular surfaces) that results in rapid joint breakdown.

Transection of the anterior cruciate ligament in the dog leads to osteoarthritis. This study defines the kinematic changes in the unstable knee after transection of the cruciate ligament (six dogs) and after a sham operation (four dogs). In the dogs that were anterior cruciate ligament-deficient (ACL-D), the duration of stance 1 week postoperatively decreased 38% from the preoperative value, but only a 4% decrease was seen at 6 weeks. The duration of double hindlimb support increased from 6 to 19% of the entire cycle 1 week after surgery but returned to the baseline value by 18 weeks. As the unstable limb contacted the treadmill belt, the initial flexion (yield) and subsequent extension (propulsive) phases were not evident or were markedly attenuated in every ACL-D dog throughout the 26-week period of observation. The angular velocity patterns were characterized by a slight extension velocity at touchdown (compared with a zero value preoperatively) and a decrease in the peak velocities (both flexion and extension) during the remainder of the stance phase. None of these changes was observed in the animals that had a sham operation. These data indicate that, in the dog, the nervous system compensates for instability of the knee by altering angular, but not temporal, parameters. The extension velocity at touchdown and the reduction in peak flexion velocity during the yield component of the stance phase may reduce the ability of the limb to absorb impact forces and lead to the development of osteoarthritis of the knee. Alternatively, the reductions in flexion and extension of the knee during the stance phase and in the values for peak velocity presumably reflect protective muscular reflexes that may slow the rate of progression of joint breakdown.

A wearable cardiopulmonary monitoring system, a LifeShirt®, was used to acquire continuous electrocardiograms (ECGs) from ambulatory swine. The animals received intracoronary injections of autologous mesenchymal stem cells, and the LifeShirt® was used for long-duration ECG monitoring in pre-defined periods post cell infusion. The system used here was developed for measurements from non-human primates and canines; however, we demonstrated that it could be used to non-invasively measure ECGs from swine without creating undue stress or restricting movement. A MATLAB-based analysis algorithm was developed to automatically detect premature ventricular contractions (PVCs) that arose 8-10 hours after cell delivery with spontaneous resolution 2-3 days post-infusion. Template based cross-correlation was used to detect the PVCs and identify regions of consecutive ventricular rhythm. The final algorithm was highly specific and sensitive when tested on records from the MIT-BIH arrhythmia database. The algorithm was subsequently used to automatically identify and quantify PVCs from over 200 hours of ECG data obtained from nine ambulatory swine.

An in vivo study was conducted to investigate the feasibility, mechanical function, and chronic biocompatibility of a new light-activated surgical adhesive for achieving rapid hemostasis of the puncture site following diagnostic catheterization and interventional cardiac procedures. Porcine carotid arteries (nequals6) and femoral arteries (nequals6) were exposed, and an incision was made in the arterial walls using a 16G needle. The surgical adhesive, composed of a poly(L-lactic-co-glycolic acid) scaffold doped with the traditional protein solder mix of serum albumin and indocyanine green dye, was used to close the incisions in conjunction with an 805-nm diode laser. Blood flow was restored to the vessels immediately after the procedure and the incision sites were checked for patency. The strength and hemostatic abilities of the new surgical adhesive were evaluated in the context of arterial pressure, persistence of hemostatis and presence of any inflammatory reaction after 3 days. After this evaluation period, the surgical procedure was repeated on the carotid arteries (nequals6) and femoral arteries (nequals6) of three additional animals that had been heparinized prior to surgery to closer approximate the conditions seen in a typical vascular surgical setting.

Following unilateral L4-S1 dorsal root ganglionectomy to deafferent the hind limb, each of six dogs showed increased extension of the ipsilateral hip, knee and ankle joints during most of the gait cycle throughout a 26-week period of observation. The contralateral hind limb joints initially exhibited increased flexion during gait (which presumably compensated for the increased extension of the deafferented limb), but over time contralateral joint extension gradually increased, i.e. the movement of the joints of the contralateral limb progressively began to resemble that of the ipsilateral joints. We suggest that the long-term kinematic changes in both limbs (increased extension) occurred because of neurological changes in spinal cord structure, associated with death of sensory neurons and an associated increase in the influence of descending systems (e.g. vestibulospinal) on motoneurons. These results emphasize the importance of long-term observation of kinematic patterns after experimental induction of neural lesions and indicate that the contralateral limb should not, a priori, be considered a valid control in such studies.

To help elucidate how sensation-mediated kinematics modulate the rate of development of osteoarthritis in the unstable knee, we have examined the serial kinematic changes in hind- and forelimb joints, and alterations in vertical movement of the rump, in six dogs followed for 26 weeks after unilateral anterior curciate ligament transection. Although marked changes in the temporal parameters occurred in treadmill gait acutely in all four limbs, by the sixth postoperative week the stance and swing durations had returned to within ±10% of the baseline values, where they remained until sacrifice. As the cruciate-deficient limb contacted the treadmill surface, the amount of flexion (yield) of the unstable knee and ipsilateral ankle was reduced 10-20° (P0.05). In contrast, flexion of the contralateral ankle and knee increased about 10° during yield (P0.05), which was associated with a 100% increase in the extent of vertical movement of the rump (P0.05). Hip extension in the unstable limb increased about 10° during stance (P0.05), which moved the support provided by this limb away from the dog's centre of gravity. Kinematic changes in the forelimb joints were only transient, and less extensive than those in the hind limb. Before ligament transection, the joint angles of comparable joints of the different dogs were remarkably similar at touchdown. After ligament transection, the variability of the ipsilateral hip and knee joints was initially markedly increased. However, by 26 weeks after surgery the ipsilateral hip and knee touchdown joint angles of all the cruciate-deficient dogs were again similar to one another. Thus, after a period of 'trial and error', the dogs responded similarly to unilateral cruciate deficiency. This response was probably modulated by sensory nerves and reduced the trauma to the unstable knee during locomotion. Presumably, this facilitated effective, but suboptimal locomotion, while slowing the rate of progression of osteoarthritis in the unstable joint compared to dogs with a deafferented hind limb and unstable joint.