Dilip Thomas

Co-transfection of decorin and interleukin-10 modulates pro-fibrotic extracellular matrix gene expression in human tenocyte culture.

Extracellular matrix synthesis and remodelling are driven by increased activity of transforming growth factor beta 1 (TGF-β1). In tendon tissue repair, increased activity of TGF-β1 leads to progressive fibrosis. Decorin (DCN) and interleukin 10 (IL-10) antagonise pathological collagen synthesis by exerting a neutralising effect via downregulation of TGF-β1. Herein, we report that the delivery of DCN and IL-10 transgenes from a collagen hydrogel system supresses the constitutive expression of TGF-β1 and a range of pro-fibrotic extracellular matrix genes.

Scaffold and scaffold-free self-assembled systems in regenerative medicine.

Self-assembly in tissue engineering refers to the spontaneous chemical or biological association of components to form a distinct functional construct, reminiscent of native tissue. Such self-assembled systems have been widely used to develop platforms for the delivery of therapeutic and/or bioactive molecules and various cell populations. Tissue morphology and functional characteristics have been recapitulated in several self-assembled constructs, designed to incorporate stimuli responsiveness and controlled architecture through spatial confinement or field manipulation. In parallel, owing to substantial functional properties, scaffold-free cell-assembled devices have aided in the development of functional neotissues for various clinical targets. Herein, we discuss recent advancements and future aspirations in scaffold and scaffold-free self-assembled devices for regenerative medicine purposes. Biotechnol. Bioeng. 2016;113: 1155-1163.

Three-Dimensional Microgel Platform for the Production of Cell Factories Tailored for the Nucleus Pulposus

Intradiscal injection of growth factors or cells has been shown to attenuate symptoms of intervertebral disc degeneration. However, different approaches are needed to overcome limitations such as short-term efficacy and leakage of the injected solutions. The current study aims at creating a platform for the realization of functional cell factories by using in parallel cell delivery and gene therapy approaches. Superfect, a transfecting agent, was used as nonviral gene vector because of its ability to form complexes with plasmid DNA (polyplexes). Polyplexes were loaded into collagen hollow microsphere reservoirs, and their ability to transfect cells was ascertained in vitro. Adipose-derived stem cells were then embedded in three-dimensional (3D) microgels composed of type II collagen/hyaluronan, which mimics the environmental cues typical of the healthy nucleus pulposus. These were functionalized with polyplex-loaded collagen hollow spheres and the secretion of the target protein was assessed quantitatively. Delivery of polyplexes from a reservoir system lowered their toxicity significantly while maintaining high levels of transfection in a monolayer culture. In 3D microgels, lower levels of transfection were observed, however; increasing levels of luciferase were secreted from the microgels over 7 days of culture. These results indicate that 3D microgels, functionalized with polyplex-loaded reservoirs offer a reliable platform for the production of cell factories that are able to manufacture targeted therapeutic proteins for regenerative therapies that have applications in nucleus pulposus repair.

Variability in Endogenous Perfusion Recovery of Immunocompromised Mouse Models of Limb Ischemia.

Immunocompromised hind limb ischemia (HLI) murine models are essential for preclinical evaluation of human cell-based therapy or biomaterial-based interventions. These models are used to generate proof of principle that the approach is effective and also regulatory preclinical data required for translation to the clinic. However, surgical variations in creation of HLI models reported in the literature introduce variability in the pathological manifestation of the model, in consequence affecting therapeutic endpoints. This study aims to compare the extent of vascular regeneration in HLI-induced immunocompromised murine models to obtain a stable and more reproducible injury model for testing. Athymic and Balb/C nude mice underwent HLI surgery with single and double ligation of femoral artery (FA). The recovery from surgery was observed over a period of 2 weeks with respect to ischemia reperfusion using laser Doppler and clinical signs of necrosis and ambulatory impairment. Double ligation of the FA results in a more severe response to ischemia in Balb/C with endogenous perfusion recovery up to 50% ± 10% compared with 75% ± 20% in athymic nude mice. Single iliac artery (IA) and FA lead to creation of mild ischemia compared with femoral artery-vein (FAV) pair ligation in Balb/C. Microcirculatory parameters indicate significantly lower capillary numbers (26 ± 3/mm(2)) and functional capillary density (203 ± 5 cm/cm(2)) in the FAV group. In this study, we demonstrate a reproducible, arterial double ligation in an immunocompromised Balb/C nude mouse model that exhibits characteristic pathological signs of ischemia with impaired endogenous recovery.

Toward Customized Extracellular Niche Engineering: Progress in Cell‐Entrapment Technologies

The primary aim in tissue engineering is to repair, replace, and regenerate dysfunctional tissues to restore homeostasis. Cell delivery for repair and regeneration is gaining impetus with our understanding of constructing tissue-like environments. However, the perpetual challenge is to identify innovative materials or re-engineer natural materials to model cell-specific tissue-like 3D modules, which can seamlessly integrate and restore functions of the target organ. To devise an optimal functional microenvironment, it is essential to define how simple is complex enough to trigger tissue regeneration or restore cellular function. Here, the purposeful transition of cell immobilization from a cytoprotection point of view to that of a cell-instructive approach is examined, with advances in the understanding of cell-material interactions in a 3D context, and with a view to further application of the knowledge for the development of newer and complex hierarchical tissue assemblies for better examination of cell behavior and offering customized cell-based therapies for tissue engineering.

The Functional Response of Mesenchymal Stem Cells to Electron‐Beam Patterned Elastomeric Surfaces Presenting Micrometer to Nanoscale Heterogeneous Rigidity

Cells directly probe and respond to the physicomechanical properties of their extracellular environment, a dynamic process which has been shown to play a key role in regulating both cellular adhesive processes and differential cellular function. Recent studies indicate that stem cells show lineage-specific differentiation when cultured on substrates approximating the stiffness profiles of specific tissues. Although tissues are associated with a range of Youngs modulus values for bulk rigidity, at the subcellular level, tissues are comprised of heterogeneous distributions of rigidity. Lithographic processes have been widely explored in cell biology for the generation of analytical substrates to probe cellular physicomechanical responses. In this work, it is shown for the first time that that direct-write e-beam exposure can significantly alter the rigidity of elastomeric poly(dimethylsiloxane) substrates and a new class of 2D elastomeric substrates with controlled patterned rigidity ranging from the micrometer to the nanoscale is described. The mechanoresponse of human mesenchymal stem cells to e-beam patterned substrates was subsequently probed in vitro and significant modulation of focal adhesion formation and osteochondral lineage commitment was observed as a function of both feature diameter and rigidity, establishing the groundwork for a new generation of biomimetic material interfaces.

Allogeneic Mesenchymal Stromal Cells (MSCs) are of Comparable Efficacy to Syngeneic MSCs for Therapeutic Revascularization in C57BKSdb/db Mice Despite the Induction of Alloantibody

Intramuscular administration of mesenchymal stromal cells (MSCs) represents a therapeutic option for diabetic critical limb ischemia. Autologous or allogeneic approaches may be used but disease-induced cell dysfunction may limit therapeutic efficacy in the former. Our aim was to compare the efficacy of allogeneic and autologous MSC transplantation in a model of hindlimb ischemia in diabetes mellitus and to determine whether allogeneic transplantation would result in the activation of an immune response. MSCs were isolated from C57BL/6 (B6) and diabetic obese C57BKSdb/db mice. Phosphate-buffered saline (control group), and MSCs (1 × 106) from B6 (allogeneic group) or C57BKSdb/db (syngeneic group) were administered intramuscularly into the ischemic thigh of C57BKSdb/db mice following the induction of hindlimb ischemia. MSCs derived from both mouse strains secrete several angiogenic factors, suggesting that the potential therapeutic effect is due to paracrine signaling. Administration of allogeneic MSCs significantly improved blood perfusion as compared with the control group on week 2 and 3, post-operatively. In comparison with the control group, syngeneic MSCs significantly improved blood perfusion at week 2 only. There was no statistical difference in blood perfusion between allogeneic and syngeneic MSC groups at any stages. There was no statistical difference in ambulatory and necrosis score among the three groups. Amputation of toes was only observed in the control group (one out of seven animals). Alloantibody was detected in three out of the eight mice that received allogeneic MSCs but was not observed in the other groups. In summary, we demonstrated comparable efficacy after transplantation of autologous and allogeneic MSCs in a diabetic animal model despite generation of an immune response.

Publisher Correction: Stimulation of 3D osteogenesis by mesenchymal stem cells using a nanovibrational bioreactor

In the version of this Article originally published, in Fig. 4f, the asterisk was missing; in Fig. 6a–c, the labels ‘Wnt/β-catenin signalling’, ‘Wnt/Ca+ pathway’ and ‘ERK’ and their associated lines/arrows were missing; and in Fig. 6d and in the sentence beginning “In MSCs that were...”, ‘myosin’ and ‘nanostimulated’, respectively, were spelt incorrectly. These errors have now been corrected in all versions of the Article.