Fan Yang

All Publications

• Photo-crosslinkable PEG-Based Microribbons for Forming 3D Macroporous Scaffolds with Decoupled Niche Properties ADVANCED MATERIALS Han, L., Tong, X., Yang, F. 2014; 26 (11): 1757-1762

  Abstract

  PEG-based microribbons are designed and fabricated as building blocks for constructing a 3D cell niche with independently tunable biochemical, mechanical, and topographical cues. This platform supports direct cell encapsulation, allows spatial patterning of biochemical cues, and may provide a valuable tool for facilitating the analyses of how interactive niche signaling regulates cell fate in three dimensions.

  View details for DOI 10.1002/adma.201304805

  View details for Web of Science ID 000332912100016

• Engineering interpenetrating network hydrogels as biomimetic cell niche with independently tunable biochemical and mechanical properties BIOMATERIALS Tong, X., Yang, F. 2014; 35 (6): 1807-1815

  Abstract

  Hydrogels have been widely used as artificial cell niche to mimic extracellular matrix with tunable properties. However, changing biochemical cues in hydrogels developed-to-date would often induce simultaneous changes in mechanical properties, which do not support mechanistic studies on stem cell-niche interactions. Here we report the development of a PEG-based interpenetrating network (IPN), which is composed of two polymer networks that can independently and simultaneously crosslink to form hydrogels in a cell-friendly manner. The resulting IPN hydrogel allows independently tunable biochemical and mechanical properties, as well as stable and more homogeneous presentation of biochemical ligands in 3D than currently available methods. We demonstrate the potential of our IPN platform for elucidating stem cell-niche interactions by modulating osteogenic differentiation of human adipose-derived stem cells. The versatility of such IPN hydrogels is further demonstrated using three distinct and widely used polymers to form the mechanical network while keeping the biochemical network constant.

  View details for DOI 10.1016/j.biomaterials.2013.11.064

  View details for Web of Science ID 000331018700004

• A Facile Method to Fabricate Hydrogels with Microchannel-Like Porosity for Tissue Engineering TISSUE ENGINEERING PART C-METHODS Hammer, J., Han, L., Tong, X., Yang, F. 2014; 20 (2): 169-176

  Abstract

  Hydrogels are widely used as three-dimensional (3D) tissue engineering scaffolds due to their tissue-like water content, as well as their tunable physical and chemical properties. Hydrogel-based scaffolds are generally associated with nanoscale porosity, whereas macroporosity is highly desirable to facilitate nutrient transfer, vascularization, cell proliferation and matrix deposition. Diverse techniques have been developed for introducing macroporosity into hydrogel-based scaffolds. However, most of these methods involve harsh fabrication conditions that are not cell friendly, result in spherical pore structure, and are not amenable for dynamic pore formation. Human tissues contain abundant microchannel-like structures, such as microvascular network and nerve bundles, yet fabricating hydrogels containing microchannel-like pore structures remains a great challenge. To overcome these limitations, here we aim to develop a facile, cell-friendly method for engineering hydrogels with microchannel-like porosity using stimuli-responsive microfibers as porogens. Microfibers with sizes ranging 150-200 μm were fabricated using a coaxial flow of alginate and calcium chloride solution. Microfibers containing human embryonic kidney (HEK) cells were encapsulated within a 3D gelatin hydrogel, and then exposed to ethylenediaminetetraacetic acid (EDTA) solution at varying doses and duration. Scanning electron microscopy confirmed effective dissolution of alginate microfibers after EDTA treatment, leaving well-defined, interconnected microchannel structures within the 3D hydrogels. Upon release from the alginate fibers, HEK cells showed high viability and enhanced colony formation along the luminal surfaces of the microchannels. In contrast, HEK cells in non-EDTA treated control exhibited isolated cells, which remained entrapped in alginate microfibers. Together, our results showed a facile, cell-friendly process for dynamic microchannel formation within hydrogels, which may simultaneously release cells in 3D hydrogels in a spatiotemporally controlled manner. This platform may be adapted to include other cell-friendly stimuli for porogen removal, such as Matrix metalloproteinase-sensitive peptides or photodegradable gels. While we used HEK cells in this study as proof of principle, the concept described in this study may also be used for releasing clinically relevant cell types, such as smooth muscle and endothelial cells that are useful for repairing tissues involving tubular structures.

  View details for DOI 10.1089/ten.tec.2013.0176

  View details for Web of Science ID 000330310700008

  View details for PubMedID 23745610

• Modulating polymer chemistry to enhance non-viral gene delivery inside hydrogels with tunable matrix stiffness BIOMATERIALS Keeney, M., Onyiah, S., Zhang, Z., Tong, X., Han, L., Yang, F. 2013; 34 (37): 9657-9665

  Abstract

  Non-viral gene delivery holds great promise for promoting tissue regeneration, and offers a potentially safer alternative than viral vectors. Great progress has been made to develop biodegradable polymeric vectors for non-viral gene delivery in 2D culture, which generally involves isolating and modifying cells in vitro, followed by subsequent transplantation in vivo. Scaffold-mediated gene delivery may eliminate the need for the multiple-step process in vitro, and allows sustained release of nucleic acids in situ. Hydrogels are widely used tissue engineering scaffolds given their tissue-like water content, injectability and tunable biochemical and biophysical properties. However, previous attempts on developing hydrogel-mediated non-viral gene delivery have generally resulted in low levels of transgene expression inside 3D hydrogels, and increasing hydrogel stiffness further decreased such transfection efficiency. Here we report the development of biodegradable polymeric vectors that led to efficient gene delivery inside poly(ethylene glycol) (PEG)-based hydrogels with tunable matrix stiffness. Photocrosslinkable gelatin was maintained constant in the hydrogel network to allow cell adhesion. We identified a lead biodegradable polymeric vector, E6, which resulted in increased polyplex stability, DNA protection and achieved sustained high levels of transgene expression inside 3D PEG-DMA hydrogels for at least 12 days. Furthermore, we demonstrated that E6-based polyplexes allowed efficient gene delivery inside hydrogels with tunable stiffness ranging from 2 to 175 kPa, with the peak transfection efficiency observed in hydrogels with intermediate stiffness (28 kPa). The reported hydrogel-mediated gene delivery platform using biodegradable polyplexes may serve as a local depot for sustained transgene expression in situ to enhance tissue engineering across broad tissue types.

  View details for DOI 10.1016/j.biomaterials.2013.08.050

  View details for Web of Science ID 000326901200042

  View details for PubMedID 24011715

• Mutant MCP-1 protein delivery from layer-by-layer coatings on orthopedic implants to modulate inflammatory response BIOMATERIALS Keeney, M., Waters, H., Barcay, K., Jiang, X., Yao, Z., Pajarinen, J., Egashira, K., Goodman, S. B., Yang, F. 2013; 34 (38): 10287-10295

  Abstract

  Total joint replacement (TJR) is a common and effective surgical procedure for hip or knee joint reconstruction. However, the production of wear particles is inevitable for all TJRs, which activates macrophages and initiates an inflammatory cascade often resulting in bone loss, prosthetic loosening and eventual TJR failure. Macrophage Chemoattractant Protein-1 (MCP-1) is one of the most potent cytokines responsible for macrophage cell recruitment, and previous studies suggest that mutant MCP-1 proteins such as 7ND may be used as a decoy drug to block the receptor and reduce inflammatory cell recruitment. Here we report the development of a biodegradable, layer-by-layer (LBL) coating platform that allows efficient loading and controlled release of 7ND proteins from the surface of orthopedic implants using as few as 14 layers. Scanning electron microscopy and fluorescence imaging confirmed effective coating using the LBL procedure on titanium rods. 7ND protein loading concentration and release kinetics can be modulated by varying the polyelectrolytes of choice, the polymer chemistry, the pH of the polyelectrolyte solution, and the degradation rate of the LBL assembly. The released 7ND from LBL coating retained its bioactivity and effectively reduced macrophage migration towards MCP-1. Finally, the LBL coating remained intact following a femoral rod implantation procedure as determined by immunostaining of the 7ND coating. The LBL platform reported herein may be applied for in situ controlled release of 7ND protein from orthopedic implants, to reduce wear particle-induced inflammatory responses in an effort to prolong the lifetime of implants.

  View details for DOI 10.1016/j.biomaterials.2013.09.028

  View details for Web of Science ID 000328094600054

• Development of Poly(beta-amino ester)-Based Biodegradable Nanoparticles for Nonviral Delivery of Minicircle DNA ACS NANO Keeney, M., Ong, S., Padilla, A., Yao, Z., Goodman, S., Wu, J. C., Yang, F. 2013; 7 (8): 7241-7250

  Abstract

  Gene therapy provides a powerful tool for regulating cellular processes and tissue repair. Minicircle (MC) DNA are supercoiled DNA molecules free of bacterial plasmid backbone elements and have been reported to enhance prolonged gene expression compared to conventional plasmids. Despite the great promise of MC DNA for gene therapy, methods for safe and efficient MC DNA delivery remain lacking. To overcome this bottleneck, here we report the development of a poly(β-amino ester) (PBAE)-based, biodegradable nanoparticulate platform for efficient delivery of MC DNA driven by a Ubc promoter in vitro and in vivo. By synthesizing and screening a small library of 18 PBAE polymers with different backbone and end-group chemistry, we identified lead cationic PBAE structures that can complex with minicircle DNA to form nanoparticles, and delivery efficiency can be further modulated by tuning PBAE chemistry. Using human embryonic kidney 293 cells and mouse embryonic fibroblasts as model cell types, we identified a few PBAE polymers that allow efficient MC delivery at levels that are comparable or even surpassing Lipofectamine 2000. The biodegradable nature of PBAE-based nanoparticles facilitates in vivo applications and clinical translation. When injected via intraperitoneal route in vivo, MC alone resulted in high transgene expression, and a lead PBAE/MC nanoparticle formulation achieved a further 2-fold increase in protein expression compared to MC alone. Together, our results highlight the promise of PBAE-based nanoparticles as promising nonviral gene carriers for MC delivery, which may provide a valuable tool for broad applications of MC DNA-based gene therapy.

  View details for DOI 10.1021/nn402657d

  View details for Web of Science ID 000323810600089

  View details for PubMedID 23837668

• Dynamic tissue engineering scaffolds with stimuli-responsive macroporosity formation BIOMATERIALS Han, L., Lai, J. H., Yu, S., Yang, F. 2013; 34 (17): 4251-4258

  Abstract

  Macropores in tissue engineering scaffolds provide space for vascularization, cell-proliferation and cellular interactions, and is crucial for successful tissue regeneration. Modulating the size and density of macropores may promote desirable cellular processes at different stages of tissue development. Most current techniques for fabricating macroporous scaffolds produce fixed macroporosity and do not allow the control of porosity during cell culture. Most macropore-forming techniques also involve non-physiological conditions, such that cells can only be seeded in a post-fabrication process, which often leads to low cell seeding efficiency and uneven cell distribution. Here we report a process to create dynamic hydrogels as tissue engineering scaffolds with tunable macroporosity using stimuli-responsive porogens of gelatin, alginate and hyaluronic acid, which degrade in response to specific stimuli including temperature, chelating and enzymatic digestion, respectively. SEM imaging confirmed sequential pore formation in response to sequential stimulations: 37 °C on day 0, EDTA on day 7, and hyaluronidase on day 14. Bovine chondrocytes were encapsulated in the Alg porogen, which served as cell-delivery vehicles, and changes in cell viability, proliferation and tissue formation during sequential stimuli treatments were evaluated. Our results showed effective cell release from Alg porogen with high cell viability and markedly increased cell proliferation and spreading throughout the 3D hydrogels. Dynamic pore formation also led to significantly enhanced type II and X collagen production by chondrocytes. This platform provides a valuable tool to create stimuli-responsive scaffolds with dynamic macroporosity for a broad range of tissue engineering applications, and may also be used for fundamental studies to examine cell responses to dynamic niche properties.

  View details for DOI 10.1016/j.biomaterials.2013.02.051

  View details for Web of Science ID 000317700400006

  View details for PubMedID 23489920

• The future of biologic coatings for orthopaedic implants BIOMATERIALS Goodman, S. B., Yao, Z., Keeney, M., Yang, F. 2013; 34 (13): 3174-3183

  Abstract

  Implants are widely used for orthopaedic applications such as fixing fractures, repairing non-unions, obtaining a joint arthrodesis, total joint arthroplasty, spinal reconstruction, and soft tissue anchorage. Previously, orthopaedic implants were designed simply as mechanical devices; the biological aspects of the implant were a byproduct of stable internal/external fixation of the device to the surrounding bone or soft tissue. More recently, biologic coatings have been incorporated into orthopaedic implants in order to modulate the surrounding biological environment. This opinion article reviews current and potential future use of biologic coatings for orthopaedic implants to facilitate osseointegration and mitigate possible adverse tissue responses including the foreign body reaction and implant infection. While many of these coatings are still in the preclinical testing stage, bioengineers, material scientists and surgeons continue to explore surface coatings as a means of improving clinical outcome of patients undergoing orthopaedic surgery.

  View details for DOI 10.1016/j.biomaterials.2013.01.074

  View details for Web of Science ID 000316770100003

  View details for PubMedID 23391496

• CD90 (Thy-1)-Positive Selection Enhances Osteogenic Capacity of Human Adipose-Derived Stromal Cells TISSUE ENGINEERING PART A Chung, M. T., Liu, C., Hyun, J. S., Lo, D. D., Montoro, D. T., Hasegawa, M., Li, S., Sorkin, M., Rennert, R., Keeney, M., Yang, F., Quarto, N., Longaker, M. T., Wan, D. C. 2013; 19 (7-8): 989-997

  Abstract

  Stem cell-based bone tissue engineering with adipose-derived stromal cells (ASCs) has shown great promise for revolutionizing treatment of large bone deficits. However, there is still a lack of consensus on cell surface markers identifying osteoprogenitors. Fluorescence-activated cell sorting has identified a subpopulation of CD105(low) cells with enhanced osteogenic differentiation. The purpose of the present study was to compare the ability of CD90 (Thy-1) to identify osteoprogenitors relative to CD(105).Unsorted cells, CD90(+), CD90(-), CD105(high), and CD105(low) cells were treated with an osteogenic differentiation medium. For evaluation of in vitro osteogenesis, alkaline phosphatase (ALP) staining and alizarin red staining were performed at 7 days and 14 days, respectively. RNA was harvested after 7 and 14 days of differentiation, and osteogenic gene expression was examined by quantitative real-time polymerase chain reaction. For evaluation of in vivo osteogenesis, critical-sized (4-mm) calvarial defects in nude mice were treated with the hydroxyapatite-poly(lactic-co-glycolic acid) scaffold seeded with the above-mentioned subpopulations. Healing was followed using micro-CT scans for 8 weeks. Calvaria were harvested at 8 weeks postoperatively, and sections were stained with Movat's Pentachrome.Transcriptional analysis revealed that the CD90(+) subpopulation was enriched for a more osteogenic subtype relative to the CD105(low) subpopulation. Staining at day 7 for ALP was greatest in the CD90(+) cells, followed by the CD105(low) cells. Staining at day 14 for alizarin red demonstrated the greatest amount of mineralized extracellular matrix in the CD90(+) cells, again followed by the CD105(low) cells. Quantification of in vivo healing at 2, 4, 6, and 8weeks postoperatively demonstrated increased bone formation in defects treated with CD90(+) ASCs relative to all other groups. On Movat's Pentachrome-stained sections, defects treated with CD90(+) cells showed the most robust bony regeneration. Defects treated with CD90(-) cells, CD105(high) cells, and CD105(low) cells demonstrated some bone formation, but to a lesser degree when compared with the CD90(+) group.While CD105(low) cells have previously been shown to possess an enhanced osteogenic potential, we found that CD90(+) cells are more capable of forming bone both in vitro and in vivo. These data therefore suggest that CD90 may be a more effective marker than CD105 to isolate a highly osteogenic subpopulation for bone tissue engineering.

  View details for DOI 10.1089/ten.tea.2012.0370

  View details for Web of Science ID 000315951500016

• Effects of Polymer End-Group Chemistry and Order of Deposition on Controlled Protein Delivery from Layer-by-Layer Assembly BIOMACROMOLECULES Keeney, M., Mathur, M., Cheng, E., Tong, X., Yang, F. 2013; 14 (3): 794-800

  Abstract

  Layer-by-layer (LBL) assembly is an attractive platform for controlled release of biologics given its mild fabrication process and versatility in coating substrates of any shape. Proteins can be incorporated into LBL coatings by sequentially depositing oppositely charged polyelectrolytes, which self-assemble into nanoscale films on medical devices or tissue engineering scaffolds. However, previously reported LBL platforms often require the use of a few hundred layers to avoid burst release, which hinders their broad translation due to the lengthy fabrication process, cost, and batch-to-batch variability. Here we report a biodegradable LBL platform composed of only 10 layers with tunable protein release kinetics, which is an order of magnitude less than previously reported LBL platforms. We performed a combinatorial study to examine the effects of polymer chemistry and order of deposition of poly(β-amino) esters on protein release kinetics under 81 LBL assembly conditions. Using the optimal "polyelectrolyte couples" for constructing the LBL film, basic fibroblast growth factor (bFGF) was released gradually over 14 days with retained biological activity to stimulate cell proliferation. The method reported herein is applicable for coating various substrates including metals, polymers, and ceramics and may be used for a broad range of biomedical and tissue engineering applications.

  View details for DOI 10.1021/bm3018559

  View details for Web of Science ID 000316044700024

  View details for PubMedID 23360295

• The effects of interactive mechanical and biochemical niche signaling on osteogenic differentiation of adipose-derived stem cells using combinatorial hydrogels ACTA BIOMATERIALIA Nii, M., Lai, J. H., Keeney, M., Han, L., Behn, A., Imanbayev, G., Yang, F. 2013; 9 (3): 5475-5483

  Abstract

  Stem cells reside in a multi-factorial environment containing biochemical and mechanical signals. Changing biochemical signals in most scaffolds often leads to simultaneous changes in mechanical properties, which makes it difficult to elucidate the complex interplay between niche cues. Combinatorial studies on cell-material interactions have emerged as a tool to facilitate analyses of stem cell responses to various niche cues, but most studies to date have been performed on two-dimensional environments. Here we developed three-dimensional combinatorial hydrogels with independent control of biochemical and mechanical properties to facilitate analysis of interactive biochemical and mechanical signaling on adipose-derived stem cell osteogenesis in three dimensions. Our results suggest that scaffold biochemical and mechanical signals synergize only at specific combinations to promote bone differentiation. Leading compositions were identified to have intermediate stiffness (∼55kPa) and low concentration of fibronectin (10μg ml(-1)), which led to an increase in osteocalcin gene expression of over 130-fold. Our results suggest that scaffolds with independently tunable niche cues could provide a powerful tool for conducting mechanistic studies to decipher how complex niche cues regulate stem cell fate in three dimensions, and facilitate rapid identification of optimal niche cues that promote desirable cellular processes or tissue regeneration.

  View details for DOI 10.1016/j.actbio.2012.11.002

  View details for Web of Science ID 000315536000007

• Paracrine Release from Nonviral Engineered Adipose-Derived Stem Cells Promotes Endothelial Cell Survival and Migration In Vitro STEM CELLS AND DEVELOPMENT Deveza, L., Choi, J., Imanbayev, G., Yang, F. 2013; 22 (3): 483-491

  Abstract

  Stem cells hold great potential for therapeutic angiogenesis due to their ability to directly contribute to new vessel formation or secrete paracrine signals. Adipose-derived stem cells (ADSCs) are a particularly attractive autologous cell source for therapeutic angiogenesis due to their ease of isolation and relative abundance. Gene therapy may be used to further enhance the therapeutic efficacy of ADSCs by overexpressing desired therapeutic factors. Here, we developed vascular endothelial growth factor (VEGF)-overexpressing ADSCs utilizing poly(β-amino esters) (PBAEs), a hydrolytically biodegradable polymer, and examined the effects of paracrine release from nonviral modified ADSCs on the angiogenic potential of human umbilical vein endothelial cells (HUVECs) in vitro. PBAE polymeric vectors delivered DNA into ADSCs with high efficiency and low cytotoxicity, leading to an over 3-fold increase in VEGF production by ADSCs compared with Lipofectamine 2000. Paracrine release from PBAE/VEGF-transfected ADSCs enhanced HUVEC viability and decreased HUVEC apoptosis under hypoxia. Further, paracrine release from PBAE/VEGF-transfected ADSCs significantly enhanced HUVEC migration and tube formation, two critical cellular processes for effective angiogenesis. Our results demonstrate that genetically engineered ADSCs using biodegradable polymeric nanoparticles may provide a promising autologous cell source for therapeutic angiogenesis in treating cardiovascular diseases.

  View details for DOI 10.1089/scd.2012.0201

  View details for Web of Science ID 000313677000012

• Adipose-derived Stromal Cells Overexpressing Vascular Endothelial Growth Factor Accelerate Mouse Excisional Wound Healing MOLECULAR THERAPY Nauta, A., Seidel, C., Deveza, L., Montoro, D., Grova, M., Ko, S. H., Hyun, J., Gurtner, G. C., Longaker, M. T., Yang, F. 2013; 21 (2): 445-455

  Abstract

  Angiogenesis is essential to wound repair, and vascular endothelial growth factor (VEGF) is a potent factor to stimulate angiogenesis. Here, we examine the potential of VEGF-overexpressing adipose-derived stromal cells (ASCs) for accelerating wound healing using nonviral, biodegradable polymeric vectors. Mouse ASCs were transfected with DNA plasmid encoding VEGF or green fluorescent protein (GFP) using biodegradable poly (β-amino) esters (PBAE). Cells transfected using Lipofectamine 2000, a commercially available transfection reagent, were included as controls. ASCs transfected using PBAEs showed enhanced transfection efficiency and 12-15-fold higher VEGF production compared with cells transfected using Lipofectamine 2000 (*P < 0.05). When transplanted into a mouse wild-type excisional wound model, VEGF-overexpressing ASCs led to significantly accelerated wound healing, with full wound closure observed at 8 days compared to 10-12 days in groups treated with ASCs alone or saline control (*P < 0.05). Histology and polarized microscopy showed increased collagen deposition and more mature collagen fibers in the dermis of wound beds treated using PBAE/VEGF-modified ASCs than ASCs alone. Our results demonstrate the efficacy of using nonviral-engineered ASCs to accelerate wound healing, which may provide an alternative therapy for treating many diseases in which wound healing is impaired.

  View details for DOI 10.1038/mt.2012.234

  View details for Web of Science ID 000314434600021

• Microribbon-Like Elastomers for Fabricating Macroporous and Highly Flexible Scaffolds that Support Cell Proliferation in 3D ADVANCED FUNCTIONAL MATERIALS Han, L., Yu, S., Wang, T., Behn, A. W., Yang, F. 2013; 23 (3): 346-358

  View details for DOI 10.1002/adfm.201201212

  View details for Web of Science ID 000313691200011

• The Effects of Polymer End-group Chemistry and Order of Deposition on Controlled Protein Delivery from Layer-by-layer Assembly Biomacromolecules Keeney M, Mathur M, Cheng E, Yang F 2013; 14 (3): 794-800
• Stem cells catalyze cartilage formation by neonatal articular chondrocytes in 3D biomimetic hydrogels. Scientific reports Lai, J. H., Kajiyama, G., Smith, R. L., Maloney, W., Yang, F. 2013; 3: 3553-?

  Abstract

  Cartilage loss is a leading cause of disability among adults and effective therapy remains elusive. Neonatal chondrocytes (NChons) are an attractive allogeneic cell source for cartilage repair, but their clinical translation has been hindered by scarce donor availability. Here we examine the potential for catalyzing cartilage tissue formation using a minimal number of NChons by co-culturing them with adipose-derived stem cells (ADSCs) in 3D hydrogels. Using three different co-culture models, we demonstrated that the effects of co-culture on cartilage tissue formation are dependent on the intercellular distance and cell distribution in 3D. Unexpectedly, increasing ADSC ratio in mixed co-culture led to increased synergy between NChons and ADSCs, and resulted in the formation of large neocartilage nodules. This work raises the potential of utilizing stem cells to catalyze tissue formation by neonatal chondrocytes via paracrine signaling, and highlights the importance of controlling cell distribution in 3D matrices to achieve optimal synergy.

  View details for DOI 10.1038/srep03553

  View details for PubMedID 24352100

• Therapeutic angiogenesis using genetically engineered human endothelial cells JOURNAL OF CONTROLLED RELEASE Cho, S., Yang, F., Son, S. M., Park, H., Green, J. J., Bogatyrev, S., Mei, Y., Park, S., Langer, R., Anderson, D. G. 2012; 160 (3): 515-524

  Abstract

  Cell therapy holds promise as a method for the treatment of ischemic disease. However, one significant challenge to the efficacy of cell therapy is poor cell survival in vivo. Here we describe a non-viral, gene therapy approach to improve the survival and engraftment of cells transplanted into ischemic tissue. We have developed biodegradable poly(?-amino esters) (PBAE) nanoparticles as vehicles to genetically modify human umbilical vein endothelial cells (HUVECs) with vascular endothelial growth factor (VEGF). VEGF transfection using these nanoparticles significantly enhanced VEGF expression in HUVECs, compared with a commercially-available transfection reagent. Transfection resulted in the upregulation of survival factors, and improved viability under simulated ischemic conditions. In a mouse model of hindlimb ischemia, VEGF nanoparticle transfection promoted engraftment of HUVECs into mouse vasculature as well as survival of transplanted HUVECs in ischemic tissues, leading to improved angiogenesis and ischemic limb salvage. This study demonstrates that biodegradable polymer nanoparticles may provide a safe and effective method for genetic engineering of endothelial cells to enhance therapeutic angiogenesis.

  View details for DOI 10.1016/j.jconrel.2012.03.006

  View details for Web of Science ID 000305789300013

  View details for PubMedID 22450331

• Tissue Engineering: Focus on musculoskeletal system Biomaterials Science-an integrated clinical and engineering approach Keeney M, Han LH, Onyiah S, Yang F 2012
• Nanomaterials for Engineering Cell Microenvironment and Gene delivery Tissue Engineering and Regenerative Medicine: A Nano Approach. CRC Press. Lai JH, Ramasubranian A, Jeeawoody S, Yang F 2012
• Nonviral delivery of genetic medicine for therapeutic angiogenesis ADVANCED DRUG DELIVERY REVIEWS Park, H., Yang, F., Cho, S. 2012; 64 (1): 40-52

  Abstract

  Genetic medicines that induce angiogenesis represent a promising strategy for the treatment of ischemic diseases. Many types of nonviral delivery systems have been tested as therapeutic angiogenesis agents. However, their delivery efficiency, and consequently therapeutic efficacy, remains to be further improved, as few of these technologies are being used in clinical applications. This article reviews the diverse nonviral gene delivery approaches that have been applied to the field of therapeutic angiogenesis, including plasmids, cationic polymers/lipids, scaffolds, and stem cells. This article also reviews clinical trials employing nonviral gene therapy and discusses the limitations of current technologies. Finally, this article proposes a future strategy to efficiently develop delivery vehicles that might be feasible for clinically relevant nonviral gene therapy, such as high-throughput screening of combinatorial libraries of biomaterials.

  View details for DOI 10.1016/j.addr.2011.09.005

  View details for Web of Science ID 000302843300006

  View details for PubMedID 21971337

• Therapeutic Angiogenesis for Treating Cardiovascular Diseases THERANOSTICS Deveza, L., Choi, J., Yang, F. 2012; 2 (8): 801-814

  Abstract

  Cardiovascular disease is the leading cause of death worldwide and is often associated with partial or full occlusion of the blood vessel network in the affected organs. Restoring blood supply is critical for the successful treatment of cardiovascular diseases. Therapeutic angiogenesis provides a valuable tool for treating cardiovascular diseases by stimulating the growth of new blood vessels from pre-existing vessels. In this review, we discuss strategies developed for therapeutic angiogenesis using single or combinations of biological signals, cells and polymeric biomaterials. Compared to direct delivery of growth factors or cells alone, polymeric biomaterials provide a three-dimensional drug-releasing depot that is capable of facilitating temporally and spatially controlled release. Biomimetic signals can also be incorporated into polymeric scaffolds to allow environmentally-responsive or cell-triggered release of biological signals for targeted angiogenesis. Recent progress in exploiting genetically engineered stem cells and endogenous cell homing mechanisms for therapeutic angiogenesis is also discussed.

  View details for DOI 10.7150/thno.4419

  View details for Web of Science ID 000307648500006

  View details for PubMedID 22916079

• Non-viral Delivery of Inductive and Suppressive Genes to Adipose-Derived Stem Cells for Osteogenic Differentiation PHARMACEUTICAL RESEARCH Ramasubramanian, A., Shiigi, S., Lee, G. K., Yang, F. 2011; 28 (6): 1328-1337

  Abstract

  To assess the effects of co-delivering osteoinductive DNA and/or small interfering RNA in directing the osteogenic differentiation of human adipose-derived stem cells (hADSCs) using a combinatorial, non-viral gene delivery approach.hADSCs were transfected using combinations of the following genes: BMP2, siGNAS and siNoggin using poly(?-amino esters) or lipid-like molecules. A total of 15 groups were evaluated by varying DNA doses, timing of treatment, and combinations of signals. All groups were cultured in osteogenic medium for up to 37 days, and outcomes were measured using gene expression, biochemical assays, and histology.Biomaterials-mediated gene delivery led to a dose-dependent up-regulation of BMP2 and significant gene silencing of GNAS and Noggin in hADSCs. BMP2 alone slightly up-regulates osteogenic marker expression in hADSCs. In contrast, co-delivery of BMP2 and siGNAS or siNoggin significantly accelerates the hADSC differentiation towards osteogenic differentiation, with marked increase in bone marker expression and mineralization.We report a combinatorial platform for identifying synergistic interactions among multiple genetic signals associated with osteogenic differentiation of hADSCs. Our results suggest that inductive or suppressive genetic switches interact in a complex manner, and highlight the promise of combinatorial approaches towards rapidly identifying optimal signals for promoting desired stem cell differentiation.

  View details for DOI 10.1007/s11095-011-0406-9

  View details for Web of Science ID 000290804000009

  View details for PubMedID 21424160

• Preparation of Mineralized Nanofibers: Collagen Fibrils Containing Calcium Phosphate NANO LETTERS Maas, M., Guo, P., Keeney, M., Yang, F., Hsu, T. M., Fuller, G. G., Martin, C. R., Zare, R. N. 2011; 11 (3): 1383-1388

  Abstract

  We report a straightforward, bottom-up, scalable process for preparing mineralized nanofibers. Our procedure is based on flowing feed solution, containing both inorganic cations and polymeric molecules, through a nanoporous membrane into a receiver solution with anions, which leads to the formation of mineralized nanofibers at the exit of the pores. With this strategy, we were able to achieve size control of the nanofiber diameters. We illustrate this approach by producing collagen fibrils with calcium phosphate incorporated inside the fibrils. This structure, which resembles the basic constituent of bones, assembles itself without the addition of noncollagenous proteins or their polymeric substitutes. Rheological experiments demonstrated that the stiffness of gels derived from these fibrils is enhanced by mineralization. Growth experiments of human adipose derived stem cells on these gels showed the compatibility of the fibrils in a tissue-regeneration context.

  View details for DOI 10.1021/nl200116d

  View details for Web of Science ID 000288061500082

  View details for PubMedID 21280646

• Recent Progress in Cartilage Tissue Engineering Curr Opin Biotechnol Keeney M, Lai J, Yang F 2011; 22 (5): 734-740
• Combinatorial Extracellular Matrices for Human Embryonic Stem Cell Differentiation in 3D BIOMACROMOLECULES Yang, F., Cho, S., Son, S. M., Hudson, S. P., Bogatyrev, S., Keung, L., Kohane, D. S., Langer, R., Anderson, D. G. 2010; 11 (8): 1909-1914

  Abstract

  Embryonic stem cells (ESCs) are promising cell sources for tissue engineering and regenerative medicine. Scaffolds for ESC-based tissue regeneration should provide not only structural support, but also signals capable of supporting appropriate cell differentiation and tissue development. Extracellular matrix (ECM) is a key component of the stem cell niche in vivo and can influence stem cell fate via mediating cell attachment and migration, presenting chemical and physical cues, as well as binding soluble factors. Here we investigated the effects of combinatorial extracellular matrix proteins on controlled human ESC (hESC) differentiation. Varying ECM compositions in 3D markedly affects cell behavior, and optimal compositions of ECM hydrogels are identified that facilitate specific-lineage differentiation of stem cells. To our knowledge, this is the first combinatorial analysis of ECM hydrogels for their effects on hESC differentiation in 3D. The 3D matrices described herein may provide a useful platform for studying the interactive ECM signaling in influencing stem cell differentiation.

  View details for DOI 10.1021/bm100357t

  View details for Web of Science ID 000280583400002

  View details for PubMedID 20614932

• Genetic Engineering of Human Stem Cells for Enhanced Angiogenesis Using Biodegradable Polymeric Nanoparticles. Proceedings of the National Academy of Sciences Yang F, Cho SW, Son SM, Bogatyrev S, Singh D, Green JJ, Mei Y, Park S, Bhang SH, Kim BS, Langer R, Anderson DG 2010; 107 (8): 3317-22
• High-throughput Optimization of Stem Cell Microenvrionments Combinatorial Chemistry & High Throughput Screening Yang F, Mei Y, Langer R, Anderson DG 2009; 12 (6): 544-553
• Gene Delivery to Human Adult and Embryonic cell-derived Stem Cells Using Biodegradable Nanoparticulate Polymeric Vectors Gene Therapy Yang F, Green JJ, Dinio T, Keung L, Cho SW, Park H, Langer R, Anderson DG 2009; 16 (4): 533-546
• Lipid-like Nanoparticles for Small Interfering RNA Delivery to Endothelial Cells. Advanced Functional Materials Cho SW, Goldberg M, Son SM, Xu Q, Yang F, Mei Y, Bogatyrev S, Langer R, Anderson DG 2009; 19 (19): 3112-3118
• Small Molecule End Group of Linear Polymer Determines Cell-type Gene Delivery Efficacy Advanced Materials Sunshine J, Green JJ, Mahon K, Yang F, Langer R, Anderson DG 2009; 21: 1-5
• The study of abnormal bone development in the Apert syndrome Fgfr2(+/S252W) mouse using a 3D hydrogel culture model BONE Yang, F., Wang, Y., Zhang, Z., Hsu, B., Jabs, E. W., Elisseeff, J. H. 2008; 43 (1): 55-63

  Abstract

  Apert syndrome is caused by mutations in fibroblast growth factor receptor 2 (Fgfr2) and is characterized by craniosynostosis and other skeletal abnormalities. The Apert syndrome Fgfr2+/S252W mouse model exhibits perinatal lethality. A 3D hydrogel culture model, derived from tissue engineering strategies, was used to extend the study of the effect of the Fgfr2+/S252W mutation in differentiating osteoblasts postnatally. We isolated cells from the long bones of Apert Fgfr2+/S252W mice (n=6) and cells from the wild-type sibling mice (n=6) to be used as controls. During monolayer expansion, Fgfr2+/S252W cells demonstrated increased proliferation and ALP activity, as well as altered responses of these cellular functions in the presence of FGF ligands with different binding specificity (FGF2 or FGF10). To better mimic the in vivo disease development scenario, cells were also encapsulated in 3D hydrogels and their phenotype in 3D in vitro culture was compared to that of in vivo tissue specimens. After 4 weeks in 3D culture in osteogenic medium, Fgfr2+/S252W cells expressed 2.8-fold more collagen type I and 3.3-fold more osteocalcin than did wild-type controls (p<0.01). Meanwhile, Fgfr2+/S252W cells showed decreased bone matrix remodeling and expressed 87% less Metalloprotease-13 and 71% less Noggin (p<0.01). The S252W mutation also led to significantly higher production of collagen type I and II in 3D as shown by immunofluorescence staining. In situ hybridization and alizarin red S staining of postnatal day 0 (P0) mouse limb sections demonstrated significantly higher levels of osteopontin expression and mineralization in Fgfr2+/S252W mice. Complementary to in vivo findings, this 3D hydrogel culture system provides an effective in vitro venue to study the pathogenesis of Apert syndrome caused by the analogous mutation in humans.

  View details for DOI 10.1016/j.bone.2008.02.008

  View details for Web of Science ID 000257151100008

  View details for PubMedID 18407821

• Delivery of Small Interfering RNA for Inhibition of Endothelial Cell Apoptosis by Hypoxia and Serum Deprivation Biochemical and Biophysical Communications Cho SW, Hartle L, Son SM, Yang F, Goldberg M, Xu Q, Langer R, Anderson DG 2008; 376 (1): 158-163
• Tissue Engineering: The Therapeutic Strategy of the 21st Century Nanotechnology and Tissue Engineering Yang F, Neeley WL, Moore MJ, Karp JM, Shukla A, Langer R 2008
• Abnormal Tissue Development of Osteoblasts from an Apert Syndrome FGFR2+/S252W Mouse Model in 3D Hydrogels Bone Yang F, Wang YL, Zhang Z, Hsu B, Jabs EW, Elisseeff JH 2008; 43 (1): 55-63
• Metabolic changes in mesenchymal stem cells in osteogenic medium measured by autofluorescence spectroscopy STEM CELLS Reyes, J. M., Fermanian, S., Yang, F., Zhou, S., Herretes, S., Murphy, D. B., Elisseeff, J. H., Chuck, R. S. 2006; 24 (5): 1213-1217

  Abstract

  The purpose of this study was to measure metabolic changes in mesenchymal stem cells (MSCs) placed in osteogenic medium by autofluorescence spectroscopy. MSCs were plated in stem cell-supporting or osteogenic medium and imaged. Shift from the basic growth environment to the inductive osteogenic environment was confirmed by reverse transcription-polymerase chain reaction. Reduced pyridine nucleotides were detected by exciting near 366 nm and measuring fluorescence at 450 nm, and oxidized flavoproteins were detected by exciting at 460 nm and measuring fluorescence at 540 nm. The ratio of these fluorescence measurements, reduction-oxidation (redox) fluorometry, is a noninvasive measure of the cellular metabolic state. The detected pyridine nucleotide to flavoprotein ratio decreased upon transitioning from the stem cell to the differentiated state, as well as with increasing cell density and cell-cell contact. MSC metabolism increased upon placement in differentiating medium and with increasing cell density and contact. Redox fluorometry is a feasible, noninvasive technique for distinguishing MSCs from further differentiated cells.

  View details for DOI 10.1634/stemcells.2004-0324

  View details for Web of Science ID 000240639200009

  View details for PubMedID 16439616

• Cartilage Tissue Engineering Biomedical Engineering Handbook, Tissue Engineering Section Yang F, Elisseeff JH 2006
• The effect of incorporating RGD adhesive peptide in polyethylene glycol diacrylate hydrogel on osteogenesis of bone marrow stromal cells BIOMATERIALS Yang, F., Williams, C. G., Wang, D. A., LEE, H., Manson, P. N., Elisseeff, J. 2005; 26 (30): 5991-5998

  Abstract

  Advances in tissue engineering require biofunctional scaffolds that can not only provide cells with structural support, but also interact with cells in a biological manner. To achieve this goal, a frequently used cell adhesion peptide Arg-Gly-Asp (RGD) was covalently incorporated into poly(ethylene glycol) diacrylate (PEODA) hydrogel and its dosage effect (0.025, 1.25 and 2.5 mm) on osteogenesis of marrow stromal cells in a three-dimensional environment was examined. Expression of bone-related markers, osteocalcin (OCN) and Alkaline phosphatase (ALP), increased significantly as the RGD concentration increased. Compared with no RGD, 2.5 mm RGD group showed a 1344% increase in ALP production and a 277% increase in OCN accumulation in the medium. RGD helped MSCs maintain cbfa-1 expression when shifted from a two-dimensional environment to a three-dimensional environment. Soluble RGD was found to completely block the mineralization of marrow stromal cells, as manifested by quantitative calcium assay, phosphorus elemental analysis and Von Kossa staining. In conclusion, we have demonstrated that RGD-conjugated PEODA hydrogel promotes the osteogenesis of MSCs in a dosage-dependent manner, with 2.5 mm being optimal concentration.

  View details for DOI 10.1016/j.biomaterials.2005.03.018

  View details for Web of Science ID 000230538700008

  View details for PubMedID 15878198

• Abnormalities in cartilage and bone development in the Apert syndrome FGFR2(+/S252W) mouse DEVELOPMENT Wang, Y. L., Xiao, R., Yang, F., Karim, B. O., Iacovelli, A. J., Cai, J. L., Lerner, C. P., Richtsmeier, J. T., Leszl, J. M., Hill, C. A., Yu, K., Ornitz, D. M., Elisseeff, J., Huso, D. L., Jabs, E. W. 2005; 132 (15): 3537-3548

  Abstract

  Apert syndrome is an autosomal dominant disorder characterized by malformations of the skull, limbs and viscera. Two-thirds of affected individuals have a S252W mutation in fibroblast growth factor receptor 2 (FGFR2). To study the pathogenesis of this condition, we generated a knock-in mouse model with this mutation. The Fgfr2(+/S252W) mutant mice have abnormalities of the skeleton, as well as of other organs including the brain, thymus, lungs, heart and intestines. In the mutant neurocranium, we found a midline sutural defect and craniosynostosis with abnormal osteoblastic proliferation and differentiation. We noted ectopic cartilage at the midline sagittal suture, and cartilage abnormalities in the basicranium, nasal turbinates and trachea. In addition, from the mutant long bones, in vitro cell cultures grown in osteogenic medium revealed chondrocytes, which were absent in the controls. Our results suggest that altered cartilage and bone development play a significant role in the pathogenesis of the Apert syndrome phenotype.

  View details for DOI 10.1242/dev.01914

  View details for Web of Science ID 000231627800019

  View details for PubMedID 15975938

• Advances in skeletal tissue engineering with hydrogels. Orthodontics & craniofacial research Elisseeff, J., Puleo, C., Yang, F., SHARMA, B. 2005; 8 (3): 150-161

  Abstract

  Tissue engineering has the potential to make a significant impact on improving tissue repair in the craniofacial system. The general strategy for tissue engineering includes seeding cells on a biomaterial scaffold. The number of scaffold and cell choices for tissue engineering systems is continually increasing and will be reviewed.Multilayered hydrogel systems were developed to coculture different cell types and develop osteochondral tissues for applications including the temporomandibular joint.Hydrogels are one form of scaffold that can be applied to cartilage and bone repair using fully differentiated cells, adult and embryonic stem cells.Case studies represent an overview of our laboratory's investigations.Bilayered scaffolds to promote tissue development and the formation of more complex osteochondral tissues were developed and proved to be effective.Tissue engineering provides a venue to investigate tissue development of mutant or diseased cells and potential therapeutics.

  View details for PubMedID 16022717

• Bioresponsive phosphoester hydrogels for bone tissue engineering TISSUE ENGINEERING Wang, D. A., Williams, C. G., Yang, F., Cher, N., LEE, H., Elisseeff, J. H. 2005; 11 (1-2): 201-213

  Abstract

  Bioresponsive and intelligent biomaterials are a vehicle for manipulating cell function to promote tissue development and/or tissue engineering. A photopolymerized hydrogel based on a phosphoester- poly(ethylene glycol) polymer (PhosPEG) was synthesized for application to marrow-derived mesenchymal stem cell (MSC) encapsulation and tissue engineering of bone. The phosphor-containing hydrogels were hydrolytically degradable and the rate of degradation increased in the presence of a bone-derived enzyme, alkaline phosphatase. Gene expression and protein analysis of encapsulated MSCs demonstrated that PhosPEG-PEG cogels containing an intermediate concentration of phosphorus promoted the gene expression of bone-specific markers including type I collagen, alkaline phosphatase, and osteonectin, without the addition of growth factors or other biological agents, compared with pure poly(ethylene glycol)-based gels. Secretion of alkaline phosphatase, osteocalcin, and osteonectin protein was also increased in the PhosPEG cogels. Mineralization of gels increased in the presence of phosphorus in both cellular and acellular constructs compared with PEG gels. In summary, phosphate-PEG-derived hydrogels increase gene expression of bone-specific markers, secretion of bone-related matrix, and mineralization and may have a potential impact on bone-engineering therapies.

  View details for Web of Science ID 000227513600019

  View details for PubMedID 15738675

• Enhancing the tissue-biomaterial interface: Tissue-initiated integration of biomaterials ADVANCED FUNCTIONAL MATERIALS Wang, D. A., Williams, C. G., Yang, F., Elisseeff, J. H. 2004; 14 (12): 1152-1159

  View details for DOI 10.1002/adfm.200305018

  View details for Web of Science ID 000226103500002