Evaluating the polymerization effectiveness and biocompatibility of bio-sourced, visible light-based photoinitiator systems.

The use of photopolymerization is expanding across a multitude of biomedical applications, from drug delivery to bioprinting. Many of these current and emerging photopolymerization systems employ visible light, as motivated by safety and energy efficiency considerations. However, the "library" of visible light initiators is limited compared with the wealth of options available for UV polymerization. Furthermore, the synthesis of traditional photoinitiators relies on diminishing raw materials, and several traditional photoinitiators are considered emerging environmental contaminants. As such, there has been recent focus on identifying and characterizing biologically sourced, visible light-based photoinitiator systems that can be effectively used in photopolymerization applications. In this regard, several bio-sourced molecules have been shown to act as photoinitiators, primarily through Type II photoinitiation mechanisms. However, whether bio-sourced molecules can also act as effective synergists in these reactions remains unknown. In this study, we evaluated the effectiveness of bio-sourced synergist candidates, with a focus on amino acids, due to their amine functional groups, in combination with two bio-sourced photoinitiator molecules: riboflavin and curcumin. We tested the effectiveness of these photoinitiator systems under both violet (405 nm) and blue (460-475 nm) light using photo-rheology. We found that several synergist candidates, namely lysine, arginine, and histidine, increased the polymerization effectiveness of riboflavin when used with both violet and blue light. With curcumin, we found that almost all tested synergist candidates slightly decreased the polymerization effectiveness compared with curcumin alone under both light sources. These results show that bio-sourced molecules have the potential to be used as synergists with bio-sourced photoinitiators in visible light photopolymerization. However, more work must be done to fully characterize these reactions and to investigate more synergist candidates. Ultimately, this information is expected to expand the range of available visible light-based photoinitiator systems and increase their sustainability.

Influence of Substrate Stiffness on iPSC-Derived Retinal Pigmented Epithelial Cells.

Retinal degenerative diseases are a major cause of blindness involving the dysfunction of photoreceptors, retinal pigmented epithelium (RPE), or both. A promising treatment approach involves replacing these cells via surgical transplantation, and previous work has shown that cell delivery scaffolds are vital to ensure sufficient cell survival. Thus, identifying scaffold properties that are conducive to cell viability and maturation (such as suitable material and mechanical properties) is critical to ensuring a successful treatment approach. In this study, we investigated the effect of scaffold stiffness on human RPE attachment, survival, and differentiation, comparing immortalized (ARPE-19) and stem cell-derived RPE (iRPE) cells. Polydimethylsiloxane was used as a model polymer substrate, and varying stiffness (~12 to 800 kPa) was achieved by modulating the cross-link-to-base ratio. Post-attachment changes in gene and protein expression were assessed using qPCR and immunocytochemistry. We found that while ARPE-19 and iRPE exhibited significant differences in morphology and expression of RPE markers, substrate stiffness did not have a substantial impact on cell growth or maturation for either cell type. These results highlight the differences in expression between immortalized and iPSC-derived RPE cells, and also suggest that stiffnesses in this range (~12-800 kPa) may not result in significant differences in RPE growth and maturation, an important consideration in scaffold design.

Photopolymerization Parameters Influence Mechanical, Microstructural, and Cell Loading Properties of Rapidly Fabricated Cell Scaffolds.

Engineered scaffolds are commonly used to assist in cellular transplantations, providing crucial support and specific architecture for a variety of tissue engineering applications. Photopolymerization as a fabrication technique for cell scaffolds enables precise spatial and temporal control of properties and structure. One simple technique to achieve a two-dimensional structure is the use of a patterned photomask, which results in regionally selective photo-cross-linking. However, the relationships between photopolymerization parameters like light intensity and exposure time and outcomes like structural fidelity and mechanical properties are not well-established. In this work, we used photopolymerization to generate degradable polycaprolactone triacrylate (PCLTA) scaffolds with a defined microstructure. We examined the impact of light intensity and exposure time on scaffold properties such as shear modulus and micropore structure. To assess feasibility in a specific application and determine the relationship between parameter-driven properties and cell loading, we cultured retinal progenitor cells on the PCLTA scaffolds. We found that light intensity and polymerization time directly impact the scaffold stiffness and micropore structure, which in turn influenced the cell loading capacity of the scaffold. Because material stiffness and topography are known to impact cell viability and fate, understanding the effect of scaffold fabrication parameters on mechanical and structural properties is critical to optimizing cell scaffolds for specific applications.

Biocompatibility of Human Induced Pluripotent Stem Cell-Derived Retinal Progenitor Cell Grafts in Immunocompromised Rats.

Loss of photoreceptor cells is a primary feature of inherited retinal degenerative disorders including age-related macular degeneration and retinitis pigmentosa. To restore vision in affected patients, photoreceptor cell replacement will be required. The ideal donor cells for this application are induced pluripotent stem cells (iPSCs) because they can be derived from and transplanted into the same patient obviating the need for long-term immunosuppression. A major limitation for retinal cell replacement therapy is donor cell loss associated with simple methods of cell delivery such as subretinal injections of bolus cell suspensions. Transplantation with supportive biomaterials can help maintain cellular integrity, increase cell survival, and encourage proper cellular alignment and improve integration with the host retina. Using a pig model of retinal degeneration, we recently demonstrated that polycaprolactone (PCL) scaffolds fabricated with two photon lithography have excellent local and systemic tolerability. In this study, we describe rapid photopolymerization-mediated production of PCL-based bioabsorbable scaffolds, a technique for loading iPSC-derived retinal progenitor cells onto the scaffold, methods of surgical transplantation in an immunocompromised rat model and tolerability of the subretinal grafts at 1, 3, and 6 months of follow-up (n = 150). We observed no local or systemic toxicity, nor did we observe any tumor formation despite extensive clinical evaluation, clinical chemistry, hematology, gross tissue examination and detailed histopathology. Demonstrating the local and systemic compatibility of biodegradable scaffolds carrying human iPSC-derived retinal progenitor cells is an important step toward clinical safety trials of this approach in humans.

Full-Thickness Oral Mucoperiosteal Defects: Challenges and Opportunities.

Regenerative engineering strategies for the oral mucoperiosteum, as may be needed following surgeries, such as cleft palate repair and tumor resection, are underdeveloped compared with those for maxillofacial bone. However, critical-size tissue defects left to heal by secondary intention can lead to complications, such as infection, fistula formation, scarring, and midface hypoplasia. This review describes current clinical practice for replacing mucoperiosteal tissue, including autografts and allografts. Potentially paradigm-shifting experimental regenerative engineering strategies for mucoperiosteal wound healing, such as hybrid grafts and engineered matrices, are also discussed. Throughout the review, the advantages and disadvantages of each replacement or regeneration strategy are outlined in the context of clinical outcomes, quality of life for the patient, availability of materials, and cost of care. Finally, future directions for research and development in the area of mucoperiosteum repair are proposed, with an emphasis on identifying globally available and affordable solutions for promoting mucoperiosteal regeneration. Impact statement Unassisted oral mucoperiosteal wound healing can lead to severe complications such as infection, fistulae, scarring, and developmental abnormalities. Thus, strategies for promoting wound healing must be considered when mucoperiosteal defects are incident to oral surgery, as in palatoplasty or tumor resection. Emerging mucoperiosteal tissue engineering strategies, described in this study, have the potential to overcome the limitations of current standard-of-care donor tissue grafts. For example, the use of engineered mucoperiosteal biomaterials could circumvent concerns about tissue availability and immunogenicity. Moreover, employment of tissue engineering strategies may improve the equity of oral wound care by increasing global affordability and accessibility of materials.

The effect of retinal scaffold modulus on performance during surgical handling.

Emerging treatment strategies for retinal degeneration involve replacing lost photoreceptors using supportive scaffolds to ensure cells survive the implantation process. While many design aspects of these scaffolds, including material chemistry and microstructural cues, have been studied in depth, a full set of design constraints has yet to be established. For example, while known to be important in other tissues and systems, the influence of mechanical properties on surgical handling has not been quantified. In this study, photocrosslinked poly(ethylene glycol) dimethacrylate (PEGDMA) was used as a model polymer to study the effects of scaffold modulus (stiffness) on surgical handling, independent of material chemistry. This was achieved by modulating the molecular weight and concentrations of the PEGDMA in various prepolymer solutions. Scaffold modulus of each formulation was measured using photo-rheology, which enabled the collection of real-time polymerization data. In addition to measuring scaffold mechanical properties, this approach gave insight on polymerization kinetics, which were used to determine the polymerization time required for each sample. Scaffold handling characteristics were qualitatively evaluated using both in vitro and ex vivo trials that mimicked the surgical procedure. In these trials, scaffolds with shear moduli above 35 kPa performed satisfactorily, while those below this limit performed poorly. In other words, scaffolds below this modulus were too fragile for reliable transplantation. To better compare these results with literature values, the compressive modulus was measured for select samples, with the lower shear modulus limit corresponding to roughly 115 kPa compressive modulus. While an upper mechanical property limit was not readily apparent from these results, there was increased variability in surgical handling performance in samples with shear moduli above 800 kPa. Overall, the knowledge presented here provides important groundwork for future studies designed to examine additional retinal scaffold considerations, including the effect of scaffold mechanical properties on retinal progenitor cell fate.

Development of High-Resolution Three-Dimensional-Printed Extracellular Matrix Scaffolds and Their Compatibility with Pluripotent Stem Cells and Early Retinal Cells.

Purpose: Widely used approaches for retinal disease modeling and in vitro therapeutic testing can be augmented by using tissue-engineered scaffolds with a precise 3-dimensional structure. However, the materials currently used for these scaffolds are poorly matched to the biochemical and mechanical properties of the in vivo retina. Here, we create biopolymer-based scaffolds with a structure that is amenable to retinal tissue engineering and modeling. Methods: Optimal two-photon polymerization (TPP) settings, including laser power and scanning speed, are identified for 4 methacrylated biopolymer formulations: collagen, gelatin, hyaluronic acid (HA), and a 50/50 mixture of gelatin/HA, each with methylene blue as a photoinitiator. For select formulations, fabrication accuracy and swelling are determined and biocompatibility is evaluated by using human induced pluripotent stem cells and rat postnatal retinal cells. Results: TPP is feasible for each biopolymer formulation, but it is the most reliable for mixtures containing gelatin and the least reliable for HA alone. The mean size of microscaffold pores is within several microns of the intended value but the overall structure size is several times greater than the modeled volume. The addition of HA to gelatin scaffolds increases cell viability and promotes neuronal phenotype, including Tuj-1 expression and characteristic morphology. Conclusion: We successfully determined a useful range of TPP settings for 4 methacrylated biopolymer formulations. When crosslinked, these extracellular matrix-derived molecules support the growth and attachment of retinal cells. We anticipate that when combined with existing patient-specific approaches, this technique will enable more efficient and accurate retinal disease modeling and therapeutic testing in vitro than current techniques allow.

Helper-Dependent Adenovirus Transduces the Human and Rat Retina but Elicits an Inflammatory Reaction When Delivered Subretinally in Rats.

The identification of >100 genes causing inherited retinal degeneration and the promising results of recent gene augmentation trials have led to an increase in the number of studies investigating the preclinical efficacy of viral-mediated gene transfer. Despite success using adeno-associated viruses, many disease-causing genes, such as ABCA4 or USH2A, are too large to fit into these vectors. One option for large gene delivery is the family of integration-deficient helper-dependent adenoviruses (HDAds), which efficiently transduce postmitotic neurons. However, HDAds have been shown in other organ systems to elicit an immune response, and the immunogenicity of HDAds in the retina has not been characterized. In this study, HDAd serotype 5 (HDAd5) was found to successfully transduce rod and cone photoreceptors in ex vivo human retinal organ cultures. The ocular inflammatory response to subretinal injection of the HDAd5 was evaluated using a rat model. Subretinal injection of HDAd5 carrying cytomegalovirus promoter-driven enhanced green fluorescent protein (HDAd5-CMVp-eGFP) elicited a robust inflammatory response by 3 days postinjection. This reaction included vitreous infiltration of ionized calcium-binding adapter molecule 1 (Iba1)-positive monocytes and increased expression of the proinflammatory protein, intercellular adhesion molecule 1 (ICAM-1). By 7 days postinjection, most Iba1-positive infiltrates migrated into the neural retina and ICAM-1 expression was significantly increased compared with buffer-injected control eyes. At 14 days postinjection, Iba1-positive cells persisted in the retinas of HDAd5-injected eyes, and there was thinning of the outer nuclear layer. Subretinal injection of an empty HDAd5 virus was used to confirm that the inflammatory response was in response to the HDAd5 vector and not due to eGFP-induced overexpression cytotoxicity. Subretinal injection of lower doses of HDAd5 dampened the inflammatory response, but also eGFP expression. Despite their larger carrying capacity, further work is needed to elucidate the inflammatory pathways involved and to identify an immunomodulation paradigm sufficient for safe and effective transfer of large genes to the retina using HDAd5.

Two-photon polymerized poly(caprolactone) retinal cell delivery scaffolds and their systemic and retinal biocompatibility.

Cell replacement therapies are often enhanced by utilizing polymer scaffolds to improve retention or direct cell orientation and migration. Obstacles to refinement of such polymer scaffolds often include challenges in controlling the microstructure of biocompatible molecules in three dimensions at cellular scales. Two-photon polymerization of acrylated poly(caprolactone) (PCL) could offer a means of achieving precise microstructural control of a material in a biocompatible platform. In this work, we studied the effect of various formulation and two-photon polymerization parameters on minimum laser power needed to achieve polymerization, resolution, and fidelity to a target 3D model designed to be used for retinal cell replacement. Overall, we found that increasing the concentration of crosslink-able groups decreased polymerization threshold and the size of resolvable features while increasing fidelity of the scaffold to the 3D model. In general, this improvement was achieved by increasing the number of acrylate groups per prepolymer molecule, increasing the acrylated PCL concentration, or decreasing its molecular weight. Resulting two-photon polymerized PCL scaffolds successfully supported human iPSC derived retinal progenitor cells in vitro. Sub-retinal implantation of cell free scaffolds in a porcine model of retinitis pigmentosa did not cause inflammation, infection or local or systemic toxicity after one month. In addition, comprehensive ISO 10993 testing of photopolymerized scaffolds revealed a favorable biocompatibility profile. These results represent an important step towards understanding how two-photon polymerization can be applied to a wide range of biologically compatible chemistries for various biomedical applications. STATEMENT OF SIGNIFICANCE: Inherited retinal degenerative blindness results from the death of light sensing photoreceptor cells. To restore high-acuity vision a photoreceptor cell replacement strategy will likely be necessary. Unfortunately, single cell injection typically results in poor cell survival and integration post-transplantation. Polymeric biomaterial cell delivery scaffolds can be used to promote donor cell viability, control cellular polarity and increase packing density. A challenge faced in this endeavor has been developing methods suitable for generating scaffolds that can be used to deliver stem cell derived photoreceptors in an ordered columnar orientation (i.e., similar to that of the native retina). In this study we combined the biomaterial poly(caprolactone) with two-photon lithography to generate a biocompatible, clinically relevant scaffold suitable for retina cell delivery.

Two-Photon Polymerization as a Tool for Studying 3D Printed Topography-Induced Stem Cell Fate.

Geometric topographies are known to influence cellular differentiation toward specific phenotypes, but to date the range of features and type of substrates that can be easily fabricated to study these interactions is somewhat limited. In this study, an emerging technology, two-photon polymerization, is used to print topological patterns with varying feature-size and thereby study their effect on cellular differentiation. This technique offers rapid manufacturing of topographical surfaces with good feature resolution for shapes smaller than 3 µm. Human-induced pluripotent stem cells, when attached to these substrates or a non-patterned control for 1 week, express an array of genetic markers that suggest their differentiation toward a heterogeneous population of multipotent progenitors from all three germ layers. Compared to the topographically smooth control, small features (1.6 µm) encourage differentiation toward ectoderm while large features (8 µm) inhibit self-renewal. This study demonstrates the potential of using two-photon polymerization to study and control stem cell fate as a function of substrate interactions. The ability to tailor and strategically design biomaterials in this way can enable more precise and efficient generation or maintenance of desired phenotypes in vitro and in vivo.

Controlled drug delivery from 3D printed two-photon polymerized poly(ethylene glycol) dimethacrylate devices.

Controlled drug delivery systems have been utilized to enhance the therapeutic effects of many drugs by delivering drugs in a time-dependent and sustained manner. Here, with the aid of 3D printing technology, drug delivery devices were fabricated and tested using a model drug (fluorophore: rhodamine B). Poly(ethylene glycol) dimethacrylate (PEGDMA) devices were fabricated using a two-photon polymerization (TPP) system and rhodamine B was homogenously entrapped inside the polymer matrix during photopolymerization. These devices were printed with varying porosity and morphology using varying printing parameters such as slicing and hatching distance. The effects of these variables on drug release kinetics were determined by evaluating device fluorescence over the course of one week. These PEGDMA-based structures were then investigated for toxicity and biocompatibility in vitro, where MTS assays were performed using a range of cell types including induced pluripotent stem cells (iPSCs). Overall, tuning the hatching distance, slicing distance, and pore size of the fabricated devices modulated the rhodamine B release profile, in each case presumably due to resulting changes in the motility of the small molecule and its access to structure edges. In general, increased spacing provided higher drug release while smaller spacing resulted in some occlusion, preventing media infiltration and thus resulting in reduced fluorophore release. The devices had no cytotoxic effects on human embryonic kidney cells (HEK293), bone marrow stromal stem cells (BMSCs) or iPSCs. Thus, we have demonstrated the utility of two-photon polymerization to create biocompatible, complex miniature devices with fine details and tunable release of a model drug.

Effect of Molecular Weight and Functionality on Acrylated Poly(caprolactone) for Stereolithography and Biomedical Applications.

Degradable polymers are integral components in many biomedical polymer applications. The ability of these materials to decompose in situ has become a critical component for tissue engineering, allowing scaffolds to guide cell and tissue growth while facilitating gradual regeneration of native tissue. The objective of this work is to understand the role of prepolymer molecular weight and functionality of photocurable poly(caprolactone) (PCL) in determining reaction kinetics, mechanical properties, polymer degradation, biocompatibility, and suitability for stereolithography. PCL, a degradable polymer used in a number of biomedical applications, was functionalized with acrylate groups to enable photopolymerization and three-dimensional printing via stereolithography. PCL prepolymers with different molecular weights and functionalities were studied to understand the role of molecular structure in reaction kinetics, mechanical properties, and degradation rates. The mechanical properties of photocured PCL were dependent on cross-link density and directly related to the molecular weight and functionality of the prepolymers. High-molecular weight, low-functionality PCLDA prepolymers exhibited a lower modulus and a higher strain at break, while low-molecular weight, high-functionality PCLTA prepolymers exhibited a lower strain at break and a higher modulus. Additionally, degradation profiles of cross-linked PCL followed a similar trend, with low cross-link density leading to degradation times up to 2.5 times shorter than those of more highly cross-linked polymers. Furthermore, photopolymerized PCL showed biocompatibility both in vitro and in vivo, causing no observed detrimental effects on seeded murine-induced pluripotent stem cells or when implanted into pig retinas. Finally, the ability to create three-dimensional PCL structures is shown by fabrication of simple structures using digital light projection stereolithography. Low-molecular weight, high-functionality PCLTA prepolymers printed objects with feature sizes near the hardware resolution limit of 50 µm. This work lays the foundation for future work in fabricating microscale PCL structures for a wide range of tissue regeneration applications.

Feeder-free differentiation of cells exhibiting characteristics of corneal endothelium from human induced pluripotent stem cells.

The purpose of this study was to devise a strategy for the derivation of corneal endothelial cells (CEnCs) from adult fibroblast-derived induced pluripotent stem cells (iPSCs). IPSCs were generated from an adult human with normal ocular history via expression of OCT4, SOX2, KLF4 and c-MYC Neural crest cells (NCCs) were differentiated from iPSCs via addition of CHIR99021 and SB4315542. NCCs were driven toward a CEnC fate via addition of B27, PDGF-BB and DKK-2 to CEnC media. Differentiation of NCCs and CEnCs was evaluated via rt-PCR, morphological and immunocytochemical analysis. At 17 days post-NCC induction, there were notable changes in cell morphology and upregulation of the neural crest lineage transcripts PAX3, SOX9, TFAP2A, SOX10 and p75NTR and the proteins p75/NGFR and SOX10. Exposure of NCCs to B27, PDGF-BB and DKK-2 induced a shift in morphology from a spindle-shaped neural phenotype to a tightly-packed hexagonal appearance and increased expression of the transcripts ATP1A1, COL8A1, COL8A2, AQP1 and CDH2 and the proteins ZO-1, N-Cad, AQP-1 and Na+/K+ATPase. Replacement of NCC media with CEnC media on day 3, 5 or 8 reduced the differentiation time needed to yield CEnCs. IPSC-derived CEnCs could be used for evaluation of cornea endothelial disease pathophysiology and for testing of novel therapeutics.

CRISPR-Cas9 genome engineering: Treating inherited retinal degeneration.

Gene correction is a valuable strategy for treating inherited retinal degenerative diseases, a major cause of irreversible blindness worldwide. Single gene defects cause the majority of these retinal dystrophies. Gene augmentation holds great promise if delivered early in the course of the disease, however, many patients carry mutations in genes too large to be packaged into adeno-associated viral vectors and some, when overexpressed via heterologous promoters, induce retinal toxicity. In addition to the aforementioned challenges, some patients have sustained significant photoreceptor cell loss at the time of diagnosis, rendering gene replacement therapy insufficient to treat the disease. These patients will require cell replacement to restore useful vision. Fortunately, the advent of induced pluripotent stem cell and CRISPR-Cas9 gene editing technologies affords researchers and clinicians a powerful means by which to develop strategies to treat patients with inherited retinal dystrophies. In this review we will discuss the current developments in CRISPR-Cas9 gene editing in vivo in animal models and in vitro in patient-derived cells to study and treat inherited retinal degenerative diseases.

CRISPR-Cas9-Mediated Correction of the 1.02 kb Common Deletion in CLN3 in Induced Pluripotent Stem Cells from Patients with Batten Disease.

Juvenile neuronal ceroid lipofuscinosis (Batten disease) is a rare progressive neurodegenerative disorder caused by mutations in CLN3. Patients present with early-onset retinal degeneration, followed by epilepsy, progressive motor deficits, cognitive decline, and premature death. Approximately 85% of individuals with Batten disease harbor at least one allele containing a 1.02 kb genomic deletion spanning exons 7 and 8. This study demonstrates CRISPR-Cas9-based homology-dependent repair of this mutation in induced pluripotent stem cells generated from two independent patients: one homozygous and one compound heterozygous for the 1.02 kb deletion. Our strategy included delivery of a construct that carried >3 kb of DNA: wild-type CLN3 sequence and a LoxP-flanked, puromycin resistance cassette for positive selection. This strategy resulted in correction at the genomic DNA and mRNA levels in the two independent patient lines. These CRISPR-corrected isogenic cell lines will be a valuable tool for disease modeling and autologous retinal cell replacement.

Connective Tissue Growth Factor Promotes Efficient Generation of Human Induced Pluripotent Stem Cell-Derived Choroidal Endothelium.

Age-related macular degeneration (AMD) is a leading cause of irreversible blindness in the Western world. Although, the majority of stem cell research to date has focused on production of retinal pigment epithelial (RPE) and photoreceptor cells for the purpose of evaluating disease pathophysiology and cell replacement, there is strong evidence that the choroidal endothelial cells (CECs) that form the choriocapillaris vessels are the first to be lost in this disease. As such, to accurately evaluate disease pathophysiology and develop an effective treatment, production of patient-specific, stem cell-derived CECs will be required. In this study, we report for the first time a stepwise differentiation protocol suitable for generating human iPSC-derived CEC-like cells. RNA-seq analysis of the monkey CEC line, RF/6A, combined with two statistical screens allowed us to develop media comprised of various protein combinations. In both screens, connective tissue growth factor (CTGF) was identified as the key component required for driving CEC development. A second factor tumor necrosis factor (TNF)-related weak inducer of apoptosis receptor was also found to promote iPSC to CEC differentiation by inducing endogenous CTGF secretion. CTGF-driven iPSC-derived CEC-like cells formed capillary tube-like vascular networks, and expressed the EC-specific markers CD31, ICAM1, PLVAP, vWF, and the CEC-restricted marker CA4. In combination with RPE and photoreceptor cells, patient-specific iPSC derived CEC-like cells will enable scientists to accurately evaluate AMD pathophysiology and develop effective cell replacement therapies. Stem Cells Translational Medicine 2017;6:1533-1546.

Preparation and evaluation of human choroid extracellular matrix scaffolds for the study of cell replacement strategies.

Endothelial cells (ECs) of the choriocapillaris are one of the first cell types lost during age-related macular degeneration (AMD), and cell replacement therapy is currently a very promising option for patients with advanced AMD. We sought to develop a reliable method for the production of human choroidal extracellular matrix (ECM) scaffolds, which will allow for the study of choroidal EC (CEC) replacement strategies in an environment that closely resembles the native tissue. Human RPE/choroid tissue was treated sequentially with Triton X-100, SDS, and DNase to remove all native cells. While all cells were successfully removed from the tissue, collagen IV, elastin, and laminin remained, with preserved architecture of the acellular vascular tubes. The ECM scaffolds were then co-cultured with exogenous ECs to determine if the tissue can support cell growth and allow EC reintegration into the decellularized choroidal vasculature. Both monkey and human ECs took up residence in the choriocapillary tubes of the decellularized tissue. Together, these data suggest that our decellularization methods are sufficient to remove all cellular material yet gentle enough to preserve tissue structure and allow for the optimization of cell replacement strategies. STATEMENT OF SIGNIFICANCE: Age-related macular degeneration (AMD) is a devastating disease affecting more than 600 million people worldwide. Endothelial cells of the choriocapillaris (CECs) are among the first cell types lost in early AMD, and cell replacement therapy is currently the most promising option for restoring vision in patients with advanced AMD. In order to study CEC replacement strategies we have generated a 3D choroid scaffold using a novel decellularization method in human RPE/choroid tissue. To our knowledge, this is the first report describing decellularization of human RPE/choroid, as well as recellularization of a choroid scaffold with CECs. This work will aid in our development and optimization of cell replacement strategies using a tissue scaffold that is similar to the in vivo environment.

Two-photon polymerization for production of human iPSC-derived retinal cell grafts.

Recent advances in induced pluripotent stem cell (iPSC) technology have paved the way for the production of patient-specific neurons that are ideal for autologous cell replacement for treatment of neurodegenerative diseases. In the case of retinal degeneration and associated photoreceptor cell therapy, polymer scaffolds are critical for cellular survival and integration; however, prior attempts to materialize this concept have been unsuccessful in part due to the materials' inability to guide cell alignment. In this work, we used two-photon polymerization to create 180µm wide non-degradable prototype photoreceptor scaffolds with varying pore sizes, slicing distances, hatching distances and hatching types. Hatching distance and hatching type were significant factors for the error of vertical pore diameter, while slicing distance and hatching type most affected the integrity and geometry of horizontal pores. We optimized printing parameters in terms of structural integrity and printing time in order to create 1mm wide scaffolds for cell loading studies. We fabricated these larger structures directly on a porous membrane with 3µm diameter pores and seeded them with human iPSC-derived retinal progenitor cells. After two days in culture, cells nested in and extended neuronal processes parallel to the vertical pores of the scaffolds, with maximum cell loading occurring in 25µm diameter pores. These results highlight the feasibility of using this technique as part of an autologous stem cell strategy for restoring vision to patients affected with retinal degenerative diseases. STATEMENT OF SIGNIFICANCE: Cell replacement therapy is an important goal for investigators aiming to restore neural function to those suffering from neurodegenerative disease. Cell delivery scaffolds are frequently necessary for the success of such treatments, but traditional biomaterials often fail to facilitate the neuronal orientation and close packing needed to recapitulate the in vivo environment. Here, we use two-photon polymerization to create prototype cell scaffolds with densely packed vertical pores for photoreceptor cell loading and small, interconnected horizontal pores for nutrient diffusion. This study offers a thorough characterization of how two-photon polymerization parameters affect final structural outcomes and printing time. Our findings demonstrate the feasibility of using two-photon polymerization to create scaffolds that can align neuronal cells in 3D and are large enough to be used for transplantation. In future work, these scaffolds could comprise biodegradable materials with tunable microstructure, elastic modulus and degradation time; a significant step towards a promising treatment option for those suffering from late-stage neurodegeneration, including retinal degenerative blindness.

Neuronal Differentiation of Induced Pluripotent Stem Cells on Surfactant Templated Chitosan Hydrogels.

The development of effective tissue engineering materials requires careful consideration of several properties beyond biocompatibility, including permeability and mechanical stiffness. While surfactant templating has been used for over a decade to control the physical properties of photopolymer materials, the potential benefit of this technique with regard to biomaterials has yet to be fully explored. Herein we demonstrate that surfactant templating can be used to tune the water uptake and compressive modulus of photo-cross-linked chitosan hydrogels. Interestingly, templating with quaternary ammonium surfactants also hedges against property fluctuations that occur with changing pH. Further, we demonstrate that, after adequate surfactant removal, these materials are nontoxic, support the attachment of induced pluripotent stem cells and facilitate stem cell differentiation to neuronal phenotypes. These results demonstrate the utility of surfactant templating for optimizing the properties of biomaterials intended for a variety of applications, including retinal regeneration.