Translational Tissue Engineering Research Articles

Effect of tunable stiffness on immune responses in 3D-bioprinted alginate–gelatin scaffolds

Tissue engineering is an approach used to restore damaged tissues and organs using biomaterials that support cell adhesion, growth, and proliferation. However, immune responses triggered by tissue injury and biomaterial implantation can lead to undesired reactions such as foreign body response and fibrotic capsule formation. Macrophages play a critical role in these immune responses. Therefore, comprehending and controlling the immune responses to biomaterials are crucial for successful clinical translation in tissue engineering. In this experimental study, we fabricated three-dimensional-bioprinted hydrogel scaffolds with adaptable stiffness by adjusting the alginate–gelatin ratio. We examined the physical properties of these scaffolds and assessed the immune responses they provoked both in vitro and in vivo. Our results revealed that higher-stiffness implants could drive macrophage polarization toward pro-inflammatory phenotypes in vivo. Furthermore, our animal experiments demonstrated that high-stiffness hydrogels elicited elevated immune responses through the TLR4/Myd88/NF-κB signaling pathway and IL-6/JAK-STAT signaling pathway. Collectively, our study demonstrates that increased implant stiffness correlates with stronger immune responses. These findings are expected to provide novel insights for the clinical application of alginate–gelatin composite hydrogels.

Effect of Storage Time and Temperature on the Bioactivity of a Chitosan-Derived Epigenetic Modulation Scaffold.

The appropriate storage protocol is one of the main limitations of translating tissue engineering technology to commercialized clinical applications. Recently, the development of a chitosan-derived composite scaffold incorporated with bioactive molecules has been reported as an excellent material to repair a critical size bony defect in mice calvaria. This study aims to determine the storage time and appropriate storage temperature of Chitosan/Biphasic Calcium Phosphate/Trichostatin A composite scaffold (CS/BCP/TSA scaffold) in vitro. The mechanical properties and in vitro bioactivity of trichostatin A (TSA) released from CS/BCP/TSA scaffolds in different storage times and temperatures were evaluated. Different storage times (0, 14, and 28 days) and temperatures (-18, 4, and 25 °C) did not affect the porosity, compressive strength, shape memory, and amount of TSA released. However, scaffolds stored at 25 °C and 4 °C were found to lose their bioactivity after 3- and 7-day storage periods, respectively. Thus, the CS/BCP/TSA scaffold should be stored in freezing conditions to preserve the long-term stability of TSA.

Umbilical Cord Blood Cell Clearance Post-Infusion in Immune-Competent Children with Cerebral Palsy

Umbilical cord blood cells have therapeutic potential for neurological disorders, through a paracrine mechanism of action. A greater understanding of the safety and immunological effects of allogeneic donor cord blood cells in the context of a healthy recipient immune system, such as in cerebral palsy, is needed. This study aimed to determine how quickly donor cord blood cells were cleared from the circulation in children with cerebral palsy who received a single intravenous infusion of 12/12 human leucocyte antigen (HLA)-matched sibling cord blood cells. Twelve participants with cerebral palsy aged 2–12 years received cord blood cell infusions as part of a phase I trial of umbilical blood infusion for cerebral palsy. Digital droplet PCR analysis of DNA copy number variants specific to donor and recipient was used to assess donor DNA clearance at five timepoints post-infusion, a surrogate measure of cell clearance. Donor cells were cleared by 3 months post-infusion in 11/12 participants. When detected, donor DNA was at a fraction of 0.01–0.31% of total DNA with no signs of graft-versus-host disease in any participant. The donor DNA clearance times provided by this study have important implications for understanding the safety of allogeneic cord blood cell infusion for cerebral palsy and translational tissue engineering or regenerative medicine research in other disorders.

Advances in Translational 3D Printing for Cartilage, Bone, and Osteochondral Tissue Engineering.

The regeneration of 3D tissue constructs with clinically relevant sizes, structures, and hierarchical organizations for translational tissue engineering remains challenging. 3D printing, an additive manufacturing technique, has revolutionized the field of tissue engineering by fabricating biomimetic tissue constructs with precisely controlled composition, spatial distribution, and architecture that can replicate both biological and functional native tissues. Therefore, 3D printing is gaining increasing attention as a viable option to advance personalized therapy for various diseases by regenerating the desired tissues. This review outlines the recently developed 3D printing techniques for clinical translation and specifically summarizes the applications of these approaches for the regeneration of cartilage, bone, and osteochondral tissues. The current challenges and future perspectives of 3D printing technology are also discussed.

Oral stem cells, decoding and mapping the resident cells populations.

ABSTRACTThe teeth and their supporting tissues provide an easily accessible source of oral stem cells. These different stem cell populations have been extensively studied for their properties, such as high plasticity and clonogenicity, expressing stem cell markers and potency for multilineage differentiation in vitro. Such cells with stem cell properties have been derived and characterised from the dental pulp tissue, the apical papilla region of roots in development, as well as the supporting tissue of periodontal ligament that anchors the tooth within the alveolar socket and the soft gingival tissue. Studying the dental pulp stem cell populations in a continuously growing mouse incisor model, as a traceable in vivo model, enables the researchers to study the properties, origin and behaviour of mesenchymal stem cells. On the other side, the oral mucosa with its remarkable scarless wound healing phenotype, offers a model to study a well-coordinated system of healing because of coordinated actions between epithelial, mesenchymal and immune cells populations. Although described as homogeneous cell populations following their in vitro expansion, the increasing application of approaches that allow tracing of individual cells over time, along with single-cell RNA-sequencing, reveal that different oral stem cells are indeed diverse populations and there is a highly organised map of cell populations according to their location in resident tissues, elucidating diverse stem cell niches within the oral tissues. This review covers the current knowledge of diverse oral stem cells, focusing on the new approaches in studying these cells. These approaches “decode” and “map” the resident cells populations of diverse oral tissues and contribute to a better understanding of the “stem cells niche architecture and interactions. Considering the high accessibility and simplicity in obtaining these diverse stem cells, the new findings offer potential in development of translational tissue engineering approaches and innovative therapeutic solutions.

Prevention of Post-Operative Adhesions: A Comprehensive Review of Present and Emerging Strategies.

Post-operative adhesions affect patients undergoing all types of surgeries. They are associated with serious complications, including higher risk of morbidity and mortality. Given increased hospitalization, longer operative times, and longer length of hospital stay, post-surgical adhesions also pose a great financial burden. Although our knowledge of some of the underlying mechanisms driving adhesion formation has significantly improved over the past two decades, literature has yet to fully explain the pathogenesis and etiology of post-surgical adhesions. As a result, finding an ideal preventative strategy and leveraging appropriate tissue engineering strategies has proven to be difficult. Different products have been developed and enjoyed various levels of success along the translational tissue engineering research spectrum, but their clinical translation has been limited. Herein, we comprehensively review the agents and products that have been developed to mitigate post-operative adhesion formation. We also assess emerging strategies that aid in facilitating precision and personalized medicine to improve outcomes for patients and our healthcare system.

Gas foaming of electrospun poly(L-lactide-co-caprolactone)/silk fibroin nanofiber scaffolds to promote cellular infiltration and tissue regeneration

Electrospun nanofibers emulate extracellular matrix (ECM) morphology and architecture; however, small pore size and tightly-packed fibers impede their translation in tissue engineering. Here we exploited in situ gas foaming to afford three-dimensional (3D) poly(L-lactide-co-ε-caprolactone)/silk fibroin (PLCL/SF) scaffolds, which exhibited nanotopographic cues and a multilayered structure. The addition of SF improved the hydrophilicity and biocompatibility of 3D PLCL scaffolds. Three-dimensional scaffolds exhibited larger pore size (38.75 ± 9.78 μm2) and high porosity (87.1% ± 1.5%) than that of their 2D counterparts. 3D scaffolds also improved the deposition of ECM components and neo-vessel regeneration as well as exhibited more numbers of CD163+/CCR7+ cells after 2 weeks implantation in a subcutaneous model. Collectively, 3D PLCL/SF scaffolds have broad implications for regenerative medicine and tissue engineering applications.

3D bioprinting of bicellular liver lobule-mimetic structures via microextrusion of cellulose nanocrystal-incorporated shear-thinning bioink

3D bioprinting of living cellular constructs with heterogeneity in cell types and extra cellular matrices (ECMs) matching those of biological tissues remains challenging. Here, we demonstrate that, through bioink material design, microextrusion-based (ME) bioprinting techniques have the potential to address this challenge. A new bioink employing alginate (1%), cellulose nanocrystal (CNC) (3%), and gelatin methacryloyl (GelMA) (5%) (namely 135ACG hybrid ink) was formulated for the direct printing of cell-laden and acellular architectures. The 135ACG ink displayed excellent shear-thinning behavior and solid-like properties, leading to high printability without cell damage. After crosslinking, the ACG gel can also provide a stiff ECM ideal for stromal cell growth. By controlling the degree of substitution and polymer concentration, a GelMA (4%) bioink was designed to encapsulate hepatoma cells (hepG2), as GelMA gel possesses the desired low mechanical stiffness matching that of human liver tissue. Four different versions of to-scale liver lobule-mimetic constructs were fabricated via ME bioprinting, with precise positioning of two different cell types (NIH/3T3 and hepG2) embedded in matching ECMs (135ACG and GelMA, respectively). The four versions allowed us to exam effects of mechanical cues and intercellular interactions on cell behaviors. Fibroblasts thrived in stiff 135ACG matrix and aligned at the 135ACG/GelMA boundary due to durotaxis, while hepG2 formed spheroids exclusively in the soft GelMA matrix. Elevated albumin production was observed in the bicellular 3D co-culture of hepG2 and NIH/3T3, both with and without direct intercellular contact, indicating that improved hepatic cell function can be attributed to soluble chemical factors. Overall, our results showed that complex constructs with multiple cell types and varying ECMs can be bioprinted and potentially useful for both fundamental biomedical research and translational tissue engineering.

Role of organic and ceramic biomaterials on bone healing and regeneration: An experimental study with significant value in translational tissue engineering and regenerative medicine.

We investigated the role of various biomaterials on cell viability and in healing of an experimentally induced femoral bone hole model in rats. Cell viability and cytotoxicity of gelatin (Gel; 50 µg/µl), chitosan (Chi; 20 µg/µl), hydroxyapatite (HA; 50 µg/µl), nanohydroxyapatite (nHA; 10 µg/µl), three-calcium phosphate (TCP; 50 µg/µl) and strontium carbonate (Sr; 10 µg/µl) were evaluated on hADSCs via MTT assay. In vivo femoral drill-bone hole model was produced in rats that were either left untreated or treated with autograft, Gel, Chi, HA, nHA, TCP and Sr, respectively. The animals were euthanized after 30 days. Their bone holes were evaluated by gross-pathology, histopathology, SEM and radiography. Also, their dry matter, bone ash and mineral density were measured. Both the Gel and Chi showed cytotoxicity, while nHA had no role on cytotoxicity and cell-viability. All the HA, TCP and Sr significantly improved cell viability when compared to controls (P<0.05). Both the Gel and Chi had no role on osteoconduction and osteoinduction. Compared to HA, nHA showed superior role in increasing new bone formation, mineral density and ash (P<0.05). In contrast to HA and nHA, both the TCP and Sr showed superior morphological, radiographical and biochemical properties on bone healing (P<0.05). TCP and Sr showed the most effective osteoconduction and osteoinduction, respectively. In the Sr group, the most mature type of osteons formed. Various biomaterials have different in vivo efficacy during bone regeneration. TCP was found to be the best material for osteoconduction and Sr for osteoinduction.

Effect of sterilization treatment on mechanical properties, biodegradation, bioactivity and printability of GelMA hydrogels

Gelatin methacryloyl (GelMA) hydrogel scaffolds and GelMA-based bioinks are widely used in tissue engineering and bioprinting due to their ability to support cellular functions and new tissue development. Unfortunately, while terminal sterilization of the GelMA is a critical step for translational tissue engineering applications, it can potentially cause thermal or chemical modifications of GelMA. Thus, understanding the effect of terminal sterilization on GelMA properties is an important, though often overlooked, aspect of material design for translational tissue engineering applications. To this end, we characterized the effects of FDA-approved terminal sterilization methods (autoclaving, ethylene oxide treatment, and gamma (γ)-irradiation) on GelMA prepolymer (bioink) and GelMA hydrogels in terms of the relevant properties for biomedical applications, including mechanical strength, biodegradation rate, cell culture in 2D and 3D, and printability. Autoclaving and ethylene oxide treatment of the GelMA decreased the stiffness of the hydrogel, but the treatments did not modify the biodegradation rate of the hydrogel; meanwhile, γ-irradiation increased the stiffness, reduced the pore size and significantly slowed the biodegradation rate. None of the terminal sterilization methods changed the 2D fibroblast or endothelial cell adhesion and spreading. However, ethylene oxide treatment significantly lowered the fibroblast viability in 3D cell culture. Strikingly, γ-irradiation led to significantly reduced ability of the GelMA prepolymer to undergo sol–gel transition. Furthermore, printability studies showed that the bioinks prepared from γ-irradiated GelMA had significantly reduced printability as compared to the GelMA bioinks prepared from autoclaved or ethylene oxide treated GelMA. These results reveal that the choice of the terminal sterilization method can strongly influence important properties of GelMA bioink and hydrogel. Overall, this study provides further insight into GelMA-based material design with consideration of the effect of terminal sterilization.

Collagen-Supplemented Incubation Rapidly Augments Mechanical Property of Fibroblast Cell Sheets.

Cell sheet technology using UpCell™ (Thermo Fisher Scientific, Roskilde, Denmark) plates is a modern tool that enables the rapid creation of single-layered cells without using extracellular matrix (ECM) enzymatic digestion. Although this technique has the advantage of maintaining a sheet of cells without needing artificial scaffolds, these cell sheets remain extremely fragile. Collagen, the most abundant ECM component, is an attractive candidate for modulating tissue mechanical properties given its tunable property. In this study, we demonstrated rapid mechanical property augmentation of human dermal fibroblast cell sheets after incubation with bovine type I collagen for 24 h on UpCell plates. We showed that treatment with collagen resulted in increased collagen I incorporation within the cell sheet without affecting cell morphology, cell type, or cell sheet quality. Atomic force microscopy measurements for controls, and cell sheets that received 50 and 100 μg/mL collagen I treatments revealed an average Young's modulus of their respective intercellular regions: 6.6 ± 1.0, 14.4 ± 6.6, and 19.8 ± 3.8 kPa during the loading condition, and 10.3 ± 4.7, 11.7 ± 2.2, and 18.1 ± 3.4 kPa during the unloading condition. This methodology of rapid mechanical property augmentation of a cell sheet has a potential impact on cell sheet technology by improving the ease of construct manipulation, enabling new translational tissue engineering applications.

Synthetic peptide hydrogels as 3D scaffolds for tissue engineering.

The regeneration of tissues and organs poses an immense challenge due to the extreme complexity in the research work involved. Despite the tissue engineering approach being considered as a promising strategy for more than two decades, a key issue impeding its progress is the lack of ideal scaffold materials. Nature-inspired synthetic peptide hydrogels are inherently biocompatible, and its high resemblance to extracellular matrix makes peptide hydrogels suitable 3D scaffold materials. This review covers the important aspects of peptide hydrogels as 3D scaffolds, including mechanical properties, biodegradability and bioactivity, and the current approaches in creating matrices with optimized features. Many of these scaffolds contain peptide sequences that are widely reported for tissue repair and regeneration and these peptide sequences will also be discussed. Furthermore, 3D biofabrication strategies of synthetic peptide hydrogels and the recent advances of peptide hydrogels in tissue engineering will also be described to reflect the current trend in the field. In the final section, we will present the future outlook in the design and development of peptide-based hydrogels for translational tissue engineering applications.

Co-culturing porcine normal urothelial cells, urinary bladder fibroblasts and smooth muscle cells for tissue engineering research.

New strategies for culturing and co-culturing of the main types of urinary bladder cells are essential for successful establishment of biomimetic in vitro models, which could be applied for research into, and management of, diverse urological disorders. Porcine normal urothelial cells are available in nearly unlimited amounts and have many properties equivalent to human urothelial cells. In the present study, we established normal differentiated porcine urothelial cells in co-cultures with porcine urinary bladder normal fibroblasts and/or smooth muscle cells. The optimal culture medium for establishment of differentiated urothelial cells, demonstrated by positive immunofluorescence of uroplakins, cytokeratins (CK 7, CK 20), zonula occludens 1 (ZO-1), claudin 4, claudin 8, and E-cadherin, was the medium composed of equal parts of Advanced Dulbecco's modified Eagle's medium (A-DMEM) and MCDB 153 medium with physiological calcium concentration of 2.5 mM and without fetal bovine serum, named UroM (+Ca2+ - S). This medium was also proven to be suitable for culturing of bladder fibroblasts and smooth muscle cells and co-culturing of urothelial cells with these mesenchymal cells. Urothelial cell differentiation was optimal in UroM (+Ca2+ - S) medium in all co-culture conditions and when compared to all conditioned-media combinations. To summarize, these strategies for culturing and co-culturing of urinary bladder urothelial cells with mesenchymal cells could be used as new in vitro models for future basic and applicable research of the urinary bladder and thus potentially also for translational tissue engineering studies.

Semiautomated Longitudinal Microcomputed Tomography-based Quantitative Structural Analysis of a Nude Rat Osteoporosis-related Vertebral Fracture Model.

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ages. Animal OVCF models are essential to the preclinical development of translational tissue engineering strategies. While a number of models currently exist, this protocol describes an optimized method for inducing multiple highly reproducible vertebral defects in a single nude rat. A novel longitudinal semiautomated microcomputed tomography (µCT)-based quantitative structural analysis of the vertebral defects is also detailed. Briefly, rats were imaged at multiple time points post-op. The day 1 scan was reoriented to a standard position, and a standard volume of interest was defined. Subsequent µCT scans of each rat were automatically registered to the day 1 scan so the same volume of interest was then analyzed to assess for new bone formation. This versatile approach can be adapted to a variety of other models where longitudinal imaging-based analysis could benefit from precise 3D semiautomated alignment. Taken together, this protocol describes a readily quantifiable and easily reproducible system for osteoporosis and bone research. The suggested protocol takes 4 months to induce osteoporosis in nude ovariectomized rats and between 2.7 and 4 h to generate, image, and analyze two vertebral defects, depending on tissue size and equipment.

Semiautomated Longitudinal Microcomputed Tomography-based Quantitative Structural Analysis of a Nude Rat Osteoporosis-related Vertebral Fracture Model

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ages. Animal OVCF models are essential to the preclinical development of translational tissue engineering strategies. While a number of models currently exist, this protocol describes an optimized method for inducing multiple highly reproducible vertebral defects in a single nude rat. A novel longitudinal semiautomated microcomputed tomography (µCT)-based quantitative structural analysis of the vertebral defects is also detailed. Briefly, rats were imaged at multiple time points post-op. The day 1 scan was reoriented to a standard position, and a standard volume of interest was defined. Subsequent µCT scans of each rat were automatically registered to the day 1 scan so the same volume of interest was then analyzed to assess for new bone formation. This versatile approach can be adapted to a variety of other models where longitudinal imaging-based analysis could benefit from precise 3D semiautomated alignment. Taken together, this protocol describes a readily quantifiable and easily reproducible system for osteoporosis and bone research. The suggested protocol takes 4 months to induce osteoporosis in nude ovariectomized rats and between 2.7 and 4 h to generate, image, and analyze two vertebral defects, depending on tissue size and equipment.

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been 'conditioned' by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model.

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been 'conditioned' by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

An integrated theoretical-experimental approach to accelerate translational tissue engineering.

Implantable devices utilizing bioengineered tissue are increasingly showing promise as viable clinical solutions. The design of bioengineered constructs is currently directed according to the results of experiments that are used to test a wide range of different combinations and spatial arrangements of biomaterials, cells and chemical factors. There is an outstanding need to accelerate the design process and reduce financial costs, whilst minimizing the required number of animal‐based experiments. These aims could be achieved through the incorporation of mathematical modelling as a preliminary design tool. Here we focus on tissue‐engineered constructs for peripheral nerve repair, which are designed to aid nerve and blood vessel growth and repair after peripheral nerve injury. We offer insight into the role that mathematical modelling can play within tissue engineering, and motivate the use of modelling as a tool capable of improving and accelerating the design of nerve repair constructs in particular. Specific case studies are presented in order to illustrate the potential of mathematical modelling to direct construct design. Copyright © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.

The Value of Systematic Reviews in Estimating the Cost and Barriers to Translation in Tissue Engineering.

Little quantitative data exist concerning barriers that impede translation from bench to bedside. We systematically reviewed synthetic or biosynthetic polymer nerve scaffolds for peripheral nerve repair to study a defined research area that is beyond the discovery phase and has potential for clinical application. Using electronic and manual search methods, we identified published English language articles, where scaffolds were tested in preclinical animal models. A systematic review of these 416 reports estimated all costs related to the use of animals, surgery, and evaluation methods. The research studied 17 different nerves in eight animal species, with use of 65 evaluation methods at an estimated cost of $61,264,910 for the preclinical studies. A total of 127 surveys were sent to authors, of whom 12 could not be accessed electronically and 45 (39%) responded. Major causes for failure to translate included lack of a commercial partner, insufficient financial resources, a research program not involved in translation, and lack of expertise in regulatory affairs. This review emphasizes the urgent need for standardization of preclinical models and the need to establish better collaboration between laboratory investigators, clinicians, and the companies involved in commercialization. It identifies important areas for education of future investigators in the process of translation from discovery to improved health such as those funded by the National Institutes of Health Clinical and Translational Science Awards.

Stable and effective poly(ethylene glycol)-poly(beta-amino ester) nanoparticles for gene delivery

Event Abstract Back to Event Stable and effective poly(ethylene glycol)-poly(beta-amino ester) nanoparticles for gene delivery Jayoung Kim1, 2, Yechan Kang1, 2 and Jordan J. Green1, 2 1 Johns Hopkins University, Biomedical Engineering, United States 2 Johns Hopkins University, Translational Tissue Engineering Center, United States Introduction: We engineered polymeric gene delivery nanoparticles to identify enhanced formulations for cancer gene therapy. Poly(β-amino ester)s (PBAEs) are non-viral, biodegradable polymers that condense DNA to form nanoparticles. PBAEs with different structures can be synthesized from a library of monomers and small molecules to be safe and effective for in vitro gene delivery[1]. However, these nanoparticles need further optimization for in vivo applications in order to prevent particle aggregation, increase systemic half-life, enhance diffusion through biological tissues, and improve transfection[2]. To this end, we synthesized novel poly(ethylene glycol)-conjugated PBAE polymers (PEG-PBAE) and utilized a combinatorial approach to optimize PBAE/PEG-PBAE/DNA nanoparticles that exhibit enhanced stability and efficacy. Materials and Methods: A two-step Michael addition reaction was used to synthesize an array of 9 conventional PBAE polymers as previously described[3]. To synthesize four types of PEG-PBAE polymer, amine-catalyzed thiol-ene Michael addition reaction was used to conjugate PEG-thiol molecules of two different molecular weights to a specific PBAE backbone polymer of two different molecular weights (Fig. 1). Nanoparticles were formulated with a blend of PBAE and PEG-PBAE at three PBAE:PEG-PBAE (2:1, 1:1, 1:2) and three total polymer:DNA (30, 60, 90 w/w) weight ratios. These nanoparticles were sequentially measured for particle size at 0 and 24 h by Nanosight NS500 and for transfection efficacy of pEGFP by flow cytometry. MTS assay was used to compare cell death caused by delivering Herpes Simplex Virus thymidine kinase (pHSV-tk) using the most optimal formulation followed by two ganciclovir treatments to human lung cancer cells (H446) and healthy lung fibroblasts (IMR-90). Results and Discussion: First, 36 different combinations of PEG-PBAE nanoparticles were formulated using a single type of PBAE (B4S4E7) and tested for stability. The representative data showed initial particle size for most nanoparticles to be 90 - 110 nm, and four PEG-PBAE formulations that retained sub-200 nm size after 24 h in 25 mM NaAc were selected for further screening (Fig. 2). Then, 18 different PBAE nanoparticles utilizing nine PBAEs of different structures and two polymer:DNA weight ratios (60 and 75 w/w) were tested for transfection efficacy, and the four best performing PBAE structures were selected. Lastly, transfection evaluation of 16 PEG-PBAE nanoparticles formulated with polymer structures that demonstrated enhanced stability and transfection was performed. The highest transfection was from nanoparticles composed of a blend of PBAE 4 (B4S5E7) with PEG-PBAE 3 (5 kDa PEG - 4 kDa B4S4 PBAE) at a 1:1 weight ratio and with a total polymer:DNA weight ratio of 30 w/w. This nanoparticle formulation showed cancer specificity, killing 35% of the human lung cancer cells, compared to 15% in healthy lung fibroblasts with pHSV-tk and ganciclovir treatment (Fig. 3). Conclusions: A combinatorial approach was used to formulate PBAE nanoparticles with a novel PEG-PBAE polymer. The optimized formulation showed sub-300 nm in size after 24 h incubation, while maintaining high transfection efficacy and function in human small cell lung cancer cells. These results show great potential for not only its evaluation in vivo but also the combinatorial methodology toward discovering new biomaterial platforms. Samsung Scholarship; NIH 1R01EB016721; Wilmer Core Grant (P30-EY001865, Imaging and Microscopy Core Module)