Jason A Wertheim

Interests

Teaching

My teaching activities center on instructing medical trainees in the surgical care of transplant patients or those in need of organ replacement. Within my laboratory, I mentor undergraduate and graduate students in dissertation-level research and post-doctoral scientists in transitioning their research toward independence. I am also dedicated to helping faculty develop their own research programs and mentoring junior faculty in their career progression.

Research

Our research is focused on studying the development of bio-artificial liver, kidney and vascular tissue. My laboratory utilizes novel bioreactors, 3D printing, biomaterial scaffolds and organoid tissue models to study disease and develop cells for therapy or transplantation.

Courses

Scholarly Contributions

Books

• Nadig, S. N., Nadig, S. N., & Wertheim, J. A. (2018). Technological advances in organ transplantation. Springer International Publishing. doi:10.1007/978-3-319-62142-5
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  This book provides an expert view into the current technologies that are revolutionizing the field of solid organ transplantation. This unique book

Chapters

• Shetty, A., Wertheim, J. A., & Butt, Z. (2017). Health-Related Quality of Life Outcomes After Kidney Transplantation. In Kidney Transplantation, Bioengineering and Regeneration. Academic Press. doi:10.1016/b978-0-12-801734-0.00050-3
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  Abstract Health-related quality of life (HRQOL) provides a measure of patients’ perception of their symptoms, overall health or disability, and treatment. As such, HRQOL is an important patient-centered outcome and transplant-oriented clinical research endpoint. HRQOL alone, or in tandem with other objective clinical outcome measures, is useful in monitoring clinical practice, informing treatment decisions, and guiding the allocation of healthcare resources. It comprises three domains: physical, mental, and psychosocial wellbeing; and can be measured by generalized or targeted instruments depending on the degree of specificity desired by the clinician or researcher. Patients with end stage renal disease requiring dialysis report diminished quality of life compared to the general population, which in turn is associated with an increased risk of hospitalization and death, particularly in those with a high comorbidity burden. Renal transplantation confers a global quality of life benefit. Physical symptoms like pain, poor sleep quality, and fatigue are perceived to be improved after transplantation by renal transplant recipients with a functioning allograft. Depression and anxiety also improve after transplantation as does social participation and integration. However, on average, HRQOL following renal transplant remains lower than in the general population, partly due to transplant related complications like infection and malignancy and the adverse effects of immunosuppression. Other factors that negatively impact HRQOL after transplant include older recipient age, deceased donor transplantation, female gender, lack of social support, and comorbid conditions such as diabetes and depression. Improved HRQOL after kidney transplantation, especially in the physical domain, has been associated with better patient and allograft survival. Hence it is imperative for transplant clinicians to familiarize themselves with the concepts, measurement, and interpretation of HRQOL as they strive to provide comprehensive care to kidney transplant patients.
• Wang, B., & Wertheim, J. A. (2016). Experimental Cell Therapy for Liver Dysfunction. In Translating Regenerative Medicine to the Clinic. Elsevier Inc. doi:10.1016/b978-0-12-800548-4.00021-8
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  Liver transplantation is the established therapy for inherited hepatic disorders and end-stage liver failures; however, the serious organ shortage has restricted its wide application. Hepatocyte transplantation has shown potential as an alternative therapy for liver transplantation and has also been considered as a “bridge” therapy until a liver for transplantation becomes available. The current cell source for hepatocyte transplantation is usually isolated from donor livers that are not used for liver transplantation. This has led to the short supply of high-quality primary hepatocytes that limit the application of hepatocytes transplantation as a routine clinical practice. This chapter will review the latest scientific developments and major challenges that may one day lead to a wider application of hepatocyte transplantation.
• Wang, B., & Wertheim, J. A. (2015). Experimental Therapy for Liver Dysfunction. In Translating Regenerative Medicine to the Clinic. Elsevier.
• Patnaik, S. S., Wang, B., Weed, B. C., Wertheim, J. A., & Liao, J. (2014). Decellularized Scaffolds: Concepts, Methodologies, and Applications in Cardiac Tissue Engineering and Whole-Organ Regeneration. In Tissue Regeneration: Where Nanostructure Meets Biology. World Scientific Publication Company.
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  Tissue engineering research, which aims to develop tissue/organ substitutes for treating pathological disorders and organ failures, has made many breakthroughs during the past three decades. The fi eld still faces challenges such as identifying and optimizing scaffolds that must be biodegradable, non-immunogenic, and able to provide structural, mechanical, biological supports/cues for cell adhesion, proliferation, and differentiation. Recent accomplishments in tissue decellularization provide acellular tissue-derived scaffolds that retain the nature-designed structure from the whole organ level, to the microstructural scale, down to the nanoscale. The preservation of structurally organized entities such as collagen, elastin, glycosaminoglycans, and fi bronectin enables a natural template that accommodates many tissue
• Vacanti, J. P., Otte, J., & Wertheim, J. A. (2014). Introduction: Regenerative Medicine and Solid Organ Transplantation from a Historical Perspective. In Regenerative Medicine Applications in Organ Transplantation. Academic Press. doi:10.1016/b978-0-12-398523-1.00001-x
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  Regenerative medicine and transplantation are rooted in distinct but major scientific advancements during the last 15 and 50 years, respectively. To introduce this text, we review the major milestones that led to the development of solid organ transplantation and regenerative medicine. The success of transplantation has led to the need for regenerative medicine. The number of patients waiting for an organ for transplantation has surpassed 117,000. Many of these candidates may not receive an organ due to the limited supply of cadaveric grafts or those donated by altruistic living donors. It is the shared hope that regenerative medicine may one day augment organ transplantation by developing a new source of organs or potentially rehabilitating those that are not transplantable. This chapter summarizes major advancements in both fields including vascular reconstruction, organ preservation, immunosuppression, operational tolerance, novel stem cell sources, biodegradable scaffolds, and new methods to develop vascularized organ units.
• Wertheim, J. A., & Benedetto, W. (2010). ICU Care Following Liver, Kidney, and Lung Transplantation. In The Critical Care Handbook of the Massachusetts General Hospital, 5th Edition. Lippincott Williams & Wilkins.

Journals/Publications

• Abenojar, E. C., Minocha, E., Garcia Villatoro, E. A., Kirinda, V. C., Gupta, A. K., Kang, B., Cho, I. S., Zhang, V., Shin, J. W., Wertheim, J. A., & Kalow, J. A. (2025). Polyacrylamide Hydrogels with Reversibly Photocontrolled Stiffness for 2D Mechanobiology. ACS Applied Materials and Interfaces, 17(Issue 24). doi:10.1021/acsami.5c02909
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  We report the development of polyacrylamide hydrogels with photoswitchable stiffness using solely visible light and their application to cell culture. We have previously shown that azobenzenes can control the binding constants of dynamic covalent boronic ester bonds (Chem. Sci. 2018, 9, 5987; J. Am. Chem. Soc. 2020, 142, 19969). Here we show that these photoswitchable dynamic bonds can be incorporated into polyacrylamide hydrogels that are stable for at least 10 days in buffer without changes in stiffness or photoresponse. Reversible stiffening and softening are achieved with green and blue irradiation, respectively. We prepared soft (877 ± 79 Pa) and stiff (8.4 ± 0.3 kPa) hydrogels that undergo rapid, photoreversible changes in modulus over at least 3 light irradiation cycles. In vitro studies show that the hydrogels are nontoxic to HepG2 cells. The cells undergo the expected changes in morphology, actin stress fiber formation, and Yes-associated protein (YAP) subcellular localization upon stiffening and softening the hydrogel substrate with visible light. These results validate the suitability of our visible-light-controlled hydrogel as a versatile platform for cellular mechanotransduction studies.
• Gupta, A. K., Minocha, E., Koss, K. M., Naved, B. A., Safar-Boueri, L., Wertheim, J. A., & Gallon, L. (2025). A kidney organoid-based readout to assess disease activity in primary and recurrent focal segmental glomerulosclerosis. Kidney International, 107(Issue 5). doi:10.1016/j.kint.2025.01.018
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  Primary focal segmental glomerulosclerosis (pFSGS) is an acquired kidney disorder that frequently leads to kidney failure and confers an elevated risk of recurrence after kidney transplantation, termed recurrent pFSGS. Unfortunately, there is no diagnostic method to foresee recurrence of pFSGS after kidney transplantation. Progress in developing assays to test disease activity is hampered by few preclinical models to replicate disease and inability of in vitro cultured primary podocytes to remain terminally differentiated. In recent years, advancements in kidney organoid biology have led to the development of kidney tissues with glomeruli and major nephron segments including podocytes. To develop a pFSGS model, we studied the effect of plasma from patients diagnosed with pFSGS on kidney organoids differentiated from human pluripotent stem cells. The pFSGS plasma treatment induced podocytopathy, extracellular matrix protein deposition, fibrosis and apoptosis within organoids, whereas non-recurrent plasma did not affect organoid structure. pFSGS plasma also led to loss of normal expression patterns of podocyte specific proteins, nephrin and podocin within podocytes. Further, cytokine array profiling revealed that pFSGS plasma induced secretion of cytokines associated with inflammation and angiogenesis. Additionally, kidney organoids treated with plasma obtained after therapeutic plasma exchange for recurrent pFSGS led to lower cell death in organoids after sequential exchanges with the final exchange showing the least apoptotic cells without morphological abnormality. Overall, our results demonstrate the potential of kidney organoids in advancing kidney disease modeling. These insights could be applied in clinical settings to assist in gauging FSGS recurrence risk prior to kidney transplantation.
• Minocha, E., Jiang, L., Gupta, A. K., Green, R. M., Zohar, Y., & Wertheim, J. A. (2025). Microfluidic Flow Promotes a Steatotic Phenotype in Induced Pluripotent Stem Cell–Derived Hepatocytes that is Influenced by Disease State of the Donor. Gastro Hep Advances, 4(Issue 4). doi:10.1016/j.gastha.2024.100601
• Naved, B. A., Han, S., Koss, K. M., Kando, M. J., Wang, J. J., Chang, J., Weiss, C., Passman, M. G., Wertheim, J. A., Luo, Y., & Zhang, Z. J. (2025). Revealing gait as a murine biomarker of injury, disease, and age with multivariate statistics and machine learning. Scientific Reports, 15(Issue 1). doi:10.1038/s41598-025-02073-0
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  Hundreds of rodent gait studies have been published over the past two decades, according to a PubMed search. Treadmill gait data, for example from the DigiGait system, generates over 30 + spatial and temporal measures. Despite this multi-dimensional data, all but a handful of the published literature on rodent gait has conducted univariate analysis that reveals limited information on the relationships that are characteristic of different gait states. This study conducted rigorous multivariate analysis in the form of sequential feature selection and factor analysis on gait data from a variety of gait deviations (due to injury i.e. peripheral nerve transection and transplantation, disease i.e. IUGR and hyperoxia, and age-related changes) and used machine learning to train a classifier to distinguish among and score different gait states. Treadmill gait data (DigiGait) of three different types of gait deviations were collected. Data were collected from B6 mice using the DigiGait system, with gait measurements taken at standardized treadmill speeds of 10, 17, and 24 cm/s over a period of 3–4 s per observation. Each mouse underwent at least two trials at each speed. Data were collected on B6 mice that were healthy and had various types of gait deficit due to: (a) a peripheral nerve injury model with increasing degrees of damage to the neuromusculoskeletal sequence of gait i.e. nerve transection, total hind limb transplantation, (b) a central nerve injury model of increasing degrees of damage to the motor regions responsible for gait i.e. IUGR, IUGR + hyperoxia, and (c) gait changes due to increasing age. Multivariate factor analysis (using MATLAB’s factoran) and forward feature selection (with ten-fold cross-validation) were conducted to identify those features and factors most descriptive of each gait state for comparison. Various machine learning classifier models were trained with ten-fold cross-validation and evaluated (e.g. random forest, regression, discriminant analysis, support vector machine, and ensemble) in a 70 − 30 training-testing split for their accuracy, precision, recall, and F-score. The highest performing model was used to score each type of gait for direct comparison on a scale of -0.5 to 0.5. The score distributions were plotted on a histogram for direct comparisons of score populations among various gait states. Multivariate feature selection revealed that not all 30 + features were relevant to describing the gait states. Plotting misclassification error (MCE) as a function of number of features included revealed that there was a critical number of features (~ 16) that minimized MCE (0.17 via univariate feature selection vs. 0.12 via multivariate feature selection). Incorporating more than 16 features led MCE to increase linearly indicating overfitting. Relationships among the identified features were understood via factor analysis. The factor analysis results were consistent with the biological differences between the groups (e.g. total hind limb transplantation was distinguishable via features descriptive of the positioning of the paw in relation to the body while nerve transection injury alone was distinguishable via features descriptive of changes to fine motor movements). Across all gait states, there was significant conservation of features and factors. This suggests certain relationships may be fundamental to rodent gait analysis regardless of the gait pathology in question. The highest performing machine learning classifier model (ensemble) was able to distinguish between gait deficits with high performance (F-score, recall, precision, and accuracy all > 0.90). This included the ability to distinguish between peripheral vs. central gait deficit, between individual types of peripheral deficit, between individual types of central deficit, and between younger vs. older animals. Using the classifier to score individual animals and plot the scores by group revealed score distributions that were consistent with biological phenomena. For example, the multivariate gait score trends as a result of increasing central nerve injury were consistent with the trends of white matter volume loss in relevant motor regions of the brain as measured via MRI. Finally, the degrees of separation between multivariate gait scores were consistent with the degree of biological difference between gaits (e.g. central injury had greater separation from healthy vs. peripheral injury; older and younger animals had more moderate, yet still statistically significant, separation in scores vs. any of the injury / disease states did with each other). In conclusion, this study establishes a new methodology to quantify and evaluate gait deviations across a variety of different models. Its novelty is in using multivariate statistics to describe the features and factors that characterize gait states due to injury, disease, and age for use in machine learning model training. This includes statistically describing the differences in gait between diseases with vastly different etiologies of gait deficits (peripheral vs. central). In doing so the methodology’s novelty includes accounting for relationships between groupings of features in model training; something that traditional univariate analysis is unable to do. It used multivariate statistics and machine learning to reveal gait as a quantifiable, preclinical biomarker of injury, disease, and age. It collapsed a multi-dimensional biological phenomena (gait) into a single score by encoding revealed biological relationships allowing for direct, quantifiable comparisons of function as it pertains to ambulation. It revealed how these multivariate gait scores can visualize biologically consistent separation and combined effects. Finally, we demonstrate the application of this methodology to already published univariate study that is representative of the hundreds of univariate treadmill gait analysis published over the last two decades. Thereby, opening the door to a new class of multivariate gait analyses that provides greater insight and value than the current state-of-the art.
• Naved, B. A., Han, S., Koss, K. M., Kando, M. J., Wang, J. J., Weiss, C., Passman, M. G., Wertheim, J. A., Luo, Y., & Zhang, Z. J. (2025). Multivariate description of gait changes in a mouse model of peripheral nerve injury and trauma. PLoS ONE, 20(Issue 1). doi:10.1371/journal.pone.0312415
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  Objective Animal models of nerve injury are important for studying nerve injury and repair, particularly for interventions that cannot be studied in humans. However, the vast majority of gait analysis in animals has been limited to univariate analysis even though gait data is highly multidimensional. As a result, little is known about how various spatiotemporal components of the gait relate to each other in the context of peripheral nerve injury and trauma. We hypothesize that a multivariate characterization of gait will reveal relationships among spatiotemporal components of gait with biological relevance to peripheral nerve injury and trauma. We further hypothesize that legitimate relationships among said components will allow for more accurate classification among distinct gait phenotypes than if attempted with univariate analysis alone. Methods DigiGait data was collected of mice across groups representing increasing degrees of damage to the neuromusculoskeletal sequence of gait; that is (a) healthy controls, (b) nerve damage only via total nerve transection + reconnection of the femoral and sciatic nerves, and (c) nerve, muscle, and bone damage via total hind-limb transplantation. Multivariate relationships among the 30+ spatiotemporal measures were evaluated using exploratory factor analysis and forward feature selection to identify the features and latent factors that best described gait phenotypes. The identified features were then used to train classifier models and compared to a model trained with features identified using only univariate analysis. Results 10-15 features relevant to describing gait in the context of increasing degrees of traumatic peripheral nerve injury were identified. Factor analysis uncovered relationships among the identified features and enabled the extrapolation of a set of latent factors that further described the distinct gait phenotypes. The latent factors tied to biological differences among the groups (e.g. alterations to the anatomical configuration of the limb due to transplantation or aberrant fine motor function due to peripheral nerve injury). Models trained using the identified features generated values that could be used to distinguish among pathophysiological states with high statistical significance (p < .001) and accuracy (>80%) as compared to univariate analysis alone. Conclusion This is the first performance evaluation of a multivariate approach to gait analysis and the first demonstration of superior performance as compared to univariate gait analysis in animals. It is also the first study to use multivariate statistics to characterize and distinguish among different gradations of gait deficit in animals. This study contributes a comprehensive, multivariate characterization pipeline for application in the study of any pathologies in which gait is a quantitative translational outcome metric.
• Nawrocki, S., Carew, J. S., Wertheim, J. A., Chipollini, J., Jones, T., & Lee, B. R. (2023). Targeting NEDDylation is a novel strategy to attenuate cisplatin-induced nephrotoxicity. . American Association for Cancer Research.
• Koss, K. M., Son, T., Li, C., Hao, Y., Cao, J., Churchward, M. A., Zhang, Z. J., Wertheim, J. A., Derda, R., & Todd, K. G. (2024). Toward discovering a novel family of peptides targeting neuroinflammatory states of brain microglia and astrocytes. Journal of Neurochemistry, 168(Issue 10). doi:10.1111/jnc.15840
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  Microglia are immune-derived cells critical to the development and healthy function of the brain and spinal cord, yet are implicated in the active pathology of many neuropsychiatric disorders. A range of functional phenotypes associated with the healthy brain or disease states has been suggested from in vivo work and were modeled in vitro as surveying, reactive, and primed sub-types of primary rat microglia and mixed microglia/astrocytes. It was hypothesized that the biomolecular profile of these cells undergoes a phenotypical change as well, and these functional phenotypes were explored for potential novel peptide binders using a custom 7 amino acid-presenting M13 phage library (SX7) to identify unique peptides that bind differentially to these respective cell types. Surveying glia were untreated, reactive were induced with a lipopolysaccharide treatment, recovery was modeled with a potent anti-inflammatory treatment dexamethasone, and priming was determined by subsequently challenging the cells with interferon gamma. Microglial function was profiled by determining the secretion of cytokines and nitric oxide, and expression of inducible nitric oxide synthase. After incubation with the SX7 phage library, populations of SX7-positive microglia and/or astrocytes were collected using fluorescence-activated cell sorting, SX7 phage was amplified in Escherichia coli culture, and phage DNA was sequenced via next-generation sequencing. Binding validation was done with synthesized peptides via in-cell westerns. Fifty-eight unique peptides were discovered, and their potential functions were assessed using a basic local alignment search tool. Peptides potentially originated from proteins ranging in function from a variety of supportive glial roles, including synapse support and pruning, to inflammatory incitement including cytokine and interleukin activation, and potential regulation in neurodegenerative and neuropsychiatric disorders. (Figure presented.)
• Sereda, T., Beck, J., Semchuk, P., Abu Maziad, A., Wertheim, J., & Koss, K. (2024). The multifaceted helical net of amphipathic alpha-helices; the next dimension of the helical peptide wheel. Science Progress, 107(4). doi:10.1177/00368504241266357
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  The amphipathic nature of helical proteins is crucial to their binding features across a broad spectrum of physiological examples, including heat-shock proteins and hyaluronic acid (HA) receptor binding. By taking advantage of the amphipathic balance of amino acids and their presentation in helical faces, novel synthetic peptides can be designed to improve biofunctionality. We present a new approach for designing synthetic alpha helical peptides using a multifaceted analysis, which allows for new bioengineering designs of amphipathic alpha helices. Amphipathic helical peptides were presented with distinct hydrophobic and hydrophilic faces; two series of analogs, namely, peptides AX9 and AX7, were designed to contain a hydrophobic and hydrophilic face, respectively. The presence of one series of peptides exhibited a distinct hydrophobic face and the second series exhibited a distinct hydrophilic face, which was corroborated with reversed-phase chromatography (C8). Using a multifaceted approach to analyze the potential faces of an amphipathic helix, we demonstrated that these helices contain seven distinct “side-viewed” helical faces (based on the hydrophobic face of the AXP series of analogs), which provides additional spatial dimensional information beyond the averaging effect of the hydrophobic moment generated from the “top-down” view of a helical wheel. Furthermore, we cross-compared our recently published HA-binding peptide in this manner to demonstrate that the most significant binding was related to (1) balanced amphipathicity and (2) a distribution of the key HA-binding domain B1(X7)B2 presented spatially. For example, our most effective peptide binder 17x-3 has five of seven faces with B1(X7)B2 domains, while the positive control mPEP35 has three, which reflects a lower affinity. With such a tool, one is able to map helical peptides on an additional dimension to characterize and redesign fundamental amphipathic properties among other critical characteristics, such as sugar and glycan binding, which is a fundamental characteristic feature of cellular interactions in almost every biological system.
• Wertheim, J. A., Zohar, Y., Green, R. M., Gupta, A. K., Jiang, L., & Minocha, E. (2024). Microfluidic Flow Promotes a Steatotic Phenotype in Induced Pluripotent Stem Cell–Derived Hepatocytes that is Influenced by Disease State of the Donor. Gastro Hep Advances, 4(4), 100601. doi:10.1016/j.gastha.2024.100601
• White, M., Ozkan, M., Gomez-Medellin, J., Koss, K., Solanki, A., Zhang, Z., Alpar, A., Naved, B., Wertheim, J., Hubbell, J., & Rączy, M. (2024). Blocking antibodies against integrin-α3, -αM, and -αMβ2 de-differentiate myofibroblasts, and improve lung fibrosis and kidney fibrosis. Scientific Reports, 14(1). doi:10.1038/s41598-024-70737-4
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  Fibrosis is involved in 45% of deaths in the United States, and no treatment exists to reverse the progression of lung or kidney fibrosis. Myofibroblasts are key to the progression and maintenance of fibrosis. We investigated features of cell adhesion necessary for monocytes to differentiate into myofibroblasts, seeking to identify pathways key to myofibroblast differentiation. Blocking antibodies against integrins α3, αM, and αMβ2 de-differentiate myofibroblasts in vitro, lower the pro-fibrotic secretome of myofibroblasts, and treat lung fibrosis and inhibit kidney fibrosis in vivo. Decorin’s collagen-binding peptide can be used to direct functionalized blocking antibodies (against integrins-α3, -αM, -αMβ2) to both fibrotic lungs and fibrotic kidneys, reducing the dose of antibody necessary to treat fibrosis. This targeted immunotherapy blocking key integrins may be an effective therapeutic for the treatment of fibrosis.
• Jones, T. M., Espitia, C. M., Chipollini, J., Lee, B. R., Wertheim, J. A., Carew, J. S., & Nawrocki, S. T. (2023). Targeting NEDDylation is a Novel Strategy to Attenuate Cisplatin-induced Nephrotoxicity. Cancer research communications, 3(2), 245-257.
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  Although cisplatin remains a backbone of standard-of-care chemotherapy regimens for a variety of malignancies, its use is often associated with severe dose-limiting toxicities (DLT). Notably, 30%-40% of patients treated with cisplatin-based regimens are forced to discontinue treatment after experiencing nephrotoxicity as a DLT. New approaches that simultaneously prevent renal toxicity while improving therapeutic response have the potential to make a major clinical impact for patients with multiple forms of cancer. Here, we report that pevonedistat (MLN4924), a first-in-class NEDDylation inhibitor, alleviates nephrotoxicity and synergistically enhances the efficacy of cisplatin in head and neck squamous cell carcinoma (HNSCC) models. We demonstrate that pevonedistat protects normal kidney cells from injury while enhancing the anticancer activity of cisplatin through a thioredoxin-interacting protein (TXNIP)-mediated mechanism. Cotreatment with pevonedistat and cisplatin yielded dramatic HNSCC tumor regression and long-term animal survival in 100% of treated mice. Importantly, the combination decreased nephrotoxicity induced by cisplatin monotherapy as evidenced by the blockade of kidney injury molecule-1 (KIM-1) and TXNIP expression, a reduction in collapsed glomeruli and necrotic cast formation, and inhibition of cisplatin-mediated animal weight loss. Inhibition of NEDDylation represents a novel strategy to prevent cisplatin-induced nephrotoxicity while simultaneously enhancing its anticancer activity through a redox-mediated mechanism.
• Koss, K. M., Sereda, T. J., Kumirov, V. K., & Wertheim, J. A. (2023). A class of peptides designed to replicate and enhance the Receptor for Hyaluronic Acid Mediated Motility binding domain. Acta biomaterialia, 167, 293-308.
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  The extra-cellular matrix (ECM) is a complex and rich microenvironment that is exposed and over-expressed across several injury or disease pathologies. Biomaterial therapeutics are often enriched with peptide binders to target the ECM with greater specificity. Hyaluronic acid (HA) is a major component of the ECM, yet to date, few HA adherent peptides have been discovered. A class of HA binding peptides was designed using B(X)B hyaluronic acid binding domains inspired from the helical face of the Receptor for Hyaluronic Acid Mediated Motility (RHAMM). These peptides were bioengineered using a custom alpha helical net method, allowing for the enrichment of multiple B(X)B domains and the optimization of contiguous and non-contiguous domain orientations. Unexpectedly, the molecules also exhibited the behaviour of nanofiber forming self-assembling peptides and were investigated for this characteristic. Ten 23-27 amino acid residue peptides were assessed. Simple molecular modelling was used to depict helical secondary structures. Binding assays were performed with varying concentrations (1-10 mg/mL) and extra-cellular matrices (HA, collagens I-IV, elastin, and Geltrex). Concentration mediated secondary structures were assessed using circular dichroism (CD), and higher order nanostructures were visualized using transmission electron microscopy (TEM). All peptides formed the initial apparent 3/alpha-helices, yet peptides 17x-3, 4, BHP3 and BHP4 were HA specific and potent (i.e., a significant effect) binders at increasing concentrations. These peptides shifted from apparent 3/alpha-helical structures at low concentration to beta-sheets at increasing concentration and also formed nanofibers which are noted as self-assembling structures. Several of the HA binding peptides outperformed our positive control (mPEP35) at 3-4 times higher concentrations, and were enhanced by self-assembly as each of these groups had observable nanofibers. STATEMENT OF SIGNIFICANCE: Specific biomolecules or peptides have played a crucial role in developing materials or systems to deliver key drugs and therapeutics to a broad spectrum of diseases and disorders. In these diseased tissues, cells build protein/sugar networks, which are uniquely exposed and great targets to deliver drugs to. Hyaluronic acid (HA) is involved in every stage of injury and is abundant in cancer. To date, only two HA specific peptides have been discovered. In our work, we have designed a way to model and trace binding regions as they appear on the face of a helical peptide. Using this method we have created a family of peptides enriched with HA binding domains that stick with 3-4 higher affinity than those previously discovered.
• Koss, K. M., Son, T., Li, C., Hao, Y., Cao, J., Churchward, M. A., Zhang, Z. J., Wertheim, J. A., Derda, R., & Todd, K. G. (2023). Toward discovering a novel family of peptides targeting neuroinflammatory states of brain microglia and astrocytes. Journal of neurochemistry.
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  Microglia are immune-derived cells critical to the development and healthy function of the brain and spinal cord, yet are implicated in the active pathology of many neuropsychiatric disorders. A range of functional phenotypes associated with the healthy brain or disease states has been suggested from in vivo work and were modeled in vitro as surveying, reactive, and primed sub-types of primary rat microglia and mixed microglia/astrocytes. It was hypothesized that the biomolecular profile of these cells undergoes a phenotypical change as well, and these functional phenotypes were explored for potential novel peptide binders using a custom 7 amino acid-presenting M13 phage library (SX7) to identify unique peptides that bind differentially to these respective cell types. Surveying glia were untreated, reactive were induced with a lipopolysaccharide treatment, recovery was modeled with a potent anti-inflammatory treatment dexamethasone, and priming was determined by subsequently challenging the cells with interferon gamma. Microglial function was profiled by determining the secretion of cytokines and nitric oxide, and expression of inducible nitric oxide synthase. After incubation with the SX7 phage library, populations of SX7-positive microglia and/or astrocytes were collected using fluorescence-activated cell sorting, SX7 phage was amplified in Escherichia coli culture, and phage DNA was sequenced via next-generation sequencing. Binding validation was done with synthesized peptides via in-cell westerns. Fifty-eight unique peptides were discovered, and their potential functions were assessed using a basic local alignment search tool. Peptides potentially originated from proteins ranging in function from a variety of supportive glial roles, including synapse support and pruning, to inflammatory incitement including cytokine and interleukin activation, and potential regulation in neurodegenerative and neuropsychiatric disorders.
• Nawrocki, S., Carew, J. S., Wertheim, J. A., Chipollini, J., Jones, T., Lee, B. R., Nawrocki, S., Carew, J. S., Wertheim, J. A., Chipollini, J., Jones, T., & Lee, B. R. (2023). Targeting NEDDylation is a novel strategy to attenuate cisplatin-induced nephrotoxicity. . American Association for Cancer Research.
• Slepian, M. J., Arabyan, A., Brosius, F. C., & Wertheim, J. A. (2023). RENAL5: ADVANCE Kidney Health: Student, Faculty and University Engagement to Advance the Renal Workforce and Drive Innovation for the Future. ASAIO Journal, 69(Supplement 2), 99-99. doi:10.1097/01.mat.0000943788.42871.f6
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  Background: Kidney Disease (KD) is a significant cause of morbidity and morbidity on the rise in the U.S. and worldwide. A widening gap is developing between the present workforce and technology available to address KD and that which is anticipated as needed to meet future demands associated with rising KD prevalence. We developed a program - ADVANCE Kidney Health (Arizona Technology Development and Clinical Education Program for Students in Kidney Health), aimed specifically at undergraduates, to foster interest, education, engagement and research in Kidney Medicine. The program is supported by the NIH. We report on the early phase of this program. Methods: A hands-on program was developed based on four pillars: 1) science, medical and engineering education; 2) training in innovation, entrepreneurship and translation; 3) experiential learning, mentorship and clinical immersion; and 4) needs-based application and practical translation – aimed at producing motivated, trained and committed biomedical trainees interested in renal health and science. Undergraduate students were recruited as Freshman/Sophomores immersed in didactic and lab experiences, advanced in Junior year with increasing lab and clinical exposure and specific project definition, culminating in a full Senior Year Engineering Design Capstone project. Students were selected by a faculty panel based on interest, academic performance, and diversity considerations. Students were required to report and present on results and participate in science/medical meetings fostering increasing engagement in the field. Results: The program overall has attracted great undergraduate interest with increasing application rates, demand and popularity among engineering, physiology and computer science majors, In Year 1 of the program 80+ students applied with 4 students selected. In Year 2 100+ students applied with 8 selected. All students have largely continued in the program with minimal attrition, in fact attracting more adjunct students to join though related elective courses available. In Yr 1, 3 Sr. design projects advanced; in Yr 2, 5 Sr. Design projects advanced, involving 15 and 25 students, respectively. In Yr 2 individual research was submitted to national meetings including ASAIO. Conclusion: A multi-component engagement program aimed at undergraduates is an effective means of immersing students in the broad space of Kidney Medicine. To date it is apparent that students from a wide range of undergraduate majors find interest and a surprising “fit” into this space. A wide range of novel technologies and projects have emerged. Many have expressed interest in advancing to either medical school, graduate education or work in the kidney space. This approach to advance workforce development shows great promise for the future.
• Sun, J., Ince, M. N., Abraham, C., Barrett, T., Brenner, L. A., Cong, Y., Dashti, R., Dudeja, P. K., Elliott, D., Griffith, T. S., Heeger, P. S., Hoisington, A., Irani, K., Kim, T. K., Kapur, N., Leventhal, J., Mohamadzadeh, M., Mutlu, E., Newberry, R., , Peled, J. U., et al. (2023). Modulating microbiome-immune axis in the deployment-related chronic diseases of Veterans: report of an expert meeting. Gut microbes, 15(2), 2267180.
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  The present report summarizes the United States Department of Veterans Affairs (VA) field-based meeting titled "Modulating microbiome-immune axis in the deployment-related chronic diseases of Veterans." Our Veteran patient population experiences a high incidence of service-related chronic physical and mental health problems, such as infection, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), various forms of hematological and non-hematological malignancies, neurologic conditions, end-stage organ failure, requiring transplantation, and posttraumatic stress disorder (PTSD). We report the views of a group of scientists who focus on the current state of scientific knowledge elucidating the mechanisms underlying the aforementioned disorders, novel therapeutic targets, and development of new approaches for clinical intervention. In conclusion, we dovetailed on four research areas of interest: 1) microbiome interaction with immune cells after hematopoietic cell and/or solid organ transplantation, graft-versus-host disease (GVHD) and graft rejection, 2) intestinal inflammation and its modification in IBD and cancer, 3) microbiome-neuron-immunity interplay in mental and physical health, and 4) microbiome-micronutrient-immune interactions during homeostasis and infectious diseases. At this VA field-based meeting, we proposed to explore a multi-disciplinary, multi-institutional, collaborative strategy to initiate a roadmap, specifically focusing on host microbiome-immune interactions among those with service-related chronic diseases to potentially identify novel and translatable therapeutic targets.
• Ammons, M. C., Battles, J. K., Beenhouwer, D. O., Blair, S., Bolz, D. D., Bonomo, R. A., Brown, S. T., Burgoyne, H. M., Cameron, P., Chepelev, I., Clark, D. H., Curtis, J. L., Davey, V., Devine, A., Diwan, A., Epstein, L., Harley, J. B., Holodniy, M., Kaufman, K. M., , Khan, S., et al. (2022). The US Department of Veterans Affairs Science and Health Initiative to Combat Infectious and Emerging Life-Threatening Diseases (VA SHIELD): A Biorepository Addressing National Health Threats. Open Forum Infectious Diseases, 9(12). doi:10.1093/ofid/ofac641
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  Abstract Background The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has demonstrated the need to share data and biospecimens broadly to optimize clinical outcomes for US military Veterans. Methods In response, the Veterans Health Administration established VA SHIELD (Science and Health Initiative to Combat Infectious and Emerging Life-threatening Diseases), a comprehensive biorepository of specimens and clinical data from affected Veterans to advance research and public health surveillance and to improve diagnostic and therapeutic capabilities. Results VA SHIELD now comprises 12 sites collecting de-identified biospecimens from US Veterans affected by SARS-CoV-2. In addition, 2 biorepository sites, a data processing center, and a coordinating center have been established under the direction of the Veterans Affairs Office of Research and Development. Phase 1 of VA SHIELD comprises 34 157 samples. Of these, 83.8% had positive tests for SARS-CoV-2, with the remainder serving as contemporaneous controls. The samples include nasopharyngeal swabs (57.9%), plasma (27.9%), and sera (12.5%). The associated clinical and demographic information available permits the evaluation of biological data in the context of patient demographics, clinical experience and management, vaccinations, and comorbidities. Conclusions VA SHIELD is representative of US national diversity with a significant potential to impact national healthcare. VA SHIELD will support future projects designed to better understand SARS-CoV-2 and other emergent healthcare crises. To the extent possible, VA SHIELD will facilitate the discovery of diagnostics and therapeutics intended to diminish COVID-19 morbidity and mortality and to reduce the impact of new emerging threats to the health of US Veterans and populations worldwide.
• Hekman, K. E., Koss, K. M., Ivancic, D. Z., He, C., & Wertheim, J. A. (2022). Autophagy Enhances Longevity of Induced Pluripotent Stem Cell-Derived Endothelium via mTOR-Independent ULK1 Kinase. Stem cells translational medicine, 11(11), 1151-1164.
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  Stem cells are enabling an improved understanding of the peripheral arterial disease, and patient-specific stem cell-derived endothelial cells (ECs) present major advantages as a therapeutic modality. However, applications of patient-specific induced pluripotent stem cell (iPSC)-derived ECs are limited by rapid loss of mature cellular function in culture. We hypothesized that changes in autophagy impact the phenotype and cellular proliferation of iPSC-ECs. Endothelial cells were differentiated from distinct induced pluripotent stem cell lines in 2D culture and purified for CD144 positive cells. Autophagy, mitochondrial morphology, and proliferation were characterized during differentiation and over serial passages in culture. We found that autophagy activity was stimulated during differentiation but stagnated in mature iPSC-ECs. Mitochondria remodeled through mitophagy during differentiation and demonstrated increasing membrane potential and mass through serial passages; however, these plateaued, coinciding with decreased proliferation. To evaluate for oxidative damage, iPSC-ECs were alternatively grown under hypoxic culture conditions; however, hypoxia only transiently improved the proliferation. Stimulating mTOR-independent ULK1-mediated autophagy with a plant derivative AMP kinase activator Rg2 significantly improved proliferative capacity of iPSC-ECs over multiple passages. Therefore, autophagy, a known mediator of longevity, played an active role in remodeling mitochondria during maturation from pluripotency to a terminally differentiated state. Autophagy failed to compensate for increasing mitochondrial mass over serial passages, which correlated with loss of proliferation in iPSC-ECs. Stimulating ULK1-kinase-driven autophagy conferred improved proliferation and longevity over multiple passages in culture. This represents a novel approach to overcoming a major barrier limiting the use of iPSC-ECs for clinical and research applications.
• Naved, B. A., Bonventre, J. V., Hubbell, J. A., Hukriede, N. A., Humphreys, B. D., Kesselman, C., Valerius, M. T., McMahon, A. P., Shankland, S. J., Wertheim, J. A., White, M. J., de Caestecker, M. P., & Drummond, I. A. (2022). Kidney repair and regeneration: perspectives of the NIDDK (Re)Building a Kidney consortium. Kidney international, 101(5), 845-853.
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  Acute kidney injury impacts ∼13.3 million individuals and causes ∼1.7 million deaths per year globally. Numerous injury pathways contribute to acute kidney injury, including cell cycle arrest, senescence, inflammation, mitochondrial dysfunction, and endothelial injury and dysfunction, and can lead to chronic inflammation and fibrosis. However, factors enabling productive repair versus nonproductive, persistent injury states remain less understood. The (Re)Building a Kidney (RBK) consortium is a National Institute of Diabetes and Digestive and Kidney Diseases consortium focused on both endogenous kidney repair mechanisms and the generation of new kidney tissue. This short review provides an update on RBK studies of endogenous nephron repair, addressing the following questions: (i) What is productive nephron repair? (ii) What are the cellular sources and drivers of repair? and (iii) How do RBK studies promote development of therapeutics? Also, we provide a guide to RBK's open access data hub for accessing, downloading, and further analyzing data sets.
• Das, P., DiVito, M. D., Wertheim, J. A., & Tan, L. P. (2021). Bioengineered 3D electrospun nanofibrous scaffold with human liver cells to study alcoholic liver disease in vitro. Integrative biology : quantitative biosciences from nano to macro, 13(7), 184-195.
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  Alcohol injury induces hepatic fibrosis which gradually progresses to cirrhosis, sometimes may lead to liver cancer. Animal models are less efficient in mimicking responses of human liver cells, whereas in vitro models discussed so far are majorly based on rodent cells. In this work, a coculture of primary human hepatocytes (PHHs) with LX-2 cells was established on the unmodified (C:F_0:0), collagen-I modified (C:F_1:0), fibronectin modified (C:F_0:1) and 3:1 collagen-I to fibronectin modified (C:F_3:1) 3D electrospun fibrous scaffolds. The effect of alcohol injury was evaluated on this cell-scaffold model at 0-40 μl/ml alcohol concentrations over 14 days of culture period by using the gold standard sandwich culture as the control. Among all the culture groups, C:F_3:1 scaffold was able to maintain translational and transcriptional properties of human liver cells at all concentrations of alcohol treatment. The study reveals that, PHHs on C:F_3:1 were able to maintain ~4-fold and ~1.6-fold higher secretion of albumin than the gold standard sandwich culture on Day 3 and Day 7, respectively. When treated with alcohol, at concentrations of 20 and 40 μl/ml, albumin secretion was also observed to be higher (~2-fold) when compared to the gold standard sandwich culture. Again as expected, in C:F_3:1 culture group on 40 μl/ml alcohol treatment, albumin gene expression decreased by ~2-fold due to alcohol toxicity, whereas CYP2C9, CYP3A4, CYP2E1 and CYP1A2 gene expressions upregulated by ~3.5, ~~4, ~5 and ~15-fold, respectively in response to the alcohol injury. LX-2 cells also acquire more quiescent phenotype on C:F_3:1 scaffolds when compared to the gold standard sandwich culture upon alcohol treatment. Thus, C:F_3:1 scaffold with human liver cells was established as the potential platform to scan alcohol toxicity at varied alcohol concentrations. Thus, it can pave a promising path not only to support functional healthy human liver cells for liver tissue engineering but also to examine potential drugs to study the progression or inhibition of alcoholic liver fibrosis in vitro.
• Gupta, A. K., Ivancic, D. Z., Naved, B. A., Wertheim, J. A., & Oxburgh, L. (2021). An efficient method to generate kidney organoids at the air-liquid interface. Journal of biological methods, 8(2), e150.
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  The prevalence of kidney dysfunction continues to increase worldwide, driving the need to develop transplantable renal tissues. The kidney develops from four major renal progenitor populations: nephron epithelial, ureteric epithelial, interstitial and endothelial progenitors. Methods have been developed to generate kidney organoids but few or dispersed tubular clusters within the organoids hamper its use in regenerative applications. Here, we describe a detailed protocol of asynchronous mixing of kidney progenitors using organotypic culture conditions to generate kidney organoids tightly packed with tubular clusters and major renal structures including endothelial network and functional proximal tubules. This protocol provides guidance in the culture of human embryonic stem cells from a National Institute of Health-approved line and their directed differentiation into kidney organoids. Our 18-day protocol provides a rapid method to generate kidney organoids that facilitate the study of different nephrological events including tissue development, disease modeling and chemical screening. However, further studies are required to optimize the protocol to generate additional renal-specific cell types, interconnected nephron segments and physiologically functional renal tissues.
• Parsons, R. F., Baquerizo, A., Kirchner, V. A., Malek, S., Desai, C. S., Schenk, A., Finger, E. B., Brennan, T. V., Parekh, K. R., MacConmara, M., Brayman, K., Fair, J., Wertheim, J. A., & , A. C. (2021). Challenges, highlights, and opportunities in cellular transplantation: A white paper of the current landscape. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons.
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  Although cellular transplantation remains a relatively small field compared to solid organ transplantation, the prospects for advancement in basic science and clinical care remain bountiful. In this review, notable historical events and the current landscape of the field of cellular transplantation are reviewed with an emphasis on islets (allo- and xeno-), hepatocytes (including bioartificial liver), adoptive regulatory immunotherapy, and stem cells (SCs, specifically endogenous organ-specific and mesenchymal). Also, the nascent but rapidly evolving field of three-dimensional bioprinting is highlighted, including its major processing steps and latest achievements. To reach its full potential where cellular transplants are a more viable alternative than solid organ transplants, fundamental change in how the field is regulated and advanced is needed. Greater public and private investment in the development of cellular transplantation is required. Furthermore, consistent with the call of multiple national transplant societies for allo-islet transplants, the oversight of cellular transplants should mirror that of solid organ transplants and not be classified under the unsustainable, outdated model that requires licensing as a drug with the Food and Drug Administration. Cellular transplantation has the potential to bring profound benefit through progress in bioengineering and regenerative medicine, limiting immunosuppression-related toxicity, and providing markedly reduced surgical morbidity.
• Wysocki, J., Ye, M., Hassler, L., Gupta, A. K., Wang, Y., Nicoleascu, V., Randall, G., Wertheim, J. A., & Batlle, D. (2021). A Novel Soluble ACE2 Variant with Prolonged Duration of Action Neutralizes SARS-CoV-2 Infection in Human Kidney Organoids. Journal of the American Society of Nephrology : JASN.
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  There is an urgent need for approaches to prevent and treat SARS-CoV-2 infection. Administration of soluble ACE2 protein acting as a decoy to bind to SARS-CoV-2 should limit viral uptake mediated by binding to membrane-bound full-length ACE2, and further therapeutic benefit should result from ensuring enzymatic ACE2 activity to affected organs in patients with COVID-19.
• Das, P., DiVito, M. D., Wertheim, J. A., & Tan, L. P. (2020). Collagen-I and fibronectin modified three-dimensional electrospun PLGA scaffolds for long-term in vitro maintenance of functional hepatocytes. Materials science & engineering. C, Materials for biological applications, 111, 110723.
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  Extracellular matrix (ECM) proteins are important regulators of cellular behaviour in the native environment. It has been established that ECM proteins - collagen-I and fibronectin - are present in liver extracellular matrix and regulate specific functions of primary hepatocytes. While scaffolds grafted with the individual ECM protein have shown support for hepatocyte functional properties in vitro, the synergistic effects of both ECM proteins remain to be explored. Such studies are even more limited when three-dimensional (3D) scaffolds are involved. In the current work, the fabrication of a series of highly porous poly(lactic-co-glycolic acid) (PLGA) 3D electrospun scaffolds, simultaneously modified with both collagen-I and fibronectin, has been demonstrated. Different ratios of collagen-I to fibronectin were optimized to study the synergistic effects of the proteins in supporting the viability and functional properties of Huh-7.5 cells. The ratio of collagen-I to fibronectin at 3:1 was found to provide the most efficient chemisorption on the 3D scaffolds. At this ratio, the total protein content that can be grafted on the scaffolds was the highest and the most homogeous. This led to remarkable enhancement of cell seeding efficiency as well as proliferation. Most importantly, liver specific genes such as albumin and cytochrome P450 enzymes i.e. CYP3A4 and CYP3A7 were significantly upregulated by ~12.5, 7 and 4.5 fold respectively, as compared to unmodified PLGA scaffolds after 28 days of culture. Compared to single-protein modified scaffolds, scaffolds modified with 3:1 collagen to fibronectin result in a rise of the albumin gene expression of cultured cells by ~8 to 10 fold, whereas CYP3A4 gene expression improved by ~5 to 7 fold and CYP3A7 gene expression improved by ~4 to 4.5 fold after a long culture period of 28 days. Albumin secretion was improved by ~4 fold compared to unmodified PLGA scaffolds, ~3 fold compared to collagen-I modified culture groups and ~2 fold compared to fibronectin modified culture groups. The multi-protein modified scaffolds, at the optimum ratio, were able to significantly enhance functional properties of the liver cells. This simple yet highly functioning platform would be useful for in vitro culture of liver cells for both drug screening as well as translational purposes.
• Ferrer, J. R., Sinegra, A. J., Ivancic, D., Yeap, X. Y., Qiu, L., Wang, J. J., Zhang, Z. J., Wertheim, J. A., & Mirkin, C. A. (2020). Structure-Dependent Biodistribution of Liposomal Spherical Nucleic Acids. ACS nano, 14(2), 1682-1693.
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  Spherical nucleic acids (SNAs) are a class of nanomaterials with a structure defined by a radial distribution of densely packed, short DNA or RNA sequences around a nanoparticle core. This structure allows SNAs to rapidly enter mammalian cells, protects the displayed oligonucleotides from nuclease degradation, and enables co-delivery of other drug cargoes. Here, we investigate the biodistribution of liposomal spherical nucleic acid (LSNA) conjugates, SNA architectures formed from liposome templates and DNA modified with hydrophobic end groups (tails). We compared linear DNA with two types of LSNAs that differ only by the affinity of the modified DNA sequence for the liposome template. We use single-stranded DNA (ssDNA) terminated with either a low-affinity cholesterol tail (CHOL-LSNA) or a high-affinity diacylglycerol lipid tail (DPPE-LSNA). Both LSNA formulations, independent of DNA conjugation, reduce the inflammatory cytokine response to intravenously administered DNA. The difference in the affinity for the liposome template significantly affects DNA biodistribution. DNA from CHOL-LSNAs accumulates in greater amounts in the lungs than DNA from DPPE-LSNAs. In contrast, DNA from DPPE-LSNAs exhibits greater accumulation in the kidneys. Flow cytometry and fluorescence microscopy of tissue sections indicate that different cell populations-immune and nonimmune-sequester the DNA depending upon the chemical makeup of the LSNA. Taken together, these data suggest that the chemical structure of the LSNAs represents an opportunity to direct the biodistribution of nucleic acids to major tissues outside of the liver.
• He, C., Hekman, K. E., Ivancic, D. Z., Koss, K., & Wertheim, J. A. (2020). The Role of Autophagy During Differentiation and in Enhancing the Longevity of Induced Pluripotent Stem Cell-Derived Endothelial Cells. JVS: Vascular Science, 1, 254. doi:10.1016/j.jvssci.2020.11.017
• Kumar Gupta, A., Sarkar, P., Wertheim, J. A., Pan, X., Carroll, T. J., & Oxburgh, L. (2020). Asynchronous mixing of kidney progenitor cells potentiates nephrogenesis in organoids. Communications biology, 3(1), 231.
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  A fundamental challenge in emulating kidney tissue formation through directed differentiation of human pluripotent stem cells is that kidney development is iterative, and to reproduce the asynchronous mix of differentiation states found in the fetal kidney we combined cells differentiated at different times in the same organoid. Asynchronous mixing promoted nephrogenesis, and heterochronic organoids were well vascularized when engrafted under the kidney capsule. Micro-CT and injection of a circulating vascular marker demonstrated that engrafted kidney tissue was connected to the systemic circulation by 2 weeks after engraftment. Proximal tubule glucose uptake was confirmed, but despite these promising measures of graft function, overgrowth of stromal cells prevented long-term study. We propose that this is a technical feature of the engraftment procedure rather than a specific shortcoming of the directed differentiation because kidney organoids derived from primary cells and whole embryonic kidneys develop similar stromal overgrowth when engrafted under the kidney capsule.
• Zheng, F., Tully, A., Koss, K. M., Zhang, X., Qiu, L., Wang, J. J., Naved, B. A., Ivancic, D. Z., Mathew, J. M., Wertheim, J. A., & Zhang, Z. J. (2020). Taking the Next Step: a Neural Coaptation Orthotopic Hind Limb Transplant Model to Maximize Functional Recovery in Rat. Journal of visualized experiments : JoVE.
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  Limb transplant in particular and vascularized composite allotransplant (VCA) in general have wide therapeutic promise that have been stymied by current limitations in immunosuppression and functional neuromotor recovery. Many animal models have been developed for studying unique features of VCA, but here we present a robust reproducible model of orthotopic hind limb transplant in rats designed to simultaneously investigate both aspects of current VCA limitation: immunosuppression strategies and functional neuromotor recovery. At the core of the model rests a commitment to meticulous, time-tested microsurgical techniques such as hand sewn vascular anastomoses and hand sewn neural coaptation of the femoral nerve and the sciatic nerve. This approach yields durable limb reconstructions that allow for longer lived animals capable of rehabilitation, resumption of daily activities, and functional testing. With short-term treatment of conventional immunosuppressive agents, allotransplanted animals survived up to 70 days post-transplant, and isotransplanted animals provide long lived controls beyond 200 days post-operatively. Evidence of neurologic functional recovery is present by 30 days post operatively. This model not only provides a useful platform for interrogating immunological questions unique to VCA and nerve regeneration, but also allows for in vivo testing of new therapeutic strategies specifically tailored for VCA.
• Ferrer, J. R., Wertheim, J. A., & Mirkin, C. A. (2019). Dual Toll-Like Receptor Targeting Liposomal Spherical Nucleic Acids. Bioconjugate chemistry, 30(3), 944-951.
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  Liposomal spherical nucleic acids (LSNAs) are a class of nanomaterial used broadly for biomedical applications. Their intrinsic capacity to rapidly enter cells and engage cell surface and intracellular ligands stems from their unique three-dimensional architecture, which consists of densely packed and uniformly oriented oligonucleotides on the surface of a liposomal core. Such structures are promising for therapeutics because they can carry chemical cargo within the lipid core in addition to the nucleic acids that define them, in principle enabling delivery of multiple signals to a single cell. On the basis of these traits, we have designed novel dual-targeting LSNAs that deliver a nucleic acid specific for TLR9 inhibition and a small molecule (TAK-242) that inhibits TLR4. Toll-like receptors (TLRs) play a large role in pathogen recognition and disease initiation, and TLR subtypes are differentially located within the lipid membranes of the cell surface and within intracellular endosomes. Oftentimes, in acute or chronic inflammatory conditions, multiple TLRs are activated, leading to stimulation of distinct, and sometimes overlapping, downstream pathways. As such, these inflammatory conditions may respond to attenuation of more than one initiating receptor. We show that dual targeting LSNAs, comprised of unilamellar liposomal cores, the INH-18 oligonucleotide sequence, and TAK-242 robustly inhibit TLR-9 and TLR-4 respectively, in engineered TLR reporter cells and primary mouse peritoneal macrophages. Importantly, the LSNAs exhibit up to a 10- and a 1000-fold increase, respectively, in TLR inhibition compared to the linear sequence and TAK-242 alone. Moreover, the timing of delivery is shown to be a critical factor in effecting TLR-inhibition, with near-complete TLR-4 inhibition occurring when cells were pretreated with SNAs for 4 h prior to stimulation. The most pronounced effect observed from this approach is the benefit of delivering the small molecule within the SNA via the receptor-mediated internalization pathway common to SNAs.
• Manjunath, A., Koss, K., Im, C., Hekman, K. E., Manjunath*, A., & Wertheim, J. A. (2019). PD54-03 RECELLULARIZATION OF RAT KIDNEY SCAFFOLD VASCULATURE WITH HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS. The Journal of Urology, 201(Supplement 4). doi:10.1097/01.ju.0000557056.39487.da
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  INTRODUCTION AND OBJECTIVES:Bioartificial replacement renal tissues may alleviate the shortage of transplantable kidneys and may minimize the risk of rejection. In order to measure the durability o...
• Su, J., Satchell, S. C., Wertheim, J. A., & Shah, R. N. (2019). Poly(ethylene glycol)-crosslinked gelatin hydrogel substrates with conjugated bioactive peptides influence endothelial cell behavior. Biomaterials, 201, 99-112.
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  The basement membrane is a specialized extracellular matrix substrate responsible for support and maintenance of epithelial and endothelial structures. Engineered basement membrane-like hydrogel systems have the potential to advance understanding of cell-cell and cell-matrix interactions by allowing precise tuning of the substrate or matrix biochemical and biophysical properties. In this investigation, we developed tunable hydrogel substrates with conjugated bioactive peptides to modulate cell binding and growth factor signaling by endothelial cells. Hydrogels were formed by employing a poly(ethylene glycol) crosslinker to covalently crosslink gelatin polymers and simultaneously conjugate laminin-derived YIGSR peptides or vascular endothelial growth factor (VEGF)-mimetic QK peptides to the gelatin. Rheological characterization revealed rapid formation of hydrogels with similar stiffnesses across tested formulations, and swelling analysis demonstrated dependency on peptide and crosslinker concentrations in hydrogels. Levels of phosphorylated VEGF Receptor 2 in cells cultured on hydrogel substrates revealed that while human umbilical vein endothelial cells (HUVECs) responded to both soluble and conjugated forms of the QK peptide, conditionally-immortalized human glomerular endothelial cells (GEnCs) only responded to the conjugated presentation of the peptide. Furthermore, whereas HUVECs exhibited greatest upregulation in gene expression when cultured on YIGSR- and QK-conjugated hydrogel substrates after 5 days, GEnCs exhibited greatest upregulation when cultured on Matrigel control substrates at the same time point. These results indicate that conjugation of bioactive peptides to these hydrogel substrates significantly influenced endothelial cell behavior in cultures but with differential responses between HUVECs and GEnCs.
• Brown, J. H., Das, P., DiVito, M. D., Ivancic, D., Tan, L. P., & Wertheim, J. A. (2018). Nanofibrous PLGA electrospun scaffolds modified with type I collagen influence hepatocyte function and support viability in vitro. Acta biomaterialia, 73, 217-227.
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  A major challenge of maintaining primary hepatocytes in vitro is progressive loss of hepatocyte-specific functions, such as protein synthesis and cytochrome P450 (CYP450) catalytic activity. We developed a three-dimensional (3D) nanofibrous scaffold made from poly(l-lactide-co-glycolide) (PLGA) polymer using a newly optimized wet electrospinning technique that resulted in a highly porous structure that accommodated inclusion of primary human hepatocytes. Extracellular matrix (ECM) proteins (type I collagen or fibronectin) at varying concentrations were chemically linked to electrospun PLGA using amine coupling to develop an in vitro culture system containing the minimal essential ECM components of the liver micro-environment that preserve hepatocyte function in vitro. Cell-laden nanofiber scaffolds were tested in vitro to maintain hepatocyte function over a two-week period. Incorporation of type I collagen onto PLGA scaffolds (PLGA-C: 100 µg/mL) led to 10-fold greater albumin secretion, 4-fold higher urea synthesis, and elevated transcription of hepatocyte-specific CYP450 genes (CYP3A4, 3.5-fold increase and CYP2C9, 3-fold increase) in primary human hepatocytes compared to the same cells grown within unmodified PLGA scaffolds over two weeks. These indices, measured using collagen-bonded scaffolds, were also higher than scaffolds coupled to fibronectin or an ECM control sandwich culture composed of type I collagen and Matrigel. Induction of CYP2C9 activity was also higher in these same type I collagen PLGA scaffolds compared to other ECM-modified or unmodified PLGA constructs and was equivalent to the ECM control at 7 days. Together, we demonstrate a minimalist ECM-based 3D synthetic scaffold that accommodates primary human hepatocyte inclusion into the matrix, maintains long-term in vitro survival and stimulates function, which can be attributed to coupling of type I collagen.
• Friedrich, E. E., Lanier, S. T., Niknam-Bienia, S., Arenas, G. A., Rajendran, D., Wertheim, J. A., & Galiano, R. D. (2018). Residual sodium dodecyl sulfate in decellularized muscle matrices leads to fibroblast activation in vitro and foreign body response in vivo. Journal of tissue engineering and regenerative medicine, 12(3), e1704-e1715.
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  Detergents such as sodium dodecyl sulfate (SDS) are commonly used to extract cells from tissues in a process called "decellularization". Residual SDS is difficult to completely remove and may lead to an undesirable host response towards an implanted biomaterial. In this study, we developed a modification for SDS cell extraction from muscle equally efficient to previous methods but leading to significantly less residual SDS remnants in the matrices. Muscle-derived matrices were prepared via 2 SDS-based decellularization methods, which led to removal of either 81.4% or 98.4% of the SDS. In vitro, matrices were seeded with thp1 macrophages and primary human foreskin fibroblasts. By Day 2, both matrices demonstrated similar macrophage polarization; however, fibroblasts cultured on matrices with greater residual SDS expressed higher levels of mRNA associated with fibroblast activation: α-smooth muscle actin and connective tissue growth factor. In vivo, Collagen I gels spiked with increasing concentrations of SDS displayed a corresponding decrease in cell infiltration when implanted subcutaneously in rats after 4 days. Finally, as a model for muscle regeneration, matrices produced by each method were implanted in rat latissimus dorsi defects. At POD 30 greater levels of IL-1β mRNA were present in defects treated with matrices containing higher levels of SDS, indicating a more severe inflammatory response. Although matrices containing higher levels of residual SDS became encapsulated by POD 30 and showed evidence of a foreign body response, matrices with the lower levels of SDS integrated into the defect area with lower levels of inflammatory and fibrosis-related gene expression.
• Sawicki, K. T., Chang, H. C., Shapiro, J. S., Bayeva, M., De Jesus, A., Finck, B. N., Wertheim, J. A., Blackshear, P. J., & Ardehali, H. (2018). Hepatic tristetraprolin promotes insulin resistance through RNA destabilization of FGF21. JCI insight, 3(13).
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  The role of posttranscriptional metabolic gene regulatory programs in diabetes is not well understood. Here, we show that the RNA-binding protein tristetraprolin (TTP) is reduced in the livers of diabetic mice and humans and is transcriptionally induced in response to insulin treatment in murine livers in vitro and in vivo. Liver-specific Ttp-KO (lsTtp-KO) mice challenged with high-fat diet (HFD) have improved glucose tolerance and peripheral insulin sensitivity compared with littermate controls. Analysis of secreted hepatic factors demonstrated that fibroblast growth factor 21 (FGF21) is posttranscriptionally repressed by TTP. Consistent with increased FGF21, lsTtp-KO mice fed HFD have increased brown fat activation, peripheral tissue glucose uptake, and adiponectin production compared with littermate controls. Downregulation of hepatic Fgf21 via an adeno-associated virus-driven shRNA in mice fed HFD reverses the insulin-sensitizing effects of hepatic Ttp deletion. Thus, hepatic TTP posttranscriptionally regulates systemic insulin sensitivity in diabetes through liver-derived FGF21.
• Su, J., Satchell, S. C., Shah, R. N., & Wertheim, J. A. (2018). Kidney decellularized extracellular matrix hydrogels: Rheological characterization and human glomerular endothelial cell response to encapsulation. Journal of biomedical materials research. Part A, 106(9), 2448-2462.
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  Hydrogels, highly-hydrated crosslinked polymer networks, closely mimic the microenvironment of native extracellular matrix (ECM) and thus present as ideal platforms for three-dimensional cell culture. Hydrogels derived from tissue- and organ-specific decellularized ECM (dECM) may retain bioactive signaling cues from the native tissue or organ that could in turn modulate cell-material interactions and response. In this study, we demonstrate that porcine kidney dECM can be processed to form hydrogels suitable for cell culture and encapsulation studies. Scanning electron micrographs of hydrogels demonstrated a fibrous ultrastructure with interconnected pores, and rheological analysis revealed rapid gelation times with shear moduli dependent upon the protein concentration of the hydrogels. Conditionally-immortalized human glomerular endothelial cells (GEnCs) cultured on top of or encapsulated within hydrogels exhibited high cell viability and proliferation over a one-week culture period. However, gene expression analysis of GEnCs encapsulated within kidney dECM hydrogels revealed significantly lower expression of several relevant genes of interest compared to those encapsulated within hydrogels composed of only purified collagen I. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2448-2462, 2018.
• Uzarski, J. S., Bijonowski, B. M., Wang, B., Wandinger-ness, A., Miller, W. M., Wertheim, J. A., & Ward, H. H. (2018). Correction to: Dual-Purpose Bioreactors to Monitor Noninvasive Physical and Biochemical Markers of Kidney and Liver Scaffold Recellularization, by Uzarski JS, Bijonowski BM, Wang B, Ward HH, Wandinger-Ness A, Miller WM, and Wertheim JA. Tissue Eng Part C Methods 2015;21(10):1032-1043. DOI: 10.1089/ten.tec.2014.0665.. Tissue engineering. Part C, Methods, 24(7), 441-442. doi:10.1089/ten.tec.2014.0665.correx
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  [This corrects the article DOI: 10.1089/ten.tec.2014.0665.].
• Giwa, S., Lewis, J. K., Alvarez, L., Langer, R., Roth, A. E., Church, G. M., Markmann, J. F., Sachs, D. H., Chandraker, A., Wertheim, J. A., Rothblatt, M., Boyden, E. S., Eidbo, E., Lee, W. P., Pomahac, B., Brandacher, G., Weinstock, D. M., Elliott, G., Nelson, D., , Acker, J. P., et al. (2017). The promise of organ and tissue preservation to transform medicine. Nature biotechnology, 35(6), 530-542.
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  The ability to replace organs and tissues on demand could save or improve millions of lives each year globally and create public health benefits on par with curing cancer. Unmet needs for organ and tissue preservation place enormous logistical limitations on transplantation, regenerative medicine, drug discovery, and a variety of rapidly advancing areas spanning biomedicine. A growing coalition of researchers, clinicians, advocacy organizations, academic institutions, and other stakeholders has assembled to address the unmet need for preservation advances, outlining remaining challenges and identifying areas of underinvestment and untapped opportunities. Meanwhile, recent discoveries provide proofs of principle for breakthroughs in a family of research areas surrounding biopreservation. These developments indicate that a new paradigm, integrating multiple existing preservation approaches and new technologies that have flourished in the past 10 years, could transform preservation research. Capitalizing on these opportunities will require engagement across many research areas and stakeholder groups. A coordinated effort is needed to expedite preservation advances that can transform several areas of medicine and medical science.
• Jiang, B., Suen, R., Wang, J. J., Zhang, Z. J., Wertheim, J. A., & Ameer, G. A. (2017). Vascular scaffolds with enhanced antioxidant activity inhibit graft calcification. Biomaterials, 144, 166-175.
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  There is a need for off-the-shelf, small-diameter vascular grafts that are safe and exhibit high long-term patency. Decellularized tissues can potentially be used as vascular grafts; however, thrombogenic and unpredictable remodeling properties such as intimal hyperplasia and calcification are concerns that hinder their clinical use. The objective of this study was to investigate the long-term function and remodeling of extracellular matrix (ECM)-based vascular grafts composited with antioxidant poly(1, 8-octamethylene-citrate-co-cysteine) (POCC) with or without immobilized heparin. Rat aortas were decellularized to create the following vascular grafts: 1) ECM hybridized with POCC (Poly-ECM), 2) Poly-ECM subsequently functionalized with heparin (Poly-ECM-Hep), and 3) non-modified vascular ECM. Grafts were evaluated as interposition grafts in the abdominal aorta of adult rats at three months. All grafts displayed antioxidant activity, were patent, and exhibited minimal intramural cell infiltration with varying degrees of calcification. Areas of calcification co-localized with osteochondrogenic differentiation of vascular smooth muscle cells, lipid peroxidation, oxidized DNA damage, and cell apoptosis, suggesting an important role for oxidative stress in the calcification of grafts. The extent of calcification within grafts was inversely proportional to their antioxidant activity: Poly-ECM-Hep > ECM > Poly-ECM. The incorporation of antioxidants into vascular grafts may be a viable strategy to inhibit degenerative changes.
• Oxburgh, L., Carroll, T. J., Cleaver, O., Gossett, D. R., Hoshizaki, D. K., Hubbell, J. A., Humphreys, B. D., Jain, S., Jensen, J., Kaplan, D. L., Kesselman, C., Ketchum, C. J., Little, M. H., McMahon, A. P., Shankland, S. J., Spence, J. R., Valerius, M. T., Wertheim, J. A., Wessely, O., , Zheng, Y., et al. (2017). (Re)Building a Kidney. Journal of the American Society of Nephrology : JASN, 28(5), 1370-1378.
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  (Re)Building a Kidney is a National Institute of Diabetes and Digestive and Kidney Diseases-led consortium to optimize approaches for the isolation, expansion, and differentiation of appropriate kidney cell types and the integration of these cells into complex structures that replicate human kidney function. The ultimate goals of the consortium are two-fold: to develop and implement strategies for engineering of replacement kidney tissue, and to devise strategies to stimulate regeneration of nephrons to restore failing kidney function. Projects within the consortium will answer fundamental questions regarding human gene expression in the developing kidney, essential signaling crosstalk between distinct cell types of the developing kidney, how to derive the many cell types of the kidney through directed differentiation of human pluripotent stem cells, which bioengineering or scaffolding strategies have the most potential for kidney tissue formation, and basic parameters of the regenerative response to injury. As these projects progress, the consortium will incorporate systematic investigations in physiologic function of and differentiated kidney tissue, strategies for engraftment in experimental animals, and development of therapeutic approaches to activate innate reparative responses.
• Uzarski, J. S., DiVito, M. D., Wertheim, J. A., & Miller, W. M. (2017). Essential design considerations for the resazurin reduction assay to noninvasively quantify cell expansion within perfused extracellular matrix scaffolds. Biomaterials, 129, 163-175.
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  Precise measurement of cellularity within bioartificial tissues and extracellular matrix (ECM) scaffolds is necessary to augment rigorous characterization of cellular behavior, as accurate benchmarking of tissue function to cell number allows for comparison of data across experiments and between laboratories. Resazurin, a soluble dye that is reduced to highly fluorescent resorufin in proportion to the metabolic activity of a cell population, is a valuable, noninvasive tool to measure cell number. We investigated experimental conditions in which resazurin reduction is a reliable indicator of cellularity within three-dimensional (3D) ECM scaffolds. Using three renal cell populations, we demonstrate that correlation of viable cell numbers with the rate of resorufin generation may deviate from linearity at higher cell densities, lower resazurin working volumes, or longer incubation times that all contribute to depleting the pool of resazurin. In conclusion, while the resazurin reduction assay provides a powerful, noninvasive readout of metrics enumerating cellularity and growth within ECM scaffolds, assay conditions may strongly influence its applicability for accurate quantification of cell number. The approach and methodological recommendations presented herein may be used as a guide for application-specific optimization of this assay to obtain rigorous and accurate measurement of cellular content in bioengineered tissues.
• Jiang, B., Suen, R., Wang, J. J., Zhang, Z. J., Wertheim, J. A., & Ameer, G. A. (2016). Mechanocompatible Polymer-Extracellular-Matrix Composites for Vascular Tissue Engineering. Advanced healthcare materials, 5(13), 1594-605.
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  Small-diameter vascular grafts developed from vascular extracellular matrix (ECM) can potentially be used for bypass surgeries and other vascular reconstruction and repair procedures. The addition of heparin to the ECM improves graft hemocompatibility but often involves chemical cross-linking, which increases ECM mechanical stiffness compared to native arteries. Herein, the importance of maintaining ECM mechanocompatibility is demonstrated, and a mechanocompatible strategy to immobilize heparin onto the ECM via a biodegradable elastomer is described. Specifically, poly(1,8-octamethylene citrate)-co-cysteine is hybridized to the ECM, forming a polymer-ECM composite that allows for heparin immobilization via maleimide-thiol "click" chemistry. Heparinized composites reduce platelet adhesion by >60% in vitro, without altering the elastic modulus of the ECM. In a rat abdominal aortic interposition model, intimal hyperplasia in heparinized mechanocompatible grafts is 65% lower when compared to ECM-only control grafts at four weeks. In contrast, grafts that are heparinized with carbodiimide chemistry exhibit increased intimal hyperplasia (4.2-fold) and increased macrophage infiltration (3.5-fold) compared to ECM-only control grafts. All grafts show similar, partial endothelial cell coverage and little to no ECM remodeling. Overall, a mechanocompatible strategy to improve ECM thromboresistance is described and the importance of ECM mechanical properties for proper in vivo graft performance is highlighted.
• Jiang, B., Suen, R., Wertheim, J. A., & Ameer, G. A. (2016). Targeting Heparin to Collagen within Extracellular Matrix Significantly Reduces Thrombogenicity and Improves Endothelialization of Decellularized Tissues. Biomacromolecules, 17(12), 3940-3948.
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  Thrombosis within small-diameter vascular grafts limits the development of bioartificial, engineered vascular conduits, especially those derived from extracellular matrix (ECM). Here we describe an easy-to-implement strategy to chemically modify vascular ECM by covalently linking a collagen binding peptide (CBP) to heparin to form a heparin derivative (CBP-heparin) that selectively binds a subset of collagens. Modification of ECM with CBP-heparin leads to increased deposition of functional heparin (by ∼7.2-fold measured by glycosaminoglycan composition) and a corresponding reduction in platelet binding (>70%) and whole blood clotting (>80%) onto the ECM. Furthermore, addition of CBP-heparin to the ECM stabilizes long-term endothelial cell attachment to the lumen of ECM-derived vascular conduits, potentially through recruitment of heparin-binding growth factors that ultimately improve the durability of endothelialization in vitro. Overall, our findings provide a simple yet effective method to increase deposition of functional heparin on the surface of ECM-based vascular grafts and thereby minimize thrombogenicity of decellularized tissue, overcoming a significant challenge in tissue engineering of bioartificial vessels and vascularized organs.
• Neubert, J., Wertheim, J. A., Atala, A., Allickson, J. G., & Hunsberger, J. G. (2016). Bioengineering Priorities on a Path to Ending Organ Shortage. Stem cell reports, 2(2), 118-127. doi:10.1007/s40778-016-0038-4
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  This perspective article covers current successes in and continuing challenges remaining in eliminating the growing organ shortage. We specifically cover data from a workshop entitled “Organ Bioengineering and Banking Roadmap Workshop” funded by the National Science Foundation (NSF) and the Methuselah Foundation in Washington, D.C. on May 27, 2015, and a subsequent Roundtable held at the White House Office of Science and Technology Policy (OSTP) on May 28, 2015. We address four parallel and potentially cooperative approaches for bioengineering tissues and organs. The first approach is bioprinting of tissues and organs. The second approach encompasses recellularization strategies, which can involve either developing tissue scaffolds from non-transplantable human (or xenogenic) organs or tissues and then reconstituting these templates with human cells to create a functional tissue/organ or seeding synthetic biodegradable scaffolds with human cells. The third approach is optimization of cellular repair and regeneration with strategies that include shifting the balance away from maladaptive processes that lead to chronic scarring. The fourth approach is xenotransplantation, which involves developing functional tissues for human use in transgenic animals whose cells are modified to prevent immune rejection. Current challenges and limitations are addressed, which include mapping, cell sourcing and manufacturing, immunosuppression, integration, and vascularization. We identify commercialization strategies that will make these approaches economically feasible. We present solutions toward a vision to one day ending the current organ and tissue shortage, and the impact this will have on treating disease and providing indirect economic benefit by decreasing the disease burden on society and improving quality of life.
• Tsukamoto, A., Abbot, S. E., Kadyk, L. C., DeWitt, N. D., Schaffer, D. V., Wertheim, J. A., Whittlesey, K. J., & Werner, M. J. (2016). Challenging Regeneration to Transform Medicine. Stem cells translational medicine, 5(1), 1-7.
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  The aging population in the U.S. and other developed countries has led to a large increase in the number of patients suffering from degenerative diseases. Transplantation surgery has been a successful therapeutic option for certain patients; however, the availability of suitable donor organs and tissues significantly limits the number of patients who can benefit from this approach. Regenerative medicine has witnessed numerous recent and spectacular advances, making the repair or replacement of dysfunctional organs and tissues an achievable goal. Public-private partnerships and government policies and incentives would further catalyze the development of universally available donor tissues, resulting in broad medical and economic benefits. This article describes a Regenerative Medicine Grand Challenge that the Alliance for Regenerative Medicine recently shared with the White House's Office of Science and Technology Policy in response to a White House call to action in scientific disciplines suggesting that the development of "universal donor tissues" should be designated as a Regenerative Medicine Grand Challenge. Such a designation would raise national awareness of the potential of regenerative medicine to address the unmet needs of many diseases and would stimulate the scientific partnerships and investments in technology needed to expedite this goal. Here we outline key policy changes and technological challenges that must be addressed to achieve the promise of a major breakthrough in the treatment of degenerative disease. A nationalized effort and commitment to develop universal donor tissues could realize this goal within 10 years and along the way result in significant innovation in manufacturing technologies.
• Wang, B., Jakus, A. E., Baptista, P. M., Soker, S., Soto-Gutierrez, A., Abecassis, M. M., Shah, R. N., & Wertheim, J. A. (2016). Functional Maturation of Induced Pluripotent Stem Cell Hepatocytes in Extracellular Matrix-A Comparative Analysis of Bioartificial Liver Microenvironments. Stem cells translational medicine, 5(9), 1257-67.
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  : Induced pluripotent stem cells (iPSCs) are new diagnostic and potentially therapeutic tools to model disease and assess the toxicity of pharmaceutical medications. A common limitation of cell lineages derived from iPSCs is a blunted phenotype compared with fully developed, endogenous cells. We examined the influence of novel three-dimensional bioartificial microenvironments on function and maturation of hepatocyte-like cells differentiated from iPSCs and grown within an acellular, liver-derived extracellular matrix (ECM) scaffold. In parallel, we also compared a bioplotted poly-l-lactic acid (PLLA) scaffold that allows for cell growth in three dimensions and formation of cell-cell contacts but is infused with type I collagen (PLLA-collagen scaffold) alone as a "deconstructed" control scaffold with narrowed biological diversity. iPSC-derived hepatocytes cultured within both scaffolds remained viable, became polarized, and formed bile canaliculi-like structures; however, cells grown within ECM scaffolds had significantly higher P450 (CYP2C9, CYP3A4, CYP1A2) mRNA levels and metabolic enzyme activity compared with iPSC hepatocytes grown in either bioplotted PLLA collagen or Matrigel sandwich control culture. Additionally, the rate of albumin synthesis approached the level of primary cryopreserved hepatocytes with lower transcription of fetal-specific genes, α-fetoprotein and CYP3A7, compared with either PLLA-collagen scaffolds or sandwich culture. These studies show that two acellular, three-dimensional culture systems increase the function of iPSC-derived hepatocytes. However, scaffolds derived from ECM alone induced further hepatocyte maturation compared with bioplotted PLLA-collagen scaffolds. This effect is likely mediated by the complex composition of ECM scaffolds in contrast to bioplotted scaffolds, suggesting their utility for in vitro hepatocyte assays or drug discovery.
• Wertheim, J. A. (2016). Novel technology for liver regeneration and replacement. Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society, 22(S1), 41-46.
• Caralt, M., Uzarski, J. S., Iacob, S., Obergfell, K. P., Berg, N., Bijonowski, B. M., Kiefer, K. M., Ward, H. H., Wandinger-ness, A., Miller, W. M., Zhang, Z. J., Abecassis, M. M., & Wertheim, J. A. (2015). Optimization and critical evaluation of decellularization strategies to develop renal extracellular matrix scaffolds as biological templates for organ engineering and transplantation.. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 15(1), 64-75. doi:10.1111/ajt.12999
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  The ability to generate patient-specific cells through induced pluripotent stem cell (iPSC) technology has encouraged development of three-dimensional extracellular matrix (ECM) scaffolds as bioactive substrates for cell differentiation with the long-range goal of bioengineering organs for transplantation. Perfusion decellularization uses the vasculature to remove resident cells, leaving an intact ECM template wherein new cells grow; however, a rigorous evaluative framework assessing ECM structural and biochemical quality is lacking. To address this, we developed histologic scoring systems to quantify fundamental characteristics of decellularized rodent kidneys: ECM structure (tubules, vessels, glomeruli) and cell removal. We also assessed growth factor retention--indicating matrix biofunctionality. These scoring systems evaluated three strategies developed to decellularize kidneys (1% Triton X-100, 1% Triton X-100/0.1% sodium dodecyl sulfate (SDS) and 0.02% Trypsin-0.05% EGTA/1% Triton X-100). Triton and Triton/SDS preserved renal microarchitecture and retained matrix-bound basic fibroblast growth factor and vascular endothelial growth factor. Trypsin caused structural deterioration and growth factor loss. Triton/SDS-decellularized scaffolds maintained 3 h of leak-free blood flow in a rodent transplantation model and supported repopulation with human iPSC-derived endothelial cells and tubular epithelial cells ex vivo. Taken together, we identify an optimal Triton/SDS-based decellularization strategy that produces a biomatrix that may ultimately serve as a rodent model for kidney bioengineering.
• Ferrer, J. R., Chokechanachaisakul, A., & Wertheim, J. A. (2015). New Tools in Experimental Cellular Therapy for the Treatment of Liver Diseases.. Current transplantation reports, 2(2), 202-210. doi:10.1007/s40472-015-0059-4
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  The current standard of care for end stage liver disease is orthotopic liver transplantation (OLT). Through improvement in surgical techniques, immunosuppression, and general medical care, liver transplantation has become an effective treatment over the course of the last half-century. Unfortunately, due to the limited availability of donor organs, there is a finite limit to the number of patients who will benefit from this therapy. This review will discuss current research in experimental cellular therapies for acute, chronic, and metabolic liver failure that may be appropriate when liver transplantation is not an immediate option.
• Jiang, B., Akgun, B., Lam, R. C., Ameer, G. A., & Wertheim, J. A. (2015). A polymer-extracellular matrix composite with improved thromboresistance and recellularization properties.. Acta biomaterialia, 18(Issue), 50-8. doi:10.1016/j.actbio.2015.02.015
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  Organ engineering using decellularized scaffolds is a potential long-term solution to donor organ shortage. However, this technology is severely limited by small vessel thrombosis due to incompletely recellularized vessels, resulting in exposure of extracellular matrix (ECM) components to platelets and clotting factors in flowing blood. To address this limitation, we designed a polymer-ECM composite and demonstrated its potential to reduce thrombosis and facilitate re-endothelialization in a vascular graft model. Rat aortas were decellularized using a sequential combination of weak detergents followed by a nuclease treatment that resulted in 96.5±1.3% DNA removal, while ECM components and mechanical properties were well maintained. A biodegradable and biocompatible elastomer poly(1,8 octanediol citrate) (POC, 1wt.%) was infused throughout the ECM at mild conditions (37°C and 45°C) and was functionalized with heparin using carbodiimide chemistry. The polymer-ECM composite significantly reduced platelet adhesion (67.4±8.2% and 82.7±9.6% reduction relative to untreated ECM using one of two processing temperatures, 37°C or 45°C, respectively); inhibited whole blood clotting (85.9±4.3% and 87.0±11.9% reduction relative to untreated ECM at 37°C or 45°C processing temperature, respectively); and supported endothelial cell-and to a lesser extent smooth muscle cell-adhesion in vitro. Taken together, this novel POC composite may provide a solution for thrombosis of small vessel conduits commonly seen in decellularized scaffolds used in tissue engineering applications.
• Jiang, B., Jen, M., Perrin, L., Wertheim, J. A., & Ameer, G. A. (2015). SIRT1 Overexpression Maintains Cell Phenotype and Function of Endothelial Cells Derived from Induced Pluripotent Stem Cells.. Stem cells and development, 24(23), 2740-5. doi:10.1089/scd.2015.0191
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  Endothelial cells (ECs) that are differentiated from induced pluripotent stem cells (iPSCs) can be used in establishing disease models for personalized drug discovery or developing patient-specific vascularized tissues or organoids. However, a number of technical challenges are often associated with iPSC-ECs in culture, including instability of the endothelial phenotype and limited cell proliferative capacity over time. Early senescence is believed to be the primary mechanism underlying these limitations. Sirtuin1 (SIRT1) is an NAD(+)-dependent deacetylase involved in the regulation of cell senescence, redox state, and inflammatory status. We hypothesize that overexpression of the SIRT1 gene in iPSC-ECs will maintain EC phenotype, function, and proliferative capacity by overcoming early cell senescence. SIRT1 gene was packaged into a lentiviral vector (LV-SIRT1) and transduced into iPSC-ECs at passage 4. Beginning with passage 5, iPSC-ECs exhibited a fibroblast-like morphology, whereas iPSC-ECs overexpressing SIRT1 maintained EC cobblestone morphology. SIRT1 overexpressing iPSC-ECs also exhibited a higher percentage of canonical markers of endothelia (LV-SIRT1 61.8% CD31(+) vs. LV-empty 31.7% CD31(+), P < 0.001; LV-SIRT1 46.3% CD144(+) vs. LV-empty 20.5% CD144(+), P < 0.02), with a higher nitric oxide synthesis, lower β-galactosidase production indicating decreased senescence (3.4% for LV-SIRT1 vs. 38.6% for LV-empty, P < 0.001), enhanced angiogenesis, increased deacetylation activity, and higher proliferation rate. SIRT1 overexpressing iPSC-ECs continued to proliferate through passage 9 with high purity of EC-like characteristics, while iPSC-ECs without SIRT1 overexpression became senescent after passage 5. Taken together, SIRT1 overexpression in iPSC-ECs maintains EC phenotype, improves EC function, and extends cell lifespan, overcoming critical hurdles associated with the use of iPSC-ECs in translational research.
• Laronda, M. M., Jakus, A. E., Whelan, K. A., Wertheim, J. A., Shah, R. N., & Woodruff, T. K. (2015). Initiation of puberty in mice following decellularized ovary transplant.. Biomaterials, 50(1), 20-9. doi:10.1016/j.biomaterials.2015.01.051
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  Clinical interventions to preserve fertility and restore hormone levels in female patients with therapy-induced ovarian failure are insufficient, particularly for pediatric cancer patients. Laparoscopic isolation of cortical ovarian tissue followed by cryopreservation with subsequent autotransplantation has temporarily restored fertility in at least 27 women who survived cancer, and aided in pubertal transition for one pediatric patient. However, reintroducing cancer cells through ovarian transplantation has been a major concern. Decellularization is a process of removing cellular material, while maintaining the organ skeleton of extracellular matrices (ECM). The ECM that remains could be stripped of cancer cells and reseeded with healthy ovarian cells. We tested whether a decellularized ovarian scaffold could be created, recellularized and transplanted to initiate puberty in mice. Bovine and human ovaries were decellularized, and the ovarian skeleton microstructures were characterized. Primary ovarian cells seeded onto decellularized scaffolds produced estradiol in vitro. Moreover, the recellularized grafts initiated puberty in mice that had been ovariectomized, providing data that could be used to drive future human transplants and have broader implications on the bioengineering of other organs with endocrine function.
• Leventhal, J. R., & Wertheim, J. A. (2015). Clinical implications of basic science discoveries: induced pluripotent stem cell therapy in transplantation--a potential role for immunologic tolerance.. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 15(4), 887-90. doi:10.1111/ajt.13155
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  Induced pluripotent stem cells (iPSCs) hold the potential for future development of genetically identical tissues from almost any mature cell lineage. For clinical applications in cell therapy and transplantation, it may provide a means to one-day restore dysfunctional or damaged tissue without the need for immunosuppression. A recent study by de Almeida et al published in the journal Nature Communications indicates that iPSCs may indeed elicit an immune response that evolves as cells differentiate toward maturity to induce a state of tolerance within a recipient animal. If these early findings hold true, it suggests a possible explanation for self-recognition of mature cells derived from iPSCs for use in future therapeutic interventions in transplantation such as cellular therapy or tissue engineering.
• Li, W., Ma, G., Brazile, B., Li, N., Dai, W., Butler, J. R., Claude, A. A., Wertheim, J. A., Liao, J., & Wang, B. (2015). Investigating the Potential of Amnion-Based Scaffolds as a Barrier Membrane for Guided Bone Regeneration.. Langmuir : the ACS journal of surfaces and colloids, 31(31), 8642-53. doi:10.1021/acs.langmuir.5b02362
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  Guided bone regeneration is a new concept of large bone defect therapy, which employs a barrier membrane to afford a protected room for osteogenesis and prevent the invasion of fibroblasts. In this study, we developed a novel barrier membrane made from lyophilized multilayered acellular human amnion membranes (AHAM). After decellularization, the AHAM preserved the structural and biomechanical integrity of the amnion extracellular matrix (ECM). The AHAM also showed minimal toxic effects when cocultured with mesenchymal stem cells (MSCs), as evidenced by high cell density, good cell viability, and efficient osteogenic differentiation after 21-day culturing. The effectiveness of the multilayered AHAM in guiding bone regeneration was evaluated using an in vivo rat tibia defect model. After 6 weeks of surgery, the multilayered AHAM showed great efficiency in acting as a shield to avoid the invasion of the fibrous tissues, stabilizing the bone grafts and inducing the massive bone growth. We hence concluded that the advantages of the lyophilized multilayered AHAM barrier membrane are as follows: preservation of the structural and mechanical properties of the amnion ECM, easiness for preparation and handling, flexibility in adjusting the thickness and mechanical properties to suit the application, and efficiency in inducing bone growth and avoiding fibrous tissues invasion.
• Uzarski, J. S., Bijonowski, B. M., Wang, B., Ward, H. H., Wandinger-ness, A., Miller, W. M., & Wertheim, J. A. (2015). Dual-Purpose Bioreactors to Monitor Noninvasive Physical and Biochemical Markers of Kidney and Liver Scaffold Recellularization.. Tissue engineering. Part C, Methods, 21(10), 1032-43. doi:10.1089/ten.tec.2014.0665
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  Analysis of perfusion-based bioreactors for organ engineering and a detailed evaluation of physical and biochemical parameters that measure dynamic changes within maturing cell-laden scaffolds are critical components of ex vivo tissue development that remain understudied topics in the tissue and organ engineering literature. Intricately designed bioreactors that house developing tissue are critical to properly recapitulate the in vivo environment, deliver nutrients within perfused media, and monitor physiological parameters of tissue development. Herein, we provide an in-depth description and analysis of two dual-purpose perfusion bioreactors that improve upon current bioreactor designs and enable comparative analyses of ex vivo scaffold recellularization strategies and cell growth performance during long-term maintenance culture of engineered kidney or liver tissues. Both bioreactors are effective at maximizing cell seeding of small-animal organ scaffolds and maintaining cell survival in extended culture. We further demonstrate noninvasive monitoring capabilities for tracking dynamic changes within scaffolds as the native cellular component is removed during decellularization and model human cells are introduced into the scaffold during recellularization and proliferate in maintenance culture. We found that hydrodynamic pressure drop (ΔP) across the retained scaffold vasculature is a noninvasive measurement of scaffold integrity. We further show that ΔP, and thus resistance to fluid flow through the scaffold, decreases with cell loss during decellularization and correspondingly increases to near normal values for whole organs following recellularization of the kidney or liver scaffolds. Perfused media may be further sampled in real time to measure soluble biomarkers (e.g., resazurin, albumin, or kidney injury molecule-1) that indicate degree of cellular metabolic activity, synthetic function, or engraftment into the scaffold. Cell growth within bioreactors is validated for primary and immortalized cells, and the design of each bioreactor is scalable to accommodate any three-dimensional scaffold (e.g., synthetic or naturally derived matrix) that contains conduits for nutrient perfusion to deliver media to growing cells and monitor noninvasive parameters during scaffold repopulation, broadening the applicability of these bioreactor systems.
• Uzarski, J. S., Su, J., Xie, Y., Zhang, Z. J., Ward, H. H., Wandinger-ness, A., Miller, W. M., & Wertheim, J. A. (2015). Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development.. Journal of visualized experiments : JoVE, 2015(102), e53271. doi:10.3791/53271
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  This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 °C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 µM resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.
• Uzarski, J. S., Xia, Y., Belmonte, J. C., & Wertheim, J. A. (2014). New strategies in kidney regeneration and tissue engineering.. Current opinion in nephrology and hypertension, 23(4), 399-405. doi:10.1097/01.mnh.0000447019.66970.ea
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  The severe shortage of suitable donor kidneys limits organ transplantation to a small fraction of patients suffering from end-stage renal failure. Engineering autologous kidney grafts on-demand would potentially alleviate this shortage, thereby reducing healthcare costs, improving quality of life, and increasing longevity for patients suffering from renal failure..Over the past 2 years, several studies have demonstrated that structurally intact extracellular matrix (ECM) scaffolds can be derived from human or animal kidneys through decellularization, a process in which detergent or enzyme solutions are perfused through the renal vasculature to remove the native cells. The future clinical paradigm would be to repopulate these decellularized kidney matrices with patient-derived renal stem cells to regenerate a functional kidney graft. Recent research aiming toward this goal has focused on the optimization of decellularization protocols, design of bioreactor systems to seed cells into appropriate compartments of the renal ECM to nurture their growth to restore kidney function, and differentiation of pluripotent stem cells (PSCs) into renal progenitor lineages..New research efforts utilizing bio-mimetic perfusion bioreactor systems to repopulate decellularized kidney scaffolds, coupled with the differentiation of PSCs into renal progenitor cell populations, indicate substantial progress toward the ultimate goal of building a functional kidney graft on-demand.
• Bijonowski, B. M., Miller, W. M., & Wertheim, J. A. (2013). Bioreactor design for perfusion-based, highly-vascularized organ regeneration.. Current opinion in chemical engineering, 2(1), 32-40. doi:10.1016/j.coche.2012.12.001
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  Bioartificial or laboratory-grown organs is a growing field centered on developing replacement organs and tissues to restore body function and providing a potential solution to the shortage of donor organs for transplantation. With the entry of engineered planar tissues, such as bladder and trachea, into clinical studies, an increasing focus is being given to designing complex, three-dimensional solid organs. As tissues become larger, thicker and more complex, the vascular network becomes crucial for supplying nutrients and maintaining viability and growth of the neo-organ. Perfusion decellularization, the process of removing cells from an entire organ, leaves the matrix of the vascular network intact. Organ engineering requires a delicate process of decellularization, sterilization, reseeding with appropriate cells, and organ maturation and stimulation to ensure optimal development. The design of bioreactors to facilitate this sequence of events has been refined to the extent that some bioartificial organs grown in these systems have been transplanted into recipient animals with sustained, though limited, function. This review focuses on the state-of-art in bioreactor development for perfusion-based bioartificial organs and highlights specific design components in need of further refinement.
• Soto-gutierrez, A., & Wertheim, J. A. (2013). The regeneration of organogenesis.. Organogenesis, 9(1), 1-2. doi:10.4161/org.24545
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  We would like to welcome our readers to the regenerated Organogenesis and to share with you the reasoning behind the refocusing of this journal toward the development of tissues and organs as a future means to improve the health of patients.
• Yersiz, H., Lee, C., Kaldas, F. M., Hong, J. C., Rana, A., Schnickel, G. T., Wertheim, J. A., Zarrinpar, A., Agopian, V. G., Gornbein, J., Naini, B. V., Lassman, C. R., Busuttil, R. W., & Petrowsky, H. (2013). Assessment of hepatic steatosis by transplant surgeon and expert pathologist: a prospective, double-blind evaluation of 201 donor livers.. Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society, 19(4), 437-49. doi:10.1002/lt.23615
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  An accurate clinical assessment of hepatic steatosis before transplantation is critical for successful outcomes after liver transplantation, especially if a pathologist is not available at the time of procurement. This prospective study investigated the surgeon's accuracy in predicting hepatic steatosis and organ quality in 201 adult donor livers. A steatosis assessment by a blinded expert pathologist served as the reference gold standard. The surgeon's steatosis estimate correlated more strongly with large-droplet macrovesicular steatosis [ld-MaS; nonparametric Spearman correlation coefficient (rS ) = 0.504] versus small-droplet macrovesicular steatosis (sd-MaS; rS = 0.398). True microvesicular steatosis was present in only 2 donors (1%). Liver texture criteria (yellowness, absence of scratch marks, and round edges) were mainly associated with ld-MaS (variance = 0.619) and were less associated with sd-MaS (variance = 0.264). The prediction of ≥30% ld-MaS versus
• Soto-gutierrez, A., Wertheim, J. A., Ott, H. C., & Gilbert, T. W. (2012). Perspectives on whole-organ assembly: moving toward transplantation on demand.. The Journal of clinical investigation, 122(11), 3817-23. doi:10.1172/jci61974
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  There is an ever-growing demand for transplantable organs to replace acute and chronically damaged tissues. This demand cannot be met by the currently available donor organs. Efforts to provide an alternative source have led to the development of organ engineering, a discipline that combines cell biology, tissue engineering, and cell/organ transplantation. Over the last several years, engineered organs have been implanted into rodent recipients and have shown modest function. In this article, we summarize the most recent advances in this field and provide a perspective on the challenges of translating this promising new technology into a proven regenerative therapy.
• Wertheim, J. A., Baptista, P. M., & Soto-gutierrez, A. (2012). Cellular therapy and bioartificial approaches to liver replacement.. Current opinion in organ transplantation, 17(3), 235-40. doi:10.1097/mot.0b013e3283534ec9
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  The success of liver transplantation has increased over the past 20 years due to improved immunosuppressive medications, surgical technique and donor-recipient selection. To date, the number of patients waiting for a liver transplant exceeds the number of transplants performed yearly by over a 2 : 1 ratio. Despite efforts to expand the donor pool, mortality of patients waiting for a liver remains high due to the shortage of donor organs. Herein, we discuss options for liver replacement that are currently under development..Extracorporeal bioactive liver perfusion devices were investigated in the late 1990s and preliminarily demonstrated safety but failed to show clinical efficacy. Current research is ongoing, but the focus has shifted to xenotransplantation of whole organs, organ engineering and cell transplantation. These new modalities are limited to small and large animal studies and each present unique advantages and limitations..Discovery of new sources of organs or cells to replace a damaged liver may be the only long-term solution to provide definitive therapy to all patients who require transplantation. The past 2 years have seen notable achievements in xenotransplantation, tissue engineering and cell transplantation. Though challenges remain, now identified, they may be readily solved.
• Wertheim, J. A., Petrowsky, H., Saab, S., Kupiec-weglinski, J. W., & Busuttil, R. W. (2011). Major challenges limiting liver transplantation in the United States.. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 11(9), 1773-84. doi:10.1111/j.1600-6143.2011.03587.x
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  Liver transplantation is the gold standard of care in patients with end-stage liver disease and those with tumors of hepatic origin in the setting of liver dysfunction. From 1988 to 2009, liver transplantation in the United States grew 3.7-fold from 1713 to 6320 transplants annually. The expansion of liver transplantation is chiefly driven by scientific breakthroughs that have extended patient and graft survival well beyond those expected 50 years ago. The success of liver transplantation is now its primary obstacle, as the pool of donor livers fails to keep pace with the growing number of patients added to the national liver transplant waiting list. This review focuses on three major challenges facing liver transplantation in the United States and discusses new areas of investigation that address each issue: (1) the need for an expanded number of useable donor organs, (2) the need for improved therapies to treat recurrent hepatitis C after transplantation and (3) the need for improved detection, risk stratification based upon tumor biology and molecular inhibitors to combat hepatocellular carcinoma.
• Saidi, R. F., Wertheim, J. A., Ko, D. S., Elias, N., Yeh, H., Hertl, M., Kawai, T., & Kennealey, P. T. (2010). Donor kidney recovery methods and the incidence of lymphatic complications in kidney transplant recipients.. International journal of organ transplantation medicine, 1(1), 40-3.
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  Lymphatic leak and lymphocele are well-known complications after kidney transplantation..To determine the incidence of lymphatic complications in recipients of living donor kidneys..Among 642 kidney transplants performed between 1999 and 2007, the incidence of lymphatic complications was retrospectively analyzed in recipients of living donor kidneys procured by laparoscopic nephrectomy (LP, n=218) or by open nephrectomy (OP, n=127) and deceased donor kidneys (DD, n=297). A Jackson-Pratt drain was placed in the retroperitoneal space in all recipients and was maintained until the output became less than 30 mL/day..Although the incidence of symptomatic lymphocele, which required therapeutic intervention, was comparable in all groups, the duration of mean±SD drain placement was significantly longer in the LP group-8.6±2.7 days compared to 5.6±1.2 days in the OP group and 5.4±0.7 days in the DD group (p
• Saidi, R. F., Wertheim, J. A., Ko, D. S., Elias, N., Martin, H., Delmonico, F. L., Cosimi, A. B., Kawai, T., & Ab, C. (2008). Impact of donor kidney recovery method on lymphatic complications in kidney transplantation.. Transplantation proceedings, 40(4), 1054-5. doi:10.1016/j.transproceed.2008.04.007
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  Prolonged lymphatic drainage and lymphocele are undesirable complications following kidney transplantation. We evaluated the impact of kidney recovery methods (deceased donor vs laparoscopic nephrectomy) on the lymphatic complications of the kidney transplant recipients..The incidence of lymphatic complications was retrospectively analyzed in recipients of deceased donor kidneys (DD, n = 62) versus laparoscopically procured kidneys from living donors (LP, n = 61). A drain was placed in the retroperitoneal space in all recipients. The drain was maintained until the output became less than 30 mL/d with no evidence of fluid collection by ultrasound examination..There was no statistically significant difference in the patient demographics (age, gender, and original disease and procedure time) between two groups. The incidence of lymphocele that required therapeutic intervention was comparable in both groups (3.2%). However, the duration of drain placement was significantly longer in the LP group than in the DD group, 8.6 +/- 2.5 days versus 5.4 +/- 2.5 day, respectively (P < .05)..The recipients of laparoscopically removed kidneys had a higher incidence of prolonged lymphatic leakage. More meticulous back table preparation may be required in LP kidneys to prevent prolonged lymphatic drainage after kidney transplantation. These observations may indicate that the major source of persistent lymphatic leakage is lymphatics of the allograft rather than severed recipient lymphatics.
• Stewart, C. A., Furth, E. E., Markman, J., Shaked, A., Brensinger, C. M., Olthoff, K. M., & Wertheim, J. A. (2004). Ascites after liver transplantation--a mystery.. Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society, 10(5), 654-60. doi:10.1002/lt.20106
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  Ascites after liver transplantation, although uncommon, presents a serious clinical dilemma. The hemodynamic changes that support the development of ascites before liver transplantation are resolved after transplant; therefore, persistent ascites (PA) after liver transplantation is unexpected and poorly characterized. The aim of this study was to define the clinical factors associated with PA after liver transplantation. This was a retrospective case-control analysis of patients who underwent liver transplantation at the University of Pennsylvania. PA occurring for more than 3 months after liver transplantation was confirmed by imaging studies. PA was correlated with multiple recipient and donor variables, including etiology of liver disease, preoperative ascites, prior portosystemic shunt (PS), donor age, and cold ischemic (CI) time. There were 2 groups: group 1, cases with PA transplanted from November 1990 to July 2001, and group 2, consecutive, control subjects who underwent liver transplantation between September 1999 and December 2001. Both groups were followed to censoring, May 2002, or death. Twenty-five from group 1 had ascites after liver transplantation after a median follow-up of 2.6 years. In group 1 vs group 2 (n = 106), there was a male predominance 80% vs 61% (P =.10) with similar age 52 years; chronic hepatitis C virus (HCV) was diagnosed in 88% vs 44% (P
• Wertheim, J. A., Perera, S. A., Hammer, D. A., Ren, R., Boettiger, D., & Pear, W. S. (2003). Localization of BCR-ABL to F-actin regulates cell adhesion but does not attenuate CML development.. Blood, 102(6), 2220-8. doi:10.1182/blood-2003-01-0062
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  We have previously found that P210BCR-ABL increases the adhesion of hematopoietic cell lines to fibronectin by a mechanism that is independent of tyrosine kinase activity. To investigate the pathway(s) by which P210BCR-ABL influences cell adhesion, we used a quantitative cell adhesion device that can discern small changes in cell adhesion to assay P210BCR-ABL with mutations in several critical domains. We expressed P210BCR-ABL mutants in 32D myeloblast cells and found that binding to fibronectin is mediated primarily by the alpha5beta1 integrin. We performed a structure/function analysis to map domains important for cell adhesion. Increased adhesion was mediated by 3 domains: (1) the N-terminal coiled-coil domain that facilitates oligomerization and F-actin localization; (2) bcr sequences between aa 163 to 210; and (3) F-actin localization through the C-terminal actin-binding domain of c-abl. We compared our adhesion results with the ability of these mutants to cause a chronic myelogenous leukemia (CML)-like disease in a murine bone marrow transplantation assay and found that adhesion to fibronectin did not correlate with the ability of these mutants to cause CML. Together, our results suggest that F-actin localization may play a pivotal role in modulating adhesion but that it is dispensable for the development of CML.
• He, Y., Wertheim, J. A., Xu, L., Miller, J. P., Karnell, F. G., Choi, J. K., Ren, R., & Pear, W. S. (2002). The coiled-coil domain and Tyr177 of bcr are required to induce a murine chronic myelogenous leukemia-like disease by bcr/abl.. Blood, 99(8), 2957-68. doi:10.1182/blood.v99.8.2957
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  The bcr/abl fusion in chronic myelogenous leukemia (CML) creates a chimeric tyrosine kinase with dramatically different properties than intact c-abl. In P210 bcr/abl, the bcr portion includes a coiled-coil oligomerization domain (amino acids 1-63) and a grb2-binding site at tyrosine 177 (Tyr177) that are critical for fibroblast transformation, but give variable results in other cell lines. To investigate the role of the coiled-coil domain and Tyr177 in promoting CML, 4 P210 bcr/abl-derived mutants containing different bcr domains fused to abl were constructed. All 4 mutants, Delta(1-63) bcr/abl, (1-63) bcr/abl, Tyr177Phe bcr/abl, and (1-210) bcr/abl exhibited elevated tyrosine kinase activity and conferred factor-independent growth in cell lines. In contrast, differences in the transforming potential of the 4 mutants occurred in our mouse model, in which all mice receiving P210 bcr/abl-expressing bone marrow cells exclusively develop a myeloproliferative disease (MPD) resembling human CML. Of the 4 mutants assayed, only 1-210 bcr/abl, containing both the coiled-coil domain and Tyr177, induced MPD. Unlike full-length P210, this mutant also caused a simultaneous B-cell acute lymphocytic leukemia (ALL). The other 3 mutants, (1-63) bcr/abl, Tyr177Phe bcr/abl, and Delta(1-63) bcr/abl, failed to induce an MPD but instead caused T-cell ALL. These results show that both the bcr coiled-coil domain and Tyr177 are required for MPD induction by bcr/abl and provide the basis for investigating downstream signaling pathways that lead to CML.
• Janowska-wieczorek, A., Majka, M., Wertheim, J. A., Turner, A. R., Ratajczak, M. Z., & Marquez-curtis, L. A. (2002). Bcr-abl-positive cells secrete angiogenic factors including matrix metalloproteinases and stimulate angiogenesis in vivo in Matrigel implants.. Leukemia, 16(6), 1160-6. doi:10.1038/sj.leu.2402486
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  To further elucidate the role of angiogenesis in the pathogenesis of chronic myelogenous leukemia (CML) we evaluated the effects of the bcr-abl translocation on the secretion of the angiogenic factors VEGF, FGF-2, HGF, IL-8 and matrix metalloproteinases (MMPs) as well as on the angiogenic potential in vivo of bcr-abl+ cells. First, we examined murine FL5.12 cells transfected with the bcr-abl constructs p185, p210 and p230 and found that the transfected cells secreted as much as four-fold more VEGF (p185 > p210 >p230) than wild-type (wt) cells, as well as MMP-9 and MMP-2. When Matrigel fragments containing these bcr-abl+ cells were implanted subcutaneously in SCID or Balb-C mice they became significantly more vascularized and hemoglobinized than implants containing normal or wt cells (p185 > p210 > p230). Similarly, we found that myeloblasts expanded from bone marrow (BM) CD34+ cells derived from Philadelphia-positive CML patients secreted up to 10 times more VEGF, FGF-2, HGF and IL-8 compared to myeloblasts derived from normal donors' BM CD34+ cells and that BM mononuclear cells (MNC) isolated from CML patients induced vascularization of Matrigel implants in mice. Moreover, we found that peripheral blood MNC expressed MMP-2 and membrane-type (MT)1-MMP in about 50% of CML patients studied, and MMP-9 in all of them. Furthermore, VEGF stimulated the secretion of MMP-9 in these primary CML cells. We conclude that stimulation of angiogenesis by angiogenic factors, including MMPs, could play an important role in the pathogenesis of CML, suggesting that therapies targeting the newly formed endothelium could be developed for CML.
• Wertheim, J. A., Forsythe, K., Druker, B. J., Boettiger, D., Pear, W. S., & Hammer, D. A. (2002). BCR-ABL-induced adhesion defects are tyrosine kinase-independent.. Blood, 99(11), 4122-30. doi:10.1182/blood.v99.11.4122
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  The t(9;22) chromosomal translocation results in expression of P210(BCR-ABL), a fusion protein necessary for the development of chronic myelogenous leukemia (CML). The constitutive activation of the P210(BCR-ABL) tyrosine kinase results in phosphorylation of multiple signaling pathways leading to the transformed phenotype. Additionally, extracellular interactions between P210(BCR-ABL)-expressing progenitor cells and bone marrow stroma may provide external signals that facilitate CML development. In contrast to the intracellular signaling pathways involved in CML, little is known about how P210(BCR-ABL) expression modifies cell-cell and cell-substratum interactions. To investigate the role of P210(BCR-ABL) in modulating cellular adhesion, we used a highly sensitive and quantitative cell detachment apparatus that measures the strength of association between a population of cells and an adhesive matrix. Our findings show that P210(BCR-ABL) expression increased adhesion nearly 2-fold between the myeloblastic cell line, 32D, and fibronectin compared to a control vector. We then investigated whether abnormal adhesion due to P210(BCR-ABL) expression was caused by its tyrosine kinase activity. A quantitative analysis of cell-fibronectin adhesion found that neither expression of a kinase-inactive P210(BCR-ABL) mutant in 32D cells or attenuation of kinase activity by STI571 (imatinib mesylate) in 32D cells transduced with wild-type P210(BCR-ABL) could correct the nearly 2-fold increase in cell-fibronectin adhesion. Similarly, STI571 treatment of Meg-01 cells, a P210(BCR-ABL)-expressing cell line derived from a patient in blast crisis, failed to inhibit adhesion to fibronectin. Together, our results indicate that changes in adhesion induced by P210(BCR-ABL) are independent of its tyrosine kinase activity.
• Wertheim, J. A., Miller, J. P., Xu, L., He, Y., & Pear, W. S. (2002). The biology of chronic myelogenous leukemia:mouse models and cell adhesion.. Oncogene, 21(56), 8612-28. doi:10.1038/sj.onc.1206089
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  Chronic myelogenous leukemia (CML) is a biphasic neoplasm of the bone marrow that is precipitated by the Philadelphia chromosome, a t(9;22) balanced translocation that encodes a constitutively activated nonreceptor tyrosine kinase termed P210(BCR-ABL). This oncoprotein has several intracellular functions; however, the most important effect of P210(BCR-ABL) leading to cell transformation is phosphorylation of signaling molecules through a constitutively active tyrosine kinase domain. Despite extensive knowledge of the structure and functional domains of BCR-ABL, its precise function in transformation is not known. Progress has been hampered, in part, by the lack of relevant CML models, as cell culture and in vitro assays do not mimic the pathogenesis of CML. Recently, there has been significant progress toward improving murine models that closely resemble human CML. This has allowed researchers to evaluate critical functions of BCR-ABL and has provided a model to test the efficacy of therapeutic medications that block these pathways. Our laboratory has developed two intersecting research programs to better understand the functioning of P210(BCR-ABL) in leukemogenesis. In one approach, we have developed a murine CML model by transferring HSCs that express BCR-ABL from a retroviral vector. All recipients develop a rapidly fatal MPD that shares several important features with CML. This model has been extremely useful for studying the function of BCR-ABL in the pathogenesis of CML. A second approach utilizes a quantitative cell detachment apparatus capable of measuring small changes in cell adhesion to investigate the mechanism by which P210(BCR-ABL) causes abnormal cell binding. Altered cell adhesion may contribute to the imbalance between proliferation and self-renewal in the hematopoietic progenitor compartment. To better understand the role abnormal adhesion may play in the development of leukemia, we have attempted to correlate the effects of functional P210(BCR-ABL) mutants in regulating adhesion and oncogenicity.
• Wertheim, J., Hammer, D., Boettiger, D., & Pear, W. (2000). Quantifiable adhesion assay using a spinning disk device to measure the adherent strength of BCR/ABL transformed cells to a fibronectin matrix. Annals of Biomedical Engineering, 28(1).
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  Chronic myelogenous leukemia (CML) is a myeloproliferative disorder precipitated by the formation of bcr/abl chromosomal translocation. A novel method for studying adhesion in cell lines transformed by bcr/abl is presented.
• Pitt, C. G., Wang, C. T., Shah, S. S., & Wertheim, J. A. (1997). Polymer-drug conjugates: Manipulation of drug delivery kinetics. Macromolecular Symposia, 123(1), 225-234. doi:10.1002/masy.19971230122
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  Three methods of manipulating the kinetics of hydrolysis of polymer conjugates were evaluated. It was demonstrated that either first-order, zero-order or S-shaped kinetic profiles could be achieved by systematic changes in the chemical composition of several series of model side-chain substituted polyacrylates. The changes in kinetics were shown to arise from an increase in the rate constant during solvolysis, resulting from predictable changes in either the water content, secondary structure, or LCST of the polymer conjugate.
• Shah, S. S., Wang, C. T., Pitt, C. G., & Wertheim, J. A. (1997). Polymer-drug conjugates: manipulating drug delivery kinetics using model LCST systems. Journal of Controlled Release, 45(1), 95-101. doi:10.1016/s0168-3659(96)01550-7
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  Model studies of the kinetics of hydrolysis of side chain substituted polymers exhibiting a lower critical solution temperature (LCST) were undertaken. Copolymers of N-isopropyl acrylamide (NiPAm) and N-acryloxy succinimide (AS) were prepared by radical polymerization of the monomers or by reaction of isopropylamine with poly(N-acryloxy succinimide). The LCSTs of the copolymers were measured by DSC and shown to depend on both the NiPAm/AS ratio and the copolymer microstructure. The kinetics of hydrolysis of the succinimide side chain were measured at temperatures below, at, and above the LCST of the copolymers. The LCST increased on conversion of the acryloxy succinimide groups to acrylic acid groups during the hydrolysis process. At temperatures below the LCST, the polymers were water soluble throughout the hydrolysis and exhibited first order kinetics. Above the LCST, the polymers were initially insoluble but became soluble as hydrolysis caused a progressive increase in the LCST. The rate of hydrolysis accelerated on dissolution. As a result, the kinetics exhibited either a zero order or an S-shaped profile. In the latter case the initial slow phase was dependent on the difference between the reaction temperature and the initial LCST.

Proceedings Publications

• Su, J., Satchell, S. C., Wertheim, J. A., & Shah, R. N. (2019). Poly(Ethylene glycol)-crosslinked gelatin hydrogel substrates with conjugated bioactive peptides as basement membrane mimic for endothelial cell culture. In 42nd Society for Biomaterials Annual Meeting and Exposition 2019: The Pinnacle of Biomaterials Innovation and Excellence, 40.
• Su, J., Satchell, S. C., Wertheim, J. A., & Shah, R. N. (2019). Poly(Ethylene glycol)-crosslinked gelatin hydrogel substrates with conjugated bioactive peptides as basement membrane mimic for endothelial cell culture. In Society for Biomaterials 2019 Annual Meeting & Exposition.
• Shah, S., Wertheim, J., & Pitt, C. (1995). Polymer-drug conjugates: Dependence of the rate of hydrolysis on the LCST. In 22nd International Symposium on Controlled Release of Bioactive Materials.