David D. Krijgh

Stromal-derived factor-1 delivered via hydrogel drug-delivery vehicle accelerates wound healing in vivo

Topical treatment of superficial wounds has many advantages including decreased cost and increased ease of application compared with systemic treatments. Many of the advantages, however, are lost when it is necessary for repeated doses of topical medications to be given over an extended period of time. Therefore, a drug‐delivery vehicle that delivers biologically appropriate doses in a sustained fashion would prove valuable. In this study, an alginate hydrogel scaffold impregnated with the angiogenic chemokine stromal‐derived factor‐1 was used to provide targeted, though short‐term, delivery directly into the wound bed. Wounds were created on the dorsum of mice, and either a stromal‐derived factor‐1‐impregnated or a saline‐impregnated scaffold was applied. Wounds were explanted after 1, 3, 7 days, wound area was measured, and histology and immunohistochemistry for endothelial markers were performed. The remaining wound area in stromal‐derived factor‐1‐treated wounds vs. controls was not significant 1 day after wounding (96.7±0.1 vs. 97.5±1.1%, p=0.317), but was significant after 3 days postwounding (46.7±0.1 vs. 82.3±2.4%, p=0.046) and 7 days postwounding (2.3±1.3 vs. 32.0±4.0%, p=0.049). Immunohistochemistry revealed a greater degree of endothelial cell invasion into the wound bed infiltration compared with controls. The results of this study suggest significant clinical promise for our hydrogel‐delivery vehicle in the treatment of wounds.

Therapeutic Metabolic Inhibition: Hydrogen Sulfide Significantly Mitigates Skeletal Muscle Ischemia Reperfusion Injury In Vitro and In Vivo

Background: Recent evidence suggests that hydrogen sulfide is capable of mitigating the degree of cellular damage associated with ischemia-reperfusion injury. The purpose of this study was to determine whether it is protective in skeletal muscle. Methods: This study used both in vitro (cultured myotubes subjected to sequential anoxia and normoxia) and in vivo (mouse hind-limb ischemia followed by reperfusion) models in which hydrogen sulfide (0 to 1000 &mgr;M) was delivered before the onset of oxygen deficiency. Injury score and apoptotic index were determined by analysis of specimens stained with hematoxylin and eosin and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling, respectively. Results: In vitro, hydrogen sulfide reduced the apoptotic index by as much as 99 percent (p = 0.001), with optimal protection conferred by raising intravascular hydrogen sulfide to 10 &mgr;M. In vivo, 10 &mgr;M hydrogen sulfide delivered before 3 hours of hind-limb ischemia followed by 3 hours of reperfusion resulted in protection against ischemia-reperfusion injury–induced cellular changes, as evidenced by significant decreases in injury score and apoptotic index (by as much as 91 percent; p = 0.001). These findings were consistent at 4 weeks after injury and reperfusion. Conclusion: These findings confirm that the preischemic delivery of hydrogen sulfide limits ischemia-reperfusion injury–induced cellular damage in myotubes and skeletal muscle and suggests that, when given in the appropriate dose, this molecule may have significant therapeutic applications in multiple clinical scenarios.

Therapeutic delivery of hydrogen sulfide for salvage of ischemic skeletal muscle after the onset of critical ischemia

BACKGROUND Recent evidence suggests that hydrogen sulfide is capable of mitigating the degree of cellular damage associated with ischemia-reperfusion injury (IRI). METHODS This study evaluated the potential utility of hydrogen sulfide in preventing IRI in skeletal muscle by using in vitro (cultured myotubes subjected to sequential hypoxia and normoxia) and in vivo (mouse hind limb ischemia, followed by reperfusion) models to determine whether intravenous hydrogen sulfide delivered after the ischemic event had occurred (pharmacologic postconditioning) conferred protection against IRI. Injury score and apoptotic index were determined by analysis of specimens stained with hematoxylin and eosin and terminal deoxynucleotide transferase-mediated deoxy-uridine triphosphate nick-end labeling, respectively. RESULTS In vitro, hydrogen sulfide reduced the apoptotic index after 1, 3, or 5 hours of hypoxia by as much as 75% (P = .002), 80% (P = .006), and 83% (P < .001), respectively. In vivo, hydrogen sulfide delivered after the onset of hind limb ischemia and before reperfusion resulted in protection against IRI-induced cellular changes, which was validated by significant decreases in the injury score and apoptotic index. The timing of hydrogen sulfide delivery was crucial: when delivered 20 minutes before reperfusion, hydrogen sulfide conferred significant cytoprotection (P < .001), but treatment 1 minute before reperfusion did not provide protection (P = NS). CONCLUSIONS These findings confirm that hydrogen sulfide limits IRI-induced cellular damage in myotubes and skeletal muscle, even when delivered after the onset of ischemia in this murine model. These data suggest that when given in the appropriate dose and within the proper time frame, hydrogen sulfide may have significant therapeutic applications in multiple clinical scenarios.

Mathematical modeling and frequency gradient analysis of cellular and vascular invasion into integra and strattice: toward optimal design of tissue regeneration scaffolds.

Background: Rapid, effective host cell invasion and vascularization is essential for durable incorporation of avascular tissue-replacement scaffolds. In this study, the authors sought to qualitatively and quantitatively determine which of two commercially available products (i.e., Strattice and Integra) facilitates more rapid cellular and vascular invasion in a murine model of graft incorporation. Methods: Integra and Strattice were implanted subcutaneously into the dorsa of C57BL/6 mice; harvested after 3, 7, or 14 days; and stained with hematoxylin and eosin, 4′,6-diamidino-2-phenylindole, and immunohistochemical stains for CD31 and &agr;-smooth muscle actin. Exponential decay equations describing cellular invasion through each layer were fit to each material/time point. Mean cell density and cell frequency maps were created denoting extent of invasion by location within the scaffold. Results: Qualitative analysis demonstrated extensive cellular infiltration into Integra by 3 days and increasing over the remaining 14 days. Invasion of Strattice was sparse, even after 14 days. &agr;-Smooth muscle actin immunohistochemistry revealed blood vessel formation within Integra by 14 days but no analogous neovascularization in Strattice. Mean decay equations for Integra and Strattice were y = 76.3(0.59)x and y = 75.5(0.33)x, respectively. Both cell density measurements and frequency mapping demonstrated that, at all time points, Integra manifested a greater density/depth of cellular invasion when compared with Strattice. Conclusions: These data confirm empiric clinical observations that Integra is more rapidly invaded than Strattice when placed in a suitable host bed. A remnant microvasculature template is not sufficient for effective cellular ingrowth into an artificial tissue construct. These findings provide insight into means for improving future dermal replacement products.

Hydrogen sulfide attenuates intestinal ischemia-reperfusion injury when delivered in the post-ischemic period

Background and Aim:  To investigate whether pharmacologic post‐conditioning of intestinal tissue with hydrogen sulfide (HS) protects against ischemia reperfusion injury (IRI).

Development of an acellular bioengineered matrix with a dominant vascular pedicle.

BACKGROUND This study assessed the feasibility of creating a tissue engineering platform by decellularization of fasciocutaneous tissue. MATERIALS AND METHODS A fasciocutaneous flap based upon the superficial inferior epigastric artery was harvested from the abdominal wall of 8-wk-old male Sprague-Dawley rats. All cellular components were removed by sequential treatment with sodium azide, DNAse, and sodium deoxycholate. The degree of decellularization was qualitatively assessed by histology and quantitatively assessed by spectrophotometry. Persistence of relevant extracellular matrix proteins was shown following staining with orcein and hematoxylin. The duration of circuit patency was determined by continuous perfusion with a peristaltic perfusion pump. RESULTS Gross and histologic examination demonstrated removal of cellular constituents with preservation of tissue matrix architecture, including macrochannels and microchannels. This was confirmed by the application of spectrophotometry to DNA isolates, which showed that the decellularized flap retained 4.04 ng/μL DNA, compared with the non-processed control, which retained 37.03 ng/μL DNA, and the acellular control, which was read as having 0.65 ng/μL DNA. The extracellular matrix of vessel walls was shown to remain intact. Peristaltic perfusion of the cannulated pedicle inflow channel with phosphate buffered saline at a rate of 200 μL/min confirmed circuit patency for 6 h. CONCLUSION Fasciocutaneous flaps harvested with an intact vascular pedicle and associated tissue vascular network can be successfully decellularized and perfused ex vivo. This methodology, which is scalable to human size tissues, provides promise as a technique for the production of customizable engineered tissues.

164A: OPTIMIZING NEOVASCULARIZATION OF TISSUE REGENERATION TEMPLATES BY RATIONAL DESIGN AND MICROFABRICATION

Objective: Tissue engineering has long sought to design constructs that rapidly achieve functionality upon implantation. Current techniques, however, cannot produce pre-fabricated tissues with an intact microvascular network that is connected to macrovascular inflow and outflow vessels. Therefore, we assessed the feasibility of decellularizing tissue with an intact vascular pedicle that contains a dominant artery and vein.

7: SOLVING THE QUESTION OF PROTECTION: HYDROGEN SULFIDE CONFERS PROTECTION FROM IRI VIA ACTIVATION OF THE JAK-STAT PATHWAY

Introduction: Although there is an increasing body of evidence that demonstrates that hydrogen sulfide (HS) provides significant protection against Ischemia Reperfusion Injury (IRI), the mechanism by which this protection is conferred remains poorly understood. The JAK-STAT signaling pathway is known to regulate multiple cell processes including proliferation, differentiation and apoptosis via modulation of nuclear gene expression. Previous work has shown that this pathway is activated by mechanical preconditioning, which provides protection against IRI. We hypothesize this critical cell survival pathway would be similarly activated by treatment with HS in the setting of IR.

Therapeutic Delivery of Hydrogen Sulfide to Profoundly Ischemic Muscle: Timing Is Everything

INTRODUCTION: Hydrogen sulfide (HS) is protective effect against the detrimental effects of ischemia-reperfusion injury (IRI) when delivered either before or after an ischemic event has occurred. The optimal timing of treatment, however, remains undefined. In order to better understand the potential clinical application of HS, we sought to define the therapeutic window during which delivery of HS is protective.

40A: CHOOSING THE OPTIMAL TISSUE REGENERATION TEMPLATE: A QUANTITATIVE ANALYSIS OF CELLULAR INVASION AND MIGRATION IN INTEGRA AND STRATTICE

Background: Vascularization via host cell invasion is key to the success of dermal replacement therapy. Some tissue templates are comprised of decellularized tissue (eg. Strattice), that have as a reported advantage a retained microvasculature. Other approaches do not have an organized network of channels, and instead promote cellular invasion into a latticework of random pores (eg. Integra). This study sought to quantitatively determine which approach facilitates more rapid cellular invasion.