Evaluation of focal adhesion mediated subcellular curvature sensing in response to engineered extracellular matrix.

Fibril curvature is bioinstructive to attached cells. Similar to natural healthy tissues, an engineered extracellular matrix can be designed to stimulate cells to adopt desired phenotypes. To take full advantage of the curvature control in biomaterial fabrication methodologies, an understanding of the response to fibril subcellular curvature is required. In this work, we examined morphology, signaling, and function of human cells attached to electrospun nanofibers. We controlled curvature across an order of magnitude using nondegradable poly(methyl methacrylate) (PMMA) attached to a stiff substrate with flat PMMA as a control. Focal adhesion length and the distance of maximum intensity from the geographic center of the vinculin positive focal adhesion both peaked at a fiber curvature of 2.5 µm-1 (both ∼2× the flat surface control). Vinculin experienced slightly less tension when attached to nanofiber substrates. Vinculin expression was also more affected by a subcellular curvature than structural proteins α-tubulin or α-actinin. Among the phosphorylation sites we examined (FAK397, 576/577, 925, and Src416), FAK925 exhibited the most dependance on the nanofiber curvature. A RhoA/ROCK dependance of migration velocity across curvatures combined with an observation of cell membrane wrapping around nanofibers suggested a hybrid of migration modes for cells attached to fibers as has been observed in 3D matrices. Careful selection of nanofiber curvature for regenerative engineering scaffolds and substrates used to study cell biology is required to maximize the potential of these techniques for scientific exploration and ultimately improvement of human health.

A Safe, Fibrosis-Mitigating, and Scalable Encapsulation Device Supports Long-Term Function of Insulin-Producing Cells.

Encapsulation and transplantation of insulin-producing cells offer a promising curative treatment for type 1 diabetes (T1D) without immunosuppression. However, biomaterials used to encapsulate cells often elicit foreign body responses, leading to cellular overgrowth and deposition of fibrotic tissue, which in turn diminishes mass transfer to and from transplanted cells. Meanwhile, the encapsulation device must be safe, scalable, and ideally retrievable to meet clinical requirements. Here, a durable and safe nanofibrous device coated with a thin and uniform, fibrosis-mitigating, zwitterionically modified alginate hydrogel for encapsulation of islets and stem cell-derived beta (SC-ß) cells is reported. The device with a configuration that has cells encapsulated within the cylindrical wall, allowing scale-up in both radial and longitudinal directions without sacrificing mass transfer, is designed. Due to its facile mass transfer and low level of fibrotic reactions, the device supports long-term cell engraftment, correcting diabetes in C57BL6/J mice with rat islets for up to 399 days and SCID-beige mice with human SC-ß cells for up to 238 days. The scalability and retrievability in dogs are further demonstrated. These results suggest the potential of this new device for cell therapies to treat T1D and other diseases.

Functional hydrogels for diabetic wound management.

Diabetic wounds often have a slow healing process and become easily infected owing to hyperglycemia in wound beds. Once planktonic bacterial cells develop into biofilms, the diabetic wound becomes more resistant to treatment. Although it remains challenging to accelerate healing in a diabetic wound due to complex pathology, including bacterial infection, high reactive oxygen species, chronic inflammation, and impaired angiogenesis, the development of multifunctional hydrogels is a promising strategy. Multiple functions, including antibacterial, pro-angiogenesis, and overall pro-healing, are high priorities. Here, design strategies, mechanisms of action, performance, and application of functional hydrogels are systematically discussed. The unique properties of hydrogels, including bactericidal and wound healing promotive effects, are reviewed. Considering the clinical need, stimuli-responsive and multifunctional hydrogels that can accelerate diabetic wound healing are likely to form an important part of future diabetic wound management.

A nanofibrous encapsulation device for safe delivery of insulin-producing cells to treat type 1 diabetes.

Transplantation of stem cell-derived ß (SC-ß) cells represents a promising therapy for type 1 diabetes (T1D). However, the delivery, maintenance, and retrieval of these cells remain a challenge. Here, we report the design of a safe and functional device composed of a highly porous, durable nanofibrous skin and an immunoprotective hydrogel core. The device consists of electrospun medical-grade thermoplastic silicone-polycarbonate-urethane and is soft but tough (~15 megapascal at a rupture strain of >2). Tuning the nanofiber size to less than ~500 nanometers prevented cell penetration while maintaining maximum mass transfer and decreased cellular overgrowth on blank (cell-free) devices to as low as a single-cell layer (~3 micrometers thick) when implanted in the peritoneal cavity of mice. We confirmed device safety, indicated as continuous containment of proliferative cells within the device for 5 months. Encapsulating syngeneic, allogeneic, or xenogeneic rodent islets within the device corrected chemically induced diabetes in mice and cells remained functional for up to 200 days. The function of human SC-ß cells was supported by the device, and it reversed diabetes within 1 week of implantation in immunodeficient and immunocompetent mice, for up to 120 and 60 days, respectively. We demonstrated the scalability and retrievability of the device in dogs and observed viable human SC-ß cells despite xenogeneic immune responses. The nanofibrous device design may therefore provide a translatable solution to the balance between safety and functionality in developing stem cell-based therapies for T1D.

Nanofiber curvature with Rho GTPase activity increases mouse embryonic fibroblast random migration velocity.

Mechanotransduction arises from information encoded in the shape of materials such as curvature. It induces activation of small GTPase signaling affecting cell phenotypes including differentiation. We carried out a set of preliminary experiments to test the hypothesis that curvature (1/radius) would also affect cell motility due to signal pathway crosstalk. High molecular weight poly (methyl methacrylate) straight nanofibers were electrospun with curvature ranging from 41 to 1 µm-1 and collected on a passivated glass substrate. The fiber curvature increased mouse mesenchymal stem cell aspect ratio (P < 0.02) and decreased cell area (P < 0.01). Despite little effect on some motility patterns such as polarity and persistence, we found selected fiber curvatures can increase normalized random fibroblastic mouse embryonic cell (MEF) migration velocity close to 2.5 times compared with a flat surface (P < 0.001). A maximum in the velocity curve occurred near 2.5 µm-1 and may vary with the time since initiation of attachment to the surface (range of 0-20 h). In the middle range of fiber curvatures, the relative relationship to curvature was similar regardless of treatment with Rho-kinase inhibitor (Y27632) or cdc42 inhibitor (ML141), although it was decreased on most curvatures (P < 0.05). However, below a critical curvature threshold MEFs may not be able to distinguish shallow curvature from a flat surface, while still being affected by contact guidance. The preliminary data in this manuscript suggested the large low curvature fibers were interpreted in a manner similar to a non-curved surface. Thus, curvature is a biomaterial construct design parameter that should be considered when specific biological responses are desired. Statement of integration, innovation, and insight  Replacement of damaged or diseased tissues that cannot otherwise regenerate is transforming modern medicine. However, the extent to which we can rationally design materials to affect cellular outcomes remains low. Knowing the effect of material stiffness and diameter on stem cell differentiation, we investigated cell migration and signaling on fibrous scaffolds. By investigating diameters across orders of magnitude (50-2000 nm), we identified a velocity maximum of ~800 nm. Furthermore, the results suggest large fibers may not be interpreted by single cells as a curved surface. This work presents insight into the design of constructs for engineering tissues.

Biomaterial Applications in Islet Encapsulation and Transplantation.

The worldwide prevalence of type 1 diabetes motivates the development of different treatment options for the disease. Current clinical treatments typically require patient involvement, often resulting in stress or inconvenience to the patient due to frequent blood glucose measurements and insulin injections or infusions. Islet transplantation, a potentially curative treatment, is limited by donor availability and the need for long-term administration of immunosuppressants. Cell encapsulation may prevent graft rejection without immunosuppression, however, foreign body responses, mass transfer limitations, scalability, and safety are all significant challenges. This Spotlight paper summarizes our recent efforts to address these challenges including developing biomaterials to mitigate foreign body responses and fibrosis, engineering scalable and retrievable encapsulation devices, as well as designing oxygen supplementation and vascularization strategies.

Engineering transferrable microvascular meshes for subcutaneous islet transplantation.

The success of engineered cell or tissue implants is dependent on vascular regeneration to meet adequate metabolic requirements. However, development of a broadly applicable strategy for stable and functional vascularization has remained challenging. We report here highly organized and resilient microvascular meshes fabricated through a controllable anchored self-assembly method. The microvascular meshes are scalable to centimeters, almost free of defects and transferrable to diverse substrates, ready for transplantation. They promote formation of functional blood vessels, with a density as high as ~220 vessels mm-2, in the poorly vascularized subcutaneous space of SCID-Beige mice. We further demonstrate the feasibility of fabricating microvascular meshes from human induced pluripotent stem cell-derived endothelial cells, opening a way to engineer patient-specific microvasculature. As a proof-of-concept for type 1 diabetes treatment, we combine microvascular meshes and subcutaneously transplanted rat islets and achieve correction of chemically induced diabetes in SCID-Beige mice for 3 months.

Nanofibers as Bioinstructive Scaffolds Capable of Modulating Differentiation through Mechanosensitive Pathways for Regenerative Engineering.

Bioinstructive scaffolds encode information in the physical shape and size of materials to direct cell responses. Electrospinning nanofibers is a process that offers control over scaffold architecture and fiber diameter, while providing extended linear length of fibers. This review summarizes tissue engineering literature that has utilized nanofiber scaffolds to direct stem cell differentiation for various tissues including musculoskeletal, vascular, immunological and nervous system tissues. Nanofibers are also considered for their extracellular matrix mimetic characteristics that can preserve stem cell differentiation capacity. These topics are considered in the context of focal adhesion and integrin signaling. Regenerative engineering will be enhanced by construction of scaffolds encoded with shape information to cause an attached cell to create the intended tissue at that region. Nanofibers are likely to be a bioinstructive scaffold in future regenerative engineering development as we pursue the Grand Challenges of engineering tissues.

Engineering the vasculature for islet transplantation.

The microvasculature in the pancreatic islet is highly specialized for glucose sensing and insulin secretion. Although pancreatic islet transplantation is a potentially life-changing treatment for patients with insulin-dependent diabetes, a lack of blood perfusion reduces viability and function of newly transplanted tissues. Functional vasculature around an implant is not only necessary for the supply of oxygen and nutrients but also required for rapid insulin release kinetics and removal of metabolic waste. Inadequate vascularization is particularly a challenge in islet encapsulation. Selectively permeable membranes increase the barrier to diffusion and often elicit a foreign body reaction including a fibrotic capsule that is not well vascularized. Therefore, approaches that aid in the rapid formation of a mature and robust vasculature in close proximity to the transplanted cells are crucial for successful islet transplantation or other cellular therapies. In this paper, we review various strategies to engineer vasculature for islet transplantation. We consider properties of materials (both synthetic and naturally derived), prevascularization, local release of proangiogenic factors, and co-transplantation of vascular cells that have all been harnessed to increase vasculature. We then discuss the various other challenges in engineering mature, long-term functional and clinically viable vasculature as well as some emerging technologies developed to address them. The benefits of physiological glucose control for patients and the healthcare system demand vigorous pursuit of solutions to cell transplant challenges. STATEMENT OF SIGNIFICANCE: Insulin-dependent diabetes affects more than 1.25 million people in the United States alone. Pancreatic islets secrete insulin and other endocrine hormones that control glucose to normal levels. During preparation for transplantation, the specialized islet blood vessel supply is lost. Furthermore, in the case of cell encapsulation, cells are protected within a device, further limiting delivery of nutrients and absorption of hormones. To overcome these issues, this review considers methods to rapidly vascularize sites and implants through material properties, pre-vascularization, delivery of growth factors, or co-transplantation of vessel supporting cells. Other challenges and emerging technologies are also discussed. Proper vascular growth is a significant component of successful islet transplantation, a treatment that can provide life-changing benefits to patients.

Nanotechnology in cell replacement therapies for type 1 diabetes.

Islet transplantation is a promising long-term, compliance-free, complication-preventing treatment for type 1 diabetes. However, islet transplantation is currently limited to a narrow set of patients due to the shortage of donor islets and side effects from immunosuppression. Encapsulating cells in an immunoisolating membrane can allow for their transplantation without the need for immunosuppression. Alternatively, "open" systems may improve islet health and function by allowing vascular ingrowth at clinically attractive sites. Many processes that enable graft success in both approaches occur at the nanoscale level-in this review we thus consider nanotechnology in cell replacement therapies for type 1 diabetes. A variety of biomaterial-based strategies at the nanometer range have emerged to promote immune-isolation or modulation, proangiogenic, or insulinotropic effects. Additionally, coating islets with nano-thin polymer films has burgeoned as an islet protection modality. Materials approaches that utilize nanoscale features manipulate biology at the molecular scale, offering unique solutions to the enduring challenges of islet transplantation.

Identification of Key Signaling Pathways Orchestrating Substrate Topography Directed Osteogenic Differentiation Through High-Throughput siRNA Screening.

Fibrous scaffolds are used for bone tissue engineering purposes with great success across a variety of polymers with different physical and chemical properties. It is now evident that the correct degree of curvature promotes increased cytoskeletal tension on osteoprogenitors leading to osteogenic differentiation. However, the mechanotransductive pathways involved in this phenomenon are not fully understood. To achieve a reproducible and specific cellular response, an increased mechanistic understanding of the molecular mechanisms driving the fibrous scaffold mediated bone regeneration must be understood. High throughput siRNA mediated screening technology has been utilized for dissecting molecular targets that are important in certain cellular phenotypes. In this study, we used siRNA mediated gene silencing to understand the osteogenic differentiation observed on fibrous scaffolds. A high-throughput siRNA screen was conducted using a library collection of 863 genes including important human kinase and phosphatase targets on pre-osteoblast SaOS-2 cells. The cells were grown on electrospun poly(methyl methacrylate) (PMMA) scaffolds with a diameter of 0.938 ± 0.304 µm and a flat surface control. The osteogenic transcription factor RUNX2 was quantified with an in-cell western (ICW) assay for the primary screen and significant targets were selected via two sample t-test. After selecting the significant targets, a secondary screen was performed to identify osteoinductive markers that also effect cell shape on fibrous topography. Finally, we report the most physiologically relevant molecular signaling mechanisms that are involved in growth factor free, fibrous topography mediated osteoinduction. We identified GTPases, membrane channel proteins, and microtubule associated targets that promote an osteoinductive cell shape on fibrous scaffolds.

Correction to: Mesenchymal Stem Cell Deformability and Implications for Microvascular Sequestration.

This article was updated to correct the spelling of author Brittany L. Banik's name.

Mesenchymal Stem Cell Deformability and Implications for Microvascular Sequestration.

Mesenchymal stem cells (MSCs) have received considerable attention in regenerative medicine, particularly in light of prospects for targeted delivery by intra-arterial injection. However, little is known about the mechanics of MSC sequestration in the microvasculature and the yield pressure (PY), above which MSCs will pass through microvessels of a given diameter. The objectives of the current study were to delineate the dependency of PY on cell size and the heterogeneity of cell mechanical properties and diameters (DCELL) of cultured MSCs. To this end the transient filtration test was employed to elucidate the mean filtration pressure (〈PY〉) for an ensemble of pores of a given size (DPORE) similar to in vivo microvessels. Cultured MSCs had a log-normal distribution of cell diameters (DCELL) with a mean of 15.8 ± 0.73 SD µm. MSC clearance from track-etched polycarbonate filters was studied for pore diameters of 7.3-15.4 µm. The pressure required to clear cells from filters with 30-85 × 103 pores rose exponentially with the ratio λ = DCELL/DPORE for 1.1 ≤ λ ≤ 2.2. The clearance of cells from each filter was characterized by a log-normal distribution in PY, with a mean filtration pressure of 0.02 ≤ ã€ˆPY〉 ≤ 6.7 cmH2O. For λ ≤ 1.56, the yield pressure (PY) was well represented by the cortical shell model of a cell with a viscous interior encapsulated by a shell under cortical tension τ0 = 0.99 ± 0.42 SD dyn/cm. For λ > 1.56, the 〈PY〉 characteristic of the cell population rose exponentially with λ. Analysis of the mean filtration pressure (〈PY〉) of each sample suggested that the larger diameter cells that skewed the distribution of DCELL contributed to about 20% of the mean filtration pressure. Further, if all cells had the same deformability (i.e., PY as a function of λ) as the average cell population, then ã€ˆPY〉 would have risen an order of magnitude above the average from fivefold at λ = 1.56 to 200-fold at λ = 2.1. Comparison of ã€ˆPY〉 to published microvascular pressures suggested that ã€ˆPY〉 may exceed microvessel pressure drops for λ exceeding 2.1, and rise 14-fold above capillary pressure drop at λ = 3 leading to 100% sequestration. However, due to the large variance of in vivo microvascular pressures entrapment of MSCs may be mitigated. Thus it is suggested that selecting fractions of the MSC population according to cell deformability may permit optimization of entrapment at sites targeted for tissue regeneration.

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. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 555-568, 2018.

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.

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.

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.

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.

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.