Steven P. Schmidt

Controlled delivery of vascular endothelial growth factor promotes neovascularization and maintains limb function in a rabbit model of ischemia

PURPOSE Vascular endothelial growth factor (VEGF) modulates new blood vessel development and growth and has been suggested as a potential therapeutic agent that could alleviate debilitating claudication in patients. The objective of this study was to determine whether controlled, local delivery of a low dose of VEGF from an osmotic pump could promote neovascularization, limb perfusion, and functional improvements in the hind limbs of rabbits rendered partially ischemic by surgery. The effects of VEGF were compared with those of the vasodilator nitroglycerin (NTG) and to saline administered similarly. METHODS Thirty rabbits were randomly assigned to either VEGF (n = 10), NTG (n = 10), or saline (n = 10) treatment groups. Partial ischemia was induced in each left hind limb by surgical ligation of the common and superficial femoral arteries, leaving the internal iliac artery intact. The right limb of each animal served as a nonischemic control. Immediately after vessel ligations, a 28-day osmotic pump was implanted to deliver VEGF (0.22 microg/kg/day), NTG (17.8 microg/kg/day), or saline solution into the common iliac artery just proximal to the ligation site. Comparative vascularity between ischemic and nonischemic limbs within treatment groups and between groups was evaluated by (1) capillary counts from representative fields of hematoxylin and eosin stained muscle tissue taken from hind limbs at day 40; (2) digitized arteriograms of ischemic legs at day 40, which were used to quantify the complexity of vascular branching (fractal dimension index) and the total extent of vascularization (vascular density index); (3) measuring capillary refill times in ischemic limbs; and (4) observations of functional and trophic changes in ischemic limbs. Statistical differences between treatment groups were evaluated by one-way ANOVA. RESULTS Complexity of vascular branching and vascular density were significantly greater (p < 0.001) in VEGF-treated ischemic limbs compared with NTG- and saline-treated ischemic limbs. By postoperation day 14, all VEGF-treated ischemic limbs had restored capillary refill (p < 0.001), new hair growth, and greatly improved limb function and appearance. Saline-treated limbs exhibited ischemic changes, with poor capillary refill and negligible limb function. Capillary refill in NTG-treated ischemic limbs did not differ significantly from saline-treated limbs. Ischemic VEGF-treated limbs had significantly more capillaries compared with both ischemic and nonischemic limbs in saline-treated animals (p < 0.05). Ischemic NTG-treated limbs also had significantly more capillaries compared with ischemic limbs in saline-treated animals (p < 0.05). Because of high variability, however, capillary counts in VEGF-treated ischemic limbs did not differ significantly from those of contralateral nonischemic limbs, or from capillary counts in either ischemic or nonischemic limbs of NTG-treated rabbits. CONCLUSIONS Controlled release of microgram quantities of VEGF significantly enhanced neovascularization and vascular perfusion in ischemic limbs compared with controls in this rabbit model of partial ischemia. In addition, VEGF-treated ischemic limbs demonstrated near-normal function and appearance, whereas NTG- and saline-treated ischemic controls remained noticeably impaired. This novel approach of VEGF delivery may prove clinically useful either alone or combined with revascularization procedures.

Small-diameter vascular prostheses: two designs of PTFE and endothelial cell-seeded and nonseeded Dacron

Despite numerous advances in biomaterials design and utilization, the perfect artificial small-vessel substitute has yet to be developed. Dacron and expanded polytetrafluoroethylene (PTFE) are two materials potentially appropriate for use as small-vessel prostheses. We report the patencies of endothelial cell-seeded and nonseeded 4 mm I.D. Dacron grafts and two designs of nonseeded 4 mm I.D. PTFE (Gore-Tex and Impra) in the carotid position in dogs. All graft lengths exceeded the calculated maximum critical length for the material being tested. Dacron grafts, both endothelial cell-seeded and nonseeded, achieved higher patencies than both designs of PTFE. Endothelial cell-seeded Dacron grafts achieved the highest patencies. Endothelium was present to a significant extent only on endothelial cell-seeded Dacron grafts. There was little pannus ingrowth or midgraft pseudointima on nonseeded Dacron or on patent PTFE grafts although thrombus-free surface areas of patent PTFE grafts were high. These comparative data support the utility of endothelial cell seeding in achieving high patencies of small-diameter vascular grafts.

Material and structural characterization of human saphenous vein

Saphenous vein patch rupture after carotid endarterectomy is an infrequent but devastating complication. This study was undertaken to evaluate the material and structural properties of fresh human saphenous veins to understand the causes of this complication. Segments of saphenous veins were obtained from 22 patients from vein harvested during coronary artery bypass surgery. Ninety-three specimens, oriented in both circumferential (n = 45) and longitudinal (n = 48) directions, were prepared from the available vein segments for testing. Specimens were mounted on specially designed grips and then subjected to uniaxial tension testing. For each specimen the following material and structural parameters were determined: vessel diameter, tensile stiffness, failure and ultimate forces, and tensile modulus, failure stress, and strain. The physical properties of specimens evaluated in longitudinal orientations and thus limit the inherent strength of the vein. The physical properties of circumferentially tested vein specimens were negatively correlated to age, female gender, diabetes, and hypertension. The data obtained in this investigation suggest that age, hypertension, as well as diabetes and gender may adversely influence the circumferential tensile strength of human saphenous veins used as patch grafts.

Drug-eluting stent coatings.

This paper reviews the development of coronary stents from a polymer scientists view point, and presents the first results of an interdisciplinary team assembled for the development of new stent systems. Poly(styrene-b-isobutylene-b-styrene) block copolymer (SIBS), a nanostructured thermoplastic elastomer, is used in clinical practice as the drug-eluting polymeric coating on the Taxus coronary stent (trademark of Boston Scientific Co.). Our group has been developing new architectures comprising of arborescent (dendritic) polyisobutylene cores (D_SIBS), which were shown to be as biocompatible as SIBS. ElectroNanospray (Nanocopoeia Inc.) was used to coat test coupons and coronary stents with selected D(S)IBS polymers loaded with dexamethasone, a model drug. The surface topology varied from smooth to nanosized particulate coating. This paper will demonstrate how drug release profiles were influenced by both the molecular weight of the polyisobutylene core and spraying conditions of the polymer-drug mixture.

A nanostructured carbon-reinforced polyisobutylene-based thermoplastic elastomer

This paper presents the synthesis and characterization of a polyisobutylene (PIB)-based nanostructured carbon-reinforced thermoplastic elastomer. This thermoplastic elastomer is based on a self-assembling block copolymer having a branched PIB core carrying -OH functional groups at each branch point, flanked by blocks of poly(isobutylene-co-para-methylstyrene). The block copolymer has thermolabile physical crosslinks and can be processed as a plastic, yet retains its rubbery properties at room temperature. The carbon-reinforced thermoplastic elastomer had more than twice the tensile strength of the neat polymer, exceeding the strength of medical grade silicone rubber, while remaining significantly softer. The carbon-reinforced thermoplastic elastomer displayed a high T(g) of 126 degrees C, rendering the material steam-sterilizable. The carbon also acted as a free radical trap, increasing the onset temperature of thermal decomposition in the neat polymer from 256.6 degrees C to 327.7 degrees C. The carbon-reinforced thermoplastic elastomer had the lowest water contact angle at 82 degrees and surface nano-topography. After 180 days of implantation into rabbit soft tissues, the carbon-reinforced thermoplastic elastomer had the thinnest tissue capsule around the microdumbbell specimens, with no eosinophiles present. The material also showed excellent integration into bones.

Effects of vascular endothelial growth factor on wound closure rates in the genetically diabetic mouse model.

Impaired wound healing is characteristic of diabetic patients. Potential reasons include poor inflammatory response, granulation tissue formation, and abnormal patterns of cytokine release and response. Vascular endothelial growth factor, abnormally regulated during healing in diabetics, is the major factor stimulating angiogenesis during normal wound healing. We tested our hypothesis that topically applied vascular endothelial growth factor would improve wound closure rates in diabetic animals in a full‐thickness wound model in genetically diabetic mice (C57 BL/KsJ db/db). Animals received either 1.0 µg of vascular endothelial growth factor165 or polyethylene glycol alone topically to wounds daily between days 0 and 4 postwounding. Wound area was measured at days 0, 5, 10, 15, and 21. Data were analyzed using probit analysis and expressed as length‐of‐time (LT) to 50, 90, and 95% wound closure. Among untreated animals, nondiabetics had an LT50 of 8.5 days (fiducial limits 8.3–8.7), while diabetics had an LT50 of 15.8 days (15.6–16.1). Vascular endothelial growth factor‐treated animals had LT50 values of 7.8 (7.6–8.1) and 11.8 days (11.6–12.0) for nondiabetics and diabetics, respectively, representing a 25% improvement in time to 50% closure in treated diabetics. We conclude that topically applied vascular endothelial growth factor improves time to wound closure in the genetically diabetic mouse model.

Effects of sterilization on poly(ethylene glycol) hydrogels

The past few decades have witnessed a dramatic increase in the development of polymeric biomaterials. These biomaterials have to undergo a sterilization procedure before implantation. However, many sterilization procedures have been shown to profoundly affect polymer properties. Poly(ethylene glycol) hydrogels have gained increasing importance in the controlled delivery of therapeutics and in tissue engineering. We evaluated the effect of ethylene oxide (EtO), hydrogen peroxide (H(2)O(2)), and gamma sterilization of poly(ethylene glycol) hydrogels on properties relevant to controlled drug delivery and tissue engineering. We observed that the release of cyclosporine (CyA) (an immunosuppressive drug that is effective in combating tissue rejection following organ transplantation) was significantly affected by the type of sterilization. However, that was not the case with rhodamine B, a dye. Hence, the drug release characteristics were observed to be dependent not only on the sterilization procedure but also on the type of agent that needs to be delivered. In addition, differences in the swelling ratios for the sterilized and unsterilized hydrogels were statistically significant for 1:1 crosslinked hydrogels derived from the 8000 MW polymer. Significant differences were also observed for gamma sterilization for 1:1 crosslinked hydrogels derived from the 3350 MW polymer and also the 2:1 crosslinked hydrogels derived from the 8000 MW polymer. Atomic force microscopy (AFM) studies revealed that the roughness parameter for the unsterilized and EtO-sterilized PEG hydrogels remained similar. However, a statistically significant reduction of the roughness parameter was observed for the H(2)O(2) and gamma-sterilized samples. Electron spin resonance (ESR) studies on the unsterilized and the sterilized samples revealed the presence of the peroxy and the triphenyl methyl carbon radical in the samples. The gamma and the H(2)O(2)-sterilized samples were observed to have a much higher concentration of the radical pecies when compared with the EtO and the unsterilized samples.

Effects of antiplatelet agents in combination with endothelial cell seeding on small-diameter Dacron vascular graft performance in the canine carotid artery model

The purpose of this study was to assess the success of endothelial cell-seeded and non-seeded small-diameter vascular grafts in dogs medicated with antiplatelet agents. Eighty dogs underwent bilateral carotid artery replacements with 6 cm lengths of 4 mm I.D. double-velour Dacron grafts. In each dog one graft was seeded with enzymatically derived autologous endothelial cells; the contralateral graft was nonseeded. The following anti-platelet medications were administered beginning 4 days preoperatively: aspirin (5 grains every day); dipyridamole (50 mg twice a day); aspirin plus dipyridamole (5 grains each day plus 50 mg twice a day); aspirin (1.25 grains every other day); ibuprofen (10 mg/kg/day); U-53,059, a cyclooxygenase inhibitor (3 mg/kg/day); and U-63557A, a thromboxane synthase inhibitor (10 mg/kg/day). Grafts were harvested 5 weeks postoperatively. Graft success was evaluated by patency, thrombus-free surface area, area endothelialized, and graft production of prostacyclin. None of the medications prevented neoendothelialization of seeded grafts. Mean patencies of endothelial cell-seeded grafts from medicated dogs were significantly greater than mean patencies of endothelial cell-seeded grafts from nonmedicated dogs. The cyclooxygenase inhibitors best maintained patency in nonseeded grafts. Thrombus-free surface areas of endothelial cell-seeded grafts from medicated dogs were significantly greater than from nonseeded control grafts from the medicated dogs. All medications impaired prostacyclin synthesis. We conclude that the combination of endothelial cell seeding plus antiplatelet medication is most efficacious in small-vessel grafting success and that high levels of prostacyclin production by vascular grafts are not necessary to maintain patency in dogs medicated with antiplatelet agents.

Immunologic function in the elderly after injury--the neutrophil and innate immunity.

BACKGROUND Aging is associated with a decline in immune function. This may contribute to decreased ability of an elderly patient to mount an appropriate innate inflammatory response when injured. This study examined elderly trauma patients to determine whether there was a difference in neutrophil response to injury when compared with controls. METHODS This prospective, observational, cohort study compared neutrophil function in 24 injured elderly (older than 65 years) patients admitted to our trauma center to control groups of noninjured individuals (11 elderly and 17 young). Blood samples were also taken from the injured elderly group within 48 hours of trauma and subsequently at two periods during their hospital stay. A single blood sample was obtained from the noninjured control groups. Neutrophils were analyzed for CD18 expression, stimulated oxidative burst, apoptosis, and IL-10. Results were compared using one-way analysis of variance (alpha 0.05). This study was approved by the Institutional Review Board. RESULTS Twenty-four injured elderly subjects were enrolled: mean injury severity score 15.3, average age 74.6 years, 92% survival, 100% blunt trauma. CD18 levels in the elderly injured subjects for all three time periods were significantly higher than both control groups. When evaluated between controls, CD18 for the noninjured elderly (NIE) was also significantly higher than the noninjured young (NIY). The neutrophil stimulated oxidative burst in the injured elderly subjects at time periods 1, 2, and 3 was not significantly different from the NIY controls. However, the injured elderly had a significantly higher oxidative burst at time period 3 than the NIE controls. Apoptosis in the injured elderly subjects was significantly lower in all three time periods than the NIY. There was no difference in apoptosis between the injured elderly subjects when compared with the NIE controls. There was no significant difference in IL-10 expression among groups. CONCLUSION Injury results in differences in innate immune function in the elderly when compared with controls. The clinical significance of this is uncertain and warrants further investigation.

In vitro evaluation of electrostatic endothelial cell transplantation onto 4 mm interior diameter expanded polytetrafluoroethylene grafts

PURPOSE To perform an in vitro evaluation of electrostatic endothelial cell transplantation of human umbilical vein endothelial cells (HUVEC) onto segments of 4 mm internal diameter expanded polytetrafluoroethylene (ePTFE) vascular prostheses. METHODS This evaluation consisted of exposing vascular graft segments that had been subjected to either electrostatic or gravitation transplantation with HUVEC to a physiologic shear stress (15 dynes/cm2) under steady flow conditions within a flow loop system. Biochemical assays were performed on freshly transplanted grafts by means of radioimmunoassay for prostacyclin and thromboxane A2. RESULTS There was a 30% loss of HUVEC after 30 minutes of shear stress exposure from the grafts subjected to gravitational transplantation with no additional significant (alpha = 0.05) loss after 120 minutes. Grafts subjected to electrostatic transplantation had no significant (alpha = 0.05) loss of HUVEC during exposure to physiologic shear stress. Furthermore, after 120 minutes of shear-stress exposure, the grafts subjected to electrostatic transplantation (78,420 +/- 6274 HUVEC/cm2) retained 2.3 times more HUVEC than the counterparts subjected to gravitational transplantation (34,427 +/- 4637 HUVEC/cm2). The biochemical assay results indicated no significant (alpha = 0.05) production of prostacyclin or thromboxane A2 regardless of the method of cell transplantation. CONCLUSIONS (1) The electrostatic transplantation technique was superior to the gravitational transplantation technique in terms of cellular retention when the ePTFE grafts were exposed to physiologic shear stress. (2) Production of prostacyclin and thromboxane A2 did not differ between transplanted HUVEC subjected to gravitational or electrostatic procedures.