Jason A. Spector

High-Fidelity Tissue Engineering of Patient-Specific Auricles for Reconstruction of Pediatric Microtia and Other Auricular Deformities

Introduction Autologous techniques for the reconstruction of pediatric microtia often result in suboptimal aesthetic outcomes and morbidity at the costal cartilage donor site. We therefore sought to combine digital photogrammetry with CAD/CAM techniques to develop collagen type I hydrogel scaffolds and their respective molds that would precisely mimic the normal anatomy of the patient-specific external ear as well as recapitulate the complex biomechanical properties of native auricular elastic cartilage while avoiding the morbidity of traditional autologous reconstructions. Methods Three-dimensional structures of normal pediatric ears were digitized and converted to virtual solids for mold design. Image-based synthetic reconstructions of these ears were fabricated from collagen type I hydrogels. Half were seeded with bovine auricular chondrocytes. Cellular and acellular constructs were implanted subcutaneously in the dorsa of nude rats and harvested after 1 and 3 months. Results Gross inspection revealed that acellular implants had significantly decreased in size by 1 month. Cellular constructs retained their contour/projection from the animals dorsa, even after 3 months. Post-harvest weight of cellular constructs was significantly greater than that of acellular constructs after 1 and 3 months. Safranin O-staining revealed that cellular constructs demonstrated evidence of a self-assembled perichondrial layer and copious neocartilage deposition. Verhoeff staining of 1 month cellular constructs revealed de novo elastic cartilage deposition, which was even more extensive and robust after 3 months. The equilibrium modulus and hydraulic permeability of cellular constructs were not significantly different from native bovine auricular cartilage after 3 months. Conclusions We have developed high-fidelity, biocompatible, patient-specific tissue-engineered constructs for auricular reconstruction which largely mimic the native auricle both biomechanically and histologically, even after an extended period of implantation. This strategy holds immense potential for durable patient-specific tissue-engineered anatomically proper auricular reconstructions in the future.

Fabrication of an artificial 3-dimensional vascular network using sacrificial sugar structures

Using sacrificial sugar structures, we have formed a 3D fluidic vascular network in a polymeric matrix. Melt-spun sugar fibers (cotton candy) were used to form channels with diameters and densities similar to those of capillaries. To interface to macroscopic fluidic systems, larger sacrificial sugar structures were used to form an artificial inlet and outlet. To verify that the channel network supported flow, we used video fluorescence microscopy to image both 2 µm fluorescent polystyrene spheres in an aqueous solution and fluorescently labeled blood. This fabrication process may be applied to a wide range of polymeric materials and is rapid, inexpensive, and highly scalable.

Microstructured templates for directed growth and vascularization of soft tissue in vivo

Tissue templates for reconstruction and regeneration inxa0vivo have achieved clinical successes for homogeneous tissues in well vascularized regions. Outstanding challenges exist for applications in poorly vascularized regions and for spatially differentiated tissues. Here, we present a strategy to control the spatial patterns of cell and vascular ingrowth throughout the volume of a bioremodelable and resorbable matrix via well-defined micropores and networks of microchannels. Our presentation of this approach includes: a description of a lithographic technique to form deterministic microstructures within a matrix of native collagen; elucidation of multistep process by which microstructures drive rapid invasion and vascularization; and demonstration of long range guidance of invasion through the full thickness of patterned templates. Experiments were performed in a murine wound model. These microstructured tissue templates (MTTs) could improve outcomes in reconstructive surgery and open paths to directed regeneration of spatially heterogeneous tissues or organs.

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.

Hydrogen Sulfide Protects Against Ischemia-Reperfusion Injury in an In Vitro Model of Cutaneous Tissue Transplantation

BACKGROUNDnIschemia-reperfusion injury (IRI) is a source of morbidity and mortality in many clinical scenarios, and has as one of its many consequences the induction of cellular apoptosis. Hydrogen sulfide (H2S) may decrease cellular metabolism in a reversible, nontoxic manner. An in vitro model of cutaneous tissue transplantation was developed to assess whether H2S could ameliorate cellular injury caused by IRI.nnnMETHODSnHuman umbilical vein endothelial cells (HUVECs) were treated with media containing NaHS (0, 10 microM, 100 microM, or 1 mM) and exposed to normoxia (21% oxygen), hypoxia (1%), or anoxia (0%). Cells were then returned to normoxia, and apoptosis was quantified using a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Fibroblasts (3T3s) were treated with H2S and exposed to anoxia in a similar fashion.nnnRESULTSnTreatment with H2S resulted in a significant decrease in apoptosis in HUVECs and 3T3s subjected to IRI. Toxicity of H2S was not observed, although the protective effect was less evident at higher doses.nnnCONCLUSIONnThis is the first study to examine H2S and the cellular components of cutaneous flaps in the setting of IRI. Our results demonstrate that H2S significantly decreases apoptosis in vitro in the setting of IRI. These data suggest H2S may mitigate IRI in vivo, and, therefore, has potential as a therapy for improving tissue survivability in clinical scenarios.

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

BACKGROUNDnRecent evidence suggests that hydrogen sulfide is capable of mitigating the degree of cellular damage associated with ischemia-reperfusion injury (IRI).nnnMETHODSnThis 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.nnnRESULTSnIn 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).nnnCONCLUSIONSnThese 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.

A portable high-intensity focused ultrasound device for noninvasive venous ablation

BACKGROUNDnVaricose veins and other vascular abnormalities are common clinical entities. Treatment options include vein stripping, sclerotherapy, and endovenous laser treatment, but all involve some degree of invasive intervention. The purpose of this study was to determine ex vivo the effectiveness of a novel hand-held, battery-operated, high-intensity focused ultrasound (HIFU) device for transcutaneous venous ablation.nnnMETHODSnThe ultrasound device is 14 x 9 x 4 cm, weighs 650 g, and is powered by 4 lithium ion battery packs. An ex vivo testing platform consisting of two different models comprised of sequentially layered skin-muscle-vein or skin-fat-vein was developed, and specimens were treated with HIFU. The tissues were then disassembled, imaged, and processed for histology. The luminal cross-sectional area of vein that had been treated with HIFU and nontreated controls were measured, and the values presented as median and interquartile range (IQR). The values were compared using a Wilcoxon rank-sum test, and statistical significance was set at P < .05.nnnRESULTSnOn gross and histologic examination, veins that had been treated with HIFU showed evidence of coagulation necrosis. The surface of the muscle in direct contact with the vein had a pinpoint area of coagulation, whereas the adjacent fat appeared undisturbed; the skin, fat, and the surface of the muscle in contact with the transducer remained completely unaffected. The cross-sectional area was 3.79 mm(2) (IQR, 3.38-4.22) of the control vein lumen and 0.16 mm(2) (IQR, 0.04-0.39) in those that had been treated with HIFU (P = .0304).nnnCONCLUSIONnThis inexpensive, portable HIFU device has the potential to allow clinicians to easily perform venous ablation in a manner that is entirely noninvasive and without the expense or inconvenience of large, complicated devices. This device represents a significant step forward in the development of new applications for HIFU technology.

A rapidly resorbable hemostatic biomaterial based on dihydroxyacetone.

We have developed a rapid acting, rapidly resorbable, non-toxic, topical hemostatic agent comprised of a PEGylated, polymerized sequence of dihydroxyacetone (MPEG-pDHA) that is highly effective in vivo. Twenty-eight Sprague-Dawley rats underwent left lateral hepatectomy. To the cut edge of the liver, rats received MPEG-pDHA (50 mg), normal saline (0.5 mL), or Instat (50 mg), a commercially available hemostatic compound. Bleeding time and total blood loss were quantified. Coagulation studies and scanning electron microscopy were performed on phlebotomized blood combined with MPEG-pDHA. Rats treated with MPEG-pDHA had significantly decreased bleeding time (97 s) and total blood loss (1.35 g) compared to normal saline (464 s and 3.83 g, p < 0.05 for each), and a significantly shorter bleeding time compared to Instat (165 s, p < 0.05). Histology confirmed that all MPEG-pDHA was metabolized within 3 weeks. The addition of MPEG-pDHA to whole blood did not significantly affect prothrombin time (12.0 s vs. 13.2 s, p = 0.130), partial thromboplastin time (27.0 s vs. 21.8 s, p = 0.118), or thrombin clotting time. MPEG-pDHA is an effective and rapidly resorbable hemostatic agent that may find broad hemostatic application in a wide range of surgical procedures.

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.

Lower Lid Deformity Secondary to Autogenous Fat Transfer: A Cautionary Tale

Although autogenous fat grafting is performed with increasing frequency, its safety around the periorbital area remains ill defined. This article presents the case of a young woman whose tear troughs were treated using autogenous fat transfer (AFT), with resultant bilateral lower lid deformities. Secondary correction of this aesthetic deformity caused by AFT to the tear trough proved extremely difficult and resulted in a temporary lower lid ectropion. After resolution of her ectropion, the patient was very satisfied with her postoperative appearance. The authors recommend a judicious approach to the correction of this deformity with AFT until objective outcome studies on this technique become more widely available.