Riccardo Tamburrini

Heterogeneity of Scaffold Biomaterials in Tissue Engineering

Tissue engineering (TE) offers a potential solution for the shortage of transplantable organs and the need for novel methods of tissue repair. Methods of TE have advanced significantly in recent years, but there are challenges to using engineered tissues and organs including but not limited to: biocompatibility, immunogenicity, biodegradation, and toxicity. Analysis of biomaterials used as scaffolds may, however, elucidate how TE can be enhanced. Ideally, biomaterials should closely mimic the characteristics of desired organ, their function and their in vivo environments. A review of biomaterials used in TE highlighted natural polymers, synthetic polymers, and decellularized organs as sources of scaffolding. Studies of discarded organs supported that decellularization offers a remedy to reducing waste of donor organs, but does not yet provide an effective solution to organ demand because it has shown varied success in vivo depending on organ complexity and physiological requirements. Review of polymer-based scaffolds revealed that a composite scaffold formed by copolymerization is more effective than single polymer scaffolds because it allows copolymers to offset disadvantages a single polymer may possess. Selection of biomaterials for use in TE is essential for transplant success. There is not, however, a singular biomaterial that is universally optimal.

A step towards clinical application of acellular matrix: A clue from macrophage polarization.

The outcome of tissue engineered organ transplants depends on the capacity of the biomaterial to promote a pro-healing response once implanted in vivo. Multiple studies, including ours, have demonstrated the possibility of using the extracellular matrix (ECM) of animal organs as platform for tissue engineering and more recently, discarded human organs have also been proposed as scaffold source. In contrast to artificial biomaterials, natural ECM has the advantage of undergoing continuous remodeling which allows adaptation to diverse conditions. It is known that natural matrices present diverse immune properties when compared to artificial biomaterials. However, how these properties compare between diseased and healthy ECM and artificial scaffolds has not yet been defined. To answer this question, we used decellularized renal ECM derived from WT mice and from mice affected by Alport Syndrome at different time-points of disease progression as a model of renal failure with extensive fibrosis. We characterized the morphology and composition of these ECMs and compared their in vitro effects on macrophage activation with that of synthetic scaffolds commonly used in the clinic (collagen type I and poly-L-(lactic) acid, PLLA). We showed that ECM derived from Alport kidneys differed in fibrous protein deposition and cytokine content when compared to ECM derived from WT kidneys. Yet, both WT and Alport renal ECM induced macrophage differentiation mainly towards a reparative (M2) phenotype, while artificial biomaterials towards an inflammatory (M1) phenotype. Anti-inflammatory properties of natural ECMs were lost when homogenized, hence three-dimensional structure of ECM seems crucial for generating an anti-inflammatory response. Together, these data support the notion that natural ECM, even if derived from diseased kidneys promote a M2 protolerogenic macrophage polarization, thus providing novel insights on the applicability of ECM obtained from discarded organs as ideal scaffold for tissue engineering.

Regenerative immunology: the immunological reaction to biomaterials

Regenerative medicine promises to meet two of the most urgent needs of modern organ transplantation, namely immunosuppression‐free transplantation and an inexhaustible source of organs. Ideally, bioengineered organs would be manufactured from a patients own biomaterials—both cells and the supporting scaffolding materials in which cells would be embedded and allowed to mature to eventually regenerate the organ in question. While some groups are focusing on the feasibility of this approach, few are focusing on the immunogenicity of the scaffolds that are being developed for organ bioengineering purposes. This review will succinctly discuss progress in the understanding of immunological characteristics and behavior of different scaffolds currently under development, with emphasis on the extracellular matrix scaffolds obtained decellularized animal or human organs which seem to provide the ideal template for bioengineering purposes.

Autologous Cells for Kidney Bioengineering

Worldwide, increasing numbers of patients are developing end-stage renal disease, and at present, the only treatment options are dialysis or kidney transplantation. Dialysis is associated with increased morbidity and mortality, poor life quality and high economic costs. Transplantation is by far the better option, but there are insufficient numbers of donor kidneys available. Therefore, there is an urgent need to explore alternative approaches. In this review, we discuss how this problem could potentially be addressed by using autologous cells and appropriate scaffolds to develop ‘bioengineered’ kidneys for transplantation. In particular, we will highlight recent breakthroughs in pluripotent stem cell biology that have led to the development of autologous renal progenitor cells capable of differentiating to all renal cell types and will discuss how these cells could be combined with appropriate scaffolds to develop a bioengineered kidney.

Tissue-Engineering Approaches to Restore Kidney Function

Kidney transplantation for the treatment of chronic kidney disease has established outcome and quality of life. However, its implementation is severely limited by a chronic shortage of donor organs; consequently, most candidates remain on dialysis and on the waiting list while accruing further morbidity and mortality. Furthermore, those patients that do receive kidney transplants are committed to a life-long regimen of immunosuppressive drugs that also carry significant adverse risk profiles. The disciplines of tissue engineering and regenerative medicine have the potential to produce alternative therapies which circumvent the obstacles posed by organ shortage and immunorejection. This review paper describes some of the most promising tissue-engineering solutions currently under investigation for the treatment of acute and chronic kidney diseases. The various stem cell therapies, whole embryo transplantation, and bioengineering with ECM scaffolds are outlined and summarized.

Kidney transplantation, bioengineering and regeneration: an originally immunology-based discipline destined to transition towards ad hoc organ manufacturing and repair

ABSTRACT Kidney transplantation (KT), as a modality of renal replacement therapy (RRT), has been shown to be both economically and functionally superior to dialysis for the treatment of end-stage renal disease (ESRD). Progress in KT is limited by two major barriers: a) a chronic and burgeoning shortage of transplantable organs and b) the need for chronic immunosuppression following transplantation. Although ground-breaking advances in transplant immunology have improved patient survival and graft durability, a new pathway of innovation is needed in order to overcome current obstacles. Regenerative medicine (RM) holds the potential to shift the paradigm in RRT, through organ bioengineering. Manufactured organs represent a potentially inexhaustible source of transplantable grafts that would bypass the need for immunosuppressive drugs by using autologous cells to repopulate extracellular matrix (ECM) scaffolds. This overview discusses the current status of renal transplantation while reviewing the most promising innovations in RM therapy as applied to RRT.

Extracellular matrix scaffolds as a platform for kidney regeneration

Chronic and end stage renal disease (ESRD) have reached pandemic levels and pose a substantial public health burden. Unfortunately, available therapies lack efficacy in preventing progression to its end stage phase. Regenerative medicine promises to restore function of diseased organs among which the kidney, through two possible approaches: firstly, the maximization of the innate ability of tissues to repair or regenerate following injury; secondly, the ex vivo bio-fabrication of the organ in question. When regenerative medicine is applied to the setting of chronic or ESRD, it is intuitive that endeavors to improve renal repair, promote nephrogenesis in damaged kidneys, or the de novo engineering of transplantable kidneys, could have a major impact on the current management of this pandemic. Among the different regenerative medicine technologies currently under development, cell-on-scaffold seeding technology (CSST) - involving cells seeded throughout supporting scaffold structures made from biomaterials - is the most favorable candidate in the context of realistic clinical application. In this review, we outline and describe current investigations taking place in the field of CSST as it pertains to the restoration of kidney function.

Selective Osmotic Shock (SOS)-Based Islet Isolation for Microencapsulation.

Islet transplantation (IT) has recently been shown to be a promising alternative to pancreas transplantation for reversing diabetes. IT requires the isolation of the islets from the pancreas, and these islets can be used to fabricate a bio-artificial pancreas. Enzymatic digestion is the current gold standard procedure for islet isolation but has lingering concerns. One such concern is that it has been shown to damage the islets due to nonselective tissue digestion. This chapter provides a detailed description of a nonenzymatic method that we are exploring in our lab as an alternative to current enzymatic digestion procedures for islet isolation from human and nonhuman pancreatic tissues. This method is based on selective destruction and protection of specific cell types and has been shown to leave the extracellular matrix (ECM) of islets intact, which may thus enhance islet viability and functionality. We also show that these SOS-isolated islets can be microencapsulated for transplantation.

Glycosaminoglycans as a measure of outcome of cell-on-scaffold seeding (decellularization) technology.

IRCCS Policlinico San Matteo, Pavia, Italy; Department of Surgery, University of Pavia, Pavia, Italy; Wake Forest University School of Medicine, Winston Salem, USA; Pole de Chirurgie Experimentale et Transplantation (CHEX), Institut de Recherche Experimentale et Clinique (IREC), Université Catholique de Louvain, Brussels, Belgium; Pole de Morphologie (MORF), Institut de Recherche Experimentale et Clinique (IREC), Université catholique de Louvain, Brussels, Belgium; Department of Surgery, Wake Forest University School of Medicine, Winston Salem, USA

DCD and AKI Allografts Offer a Valuable Platform to Study Molecular Pathways Underlying Successful Adaptive Repair of the Kidney after Damage

Introduction Although the increased incidence of DGF in AKI/DCD recipients, DCD/AKI grafts typically recover and resume an excellent kidney function within one month from the surgery. Therefore, we hypothesize that comparative analysis of peripheral blood and renal allograft tissue throughout the initial 30 days of follow up after a kidney transplant will result in the identification and characterization of ongoing mechanisms of regeneration and repair (RR). Methods Peripheral blood was collected from kidney transplant (KT) patients immediately prior to transplant, immediately after transplant (for up to 5 consecutive days), twice weekly for the next 3 weeks, and at post-transplant day 30 following transplantation of living donor (LD, control group), DCD or AKI donor grafts. Total RNA was isolated from each patient sample and assayed on whole genome microarrays. Longitudinal gene expression was evaluated to identify molecular pathways and processes involved in the recovery phase following KT. Results Remarkably, comparative analysis of longitudinal peripheral blood gene expression between recipients of AKI/DCD vs. LD grafts revealed two significant gene clusters, representing a total of 141 genes that show a different expression profile between AKI/DCD and LD samples. The 77 transcripts that comprise cluster 1 show a significant over-representation of genes in several key biological pathways including mTOR signaling, Granzyme B Signaling, and Th1 and Th2 Activation and wnt Pathway. Immunological Disease is the top diseases and disorders category over-represented in this cluster. The second cluster of differentially-expressed genes (N=64) in the comparison between AKI/DCD and LD are significantly over-represented in Toll-like Receptor Signaling, Adrenergic signaling, and fMLP Signaling in neutrophils pathways. The most significant diseases and disorders category over-represented in the set of transcripts is Inflammatory Response. The 141 transcripts that are differentially expressed in the longitudinal comparison between LD and AKI/DCD include genes found to be different in LD vs AKI only (N=65), LD vs DCD only (N=63) and LD vs AKI + DCD. The 13 transcripts found to differentially-expressed in both DCD and AKI samples compared to LD may represent the most biologically relevant gene targets differentiating the two groups and warrant further investigation. Conclusions The pattern of gene expression in the peripheral blood of KT patients is significantly different, depending upon the source of donor graft. These markedly different expression patterns, particularly the coordinate elevation hundreds of expressed genes seen in DCD and AKI recipients beyond post-transplant day 1, provide further insight into mechanisms (and timing) of kidney RR that occurs following kidney transplantation.