Daniel T. Bowers

Sphingosine 1-phosphate receptor 3 regulates recruitment of anti-inflammatory monocytes to microvessels during implant arteriogenesis

Endothelial cells play significant roles in conditioning tissues after injury by the production and secretion of angiocrine factors. At least two distinct subsets of monocytes, CD45+CD11b+Gr1+Ly6C+ inflammatory and CD45+CD11b+Gr1−Ly6C− anti-inflammatory monocytes, respond differentially to these angiocrine factors and promote pathogen/debris clearance and arteriogenesis/tissue regeneration, respectively. We demonstrate here that local sphingosine 1-phosphate receptor 3 (S1P3) agonism recruits anti-inflammatory monocytes to remodeling vessels. Poly(lactic-co-glycolic acid) thin films were used to deliver FTY720, an S1P1/3 agonist, to inflamed and ischemic tissues, which resulted in a reduction in proinflammatory cytokine secretion and an increase in regenerative cytokine secretion. The altered balance of cytokine secretion results in preferential recruitment of anti-inflammatory monocytes from circulation. The chemotaxis of these cells, which express more S1P3 than inflammatory monocytes, toward SDF-1α was also enhanced with FTY720 treatment, but not in S1P3 knockout cells. FTY720 delivery enhanced arteriolar diameter expansion and increased length density of the local vasculature. This work establishes a role for S1P receptor signaling in the local conditioning of tissues by angiocrine factors that preferentially recruit regenerative monocytes that can enhance healing outcomes, tissue regeneration, and biomaterial implant functionality.

The promotion of mandibular defect healing by the targeting of S1P receptors and the recruitment of alternatively activated macrophages.

Endogenous signals originating at the site of injury are involved in the paracrine recruitment, proliferation, and differentiation of circulating progenitor and diverse inflammatory cell types. Here, we investigate a strategy to exploit endogenous cell recruitment mechanisms to regenerate injured bone by local targeting and activation of sphingosine-1-phosphate (S1P) receptors. A mandibular defect model was selected for evaluating regeneration of bone following trauma or congenital disease. The particular challenges of mandibular reconstruction are inherent in the complex anatomy and function of the bone given that the area is highly vascularized and in close proximity to muscle. Nanofibers composed of poly(DL-lactide-co-glycolide) (PLAGA) and polycaprolactone (PCL) were used to delivery FTY720, a targeted agonist of S1P receptors 1 and 3. In vitro culture of bone progenitor cells on drug-loaded constructs significantly enhanced SDF1α mediated chemotaxis of bone marrow mononuclear cells. In vivo results show that local delivery of FTY720 from composite nanofibers enhanced blood vessel ingrowth and increased recruitment of M2 alternatively activated macrophages, leading to significant osseous tissue ingrowth into critical sized defects after 12 weeks of treatment. These results demonstrate that local activation of S1P receptors is a regenerative cue resulting in recruitment of wound healing or anti-inflammatory macrophages and bone healing. Use of such small molecule therapy can provide an alternative to biological factors for the clinical treatment of critical size craniofacial defects.

Spatiotemporal Oxygen Sensing Using Dual Emissive Boron Dye–Polylactide Nanofibers

Oxygenation in tissue scaffolds continues to be a limiting factor in regenerative medicine despite efforts to induce neovascularization or to use oxygen-generating materials. Unfortunately, many established methods to measure oxygen concentration, such as using electrodes, require mechanical disturbance of the tissue structure. To address the need for scaffold-based oxygen concentration monitoring, a single-component, self-referenced oxygen sensor was made into nanofibers. Electrospinning process parameters were tuned to produce a biomaterial scaffold with specific morphological features. The ratio of an oxygen sensitive phosphorescence signal to an oxygen insensitive fluorescence signal was calculated at each image pixel to determine an oxygenation value. A single component boron dye–polymer conjugate was chosen for additional investigation due to improved resistance to degradation in aqueous media compared to a boron dye polymer blend. Standardization curves show that in fully supplemented media, the fibers are responsive to dissolved oxygen concentrations less than 15 ppm. Spatial (millimeters) and temporal (minutes) ratiometric gradients were observed in vitro radiating outward from the center of a dense adherent cell grouping on scaffolds. Sensor activation in ischemia and cell transplant models in vivo show oxygenation decreases on the scale of minutes. The nanofiber construct offers a robust approach to biomaterial scaffold oxygen sensing.

FTY720-loaded poly(DL-lactide-co-glycolide) Electrospun Scaffold Significantly Increases Microvessel Density over 7 Days in Streptozotocin-Induced Diabetic C57b16/J Mice: Preliminary Results

BACKGROUND Nanofiber scaffolds could improve islet transplant success by physically mimicking the shape of extracellular matrix and by acting as a drug-delivery vehicle. Scaffolds implanted in alternate transplant sites must be prevascularized or very quickly vascularized following transplantation to prevent hypoxia-induced islet necrosis. The local release of the S1P prodrug FTY720 induces diameter enlargement and increases in length density. The objective of this preliminary study was to evaluate length and diameter differences between diabetic and nondiabetic animals implanted with FTY720-containing electrospun scaffolds using intravital imaging of dorsal skinfold window chambers. METHODS Electrospun mats of randomly oriented fibers we created from polymer solutions of PLAGA (50:50 LA:GA) with and without FTY720 loaded at a ratio of 1:200 (FTY720:PLAGA by wt). The implanted fiber mats were 4 mm in diameter and ∼0.2 mm thick. Increases in length density and vessel diameter were assessed by automated analysis of images over 7 days in RAVE, a Matlab program. RESULTS Image analysis of repeated measures of microvessel metrics demonstrated a significant increase in the length density from day 0 to day 7 in the moderately diabetic animals of this preliminary study (P < .05). Furthermore, significant differences in length density at day 0 and day 3 were found between recently STZ-induced moderately diabetic and nondiabetic animals in response to FTY720 local release (P < .05, Student t test). CONCLUSIONS Driving the islet revascularization process using local release of factors, such as FTY720, from biodegradable polymers makes an attractive system for the improvement of islet transplant success. Preliminary study results suggest that a recently induced moderately diabetic state may potentiate the mechanism by which local release of FTY720 from polymer fibers increases length density of microvessels. Therefore, local release of S1P receptor-targeted drugs is under further investigation for improvement of transplanted islet function.

Multilayer Cell-Seeded Polymer Nanofiber Constructs for Soft-Tissue Reconstruction

IMPORTANCE Cell seeding throughout the thickness of a nanofiber construct allows for patient-specific implant alternatives with long-lasting effects, earlier integration, and reduced inflammation when compared with traditional implants. Cell seeding may improve implant integration with host tissue; however, the effect of cell seeding on thick nanofiber constructs has not been studied. OBJECTIVE To use a novel cell-preseeded nanofiber tissue engineering technique to create a 3-dimensional biocompatible implant alternative to decellularized extracellular matrix. DESIGN Animal study with mammalian cell culture to study tissue engineered scaffolds. SETTING Academic research laboratory. PARTICIPANTS Thirty-six Sprague-Dawley rats. INTERVENTIONS The rats each received 4 implant types. The grafts included rat primary (enhanced green fluorescent protein-positive [eGFP+]) fibroblast-seeded polycaprolactone (PCL)/collagen nanofiber scaffold, PCL/collagen cell-free nanofiber scaffold, acellular human cadaveric dermis (AlloDerm), and acellular porcine dermis (ENDURAGen). Rats were monitored postoperatively and received enrofloxacin in the drinking water for 4 days prophylactically and buprenorphine (0.2-0.5 mg/kg administered subcutaneously twice a day postoperatively for pain for 48 hours). MAIN OUTCOMES AND MEASURES The viability of NIH/3T3 fibroblasts cultured on PCL electrospun nanofibers was evaluated using fluorescence microscopy. Soft-tissue remodeling was examined histologically and with novel ex vivo volume determinations of implants using micro-computed tomography of cell-seeded implants relative to nanofibers without cells and commonly used dermal grafts of porcine and human origin (ENDURAGen and AlloDerm, respectively). The fate and distribution of eGFP+ seeded donor fibroblasts were assessed using immunohistochemistry. RESULTS Fibroblasts migrated across nanofiber layers within 12 hours and remained viable on a single layer for up to 14 days. Scanning electron microscopy confirmed a nanoscale structure with a mean (SD) diameter of 158 (72) nm. Low extrusion rates demonstrated the excellent biocompatibility in vivo. Histological examination of the scaffolds demonstrated minimal inflammation. Cell seeding encouraged rapid vascularization of the nanofiber implants. Cells of donor origin (eGFP+) declined with the duration of implantation. Implant volume was not significantly affected for up to 8 weeks by the preseeding of cells (P > .05). CONCLUSIONS AND RELEVANCE Polymer nanofiber-based scaffolds mimic natural extracellular matrix. Preseeding the nanofiber construct with cells improved vascularization without notable effects on volume. An effect of cell preseeding on scaffold vascularization was evident beyond the presence of preseeded cells. This 3-dimensional, multilayer method of cell seeding throughout a 1-mm-thick construct is simple and feasible for clinical application. Further development of this technique may affect the clinical practice of facial plastic and reconstructive surgeons.

An engineered macroencapsulation membrane releasing FTY720 to precondition pancreatic islet transplantation

Macroencapsulation is a powerful approach to increase the efficiency of extrahepatic pancreatic islet transplant. FTY720, a small molecule that activates signaling through sphingosine-1-phosphate receptors, is immunomodulatory and pro-angiogenic upon sustained delivery from biomaterials. While FTY720 (fingolimod, Gilenya) has been explored for organ transplantation, in the present work the effect of locally released FTY720 from novel nanofiber-based macroencapsulation membranes is explored for islet transplantation. We screened islet viability during culture with FTY720 and various biodegradable polymers. Islet viability is significantly reduced by the addition of high doses (≥500 ng/mL) of soluble FTY720. Among the polymers screened, islets have the highest viability when cultured with poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). Therefore, PHBV was blended with polycaprolactone (PCL) for mechanical stability and electrospun into nanofibers. Islets had no detectable function ex vivo following 5 days or 12 h of subcutaneous implantation within our engineered device. Subsequently, we explored a preconditioning scheme in which islets are transplanted 2 weeks after FTY720-loaded nanofibers are implanted. This allows FTY720 to orchestrate a local regenerative milieu while preventing premature transplantation into avascular sites that contain high concentrations of FTY720. These results provide a foundation and motivation for further investigation into the use of FTY720 in preconditioning sites for efficacious islet transplantation.

Advances in Local Drug Release and Scaffolding Design to Enhance Cell Therapy for Diabetes.

Islet transplant is a curative treatment for insulin-dependent diabetes. However, challenges, including poor tissue survival and a lack of efficient engraftment, must be overcome. An encapsulating or scaffolding material can act as a vehicle for agents carefully chosen for the islet transplant application. From open porous scaffolds to spherical capsules and conformal coatings, greater immune protection is often accompanied by greater distances to microvasculature. Generating a local oxygen supply from the implant material or encouraging vessel growth through the release of local factors can create an oxygenated engraftment site. Intricately related to the vascularization response, inflammatory interaction with the cell supporting implant is a long-standing hurdle to material-based islet transplant. Modulation of the immune responses to the islets as well as the material itself must be considered. To match the post-transplant complexity, the release rate can be tuned to orchestrate temporal responses. Material degradation properties can be utilized in passive approaches or external stimuli and biological cues in active approaches. A combination of multiple carefully chosen factors delivered in an agent-specialized manner is considered by this review to improve the long-term function of islets transplanted in scaffolding and encapsulating materials.