Pro-regenerative Environment Research Articles

Design, clinical translation and immunological response of biomaterials in regenerative medicine

The field of regenerative medicine aims to replace tissues lost as a consequence of disease, trauma or congenital abnormalities. Biomaterials serve as scaffolds for regenerative medicine to deliver cells, provide biological signals and physical support, and mobilize endogenous cells to repair tissues. Sophisticated chemistries are used to synthesize materials that mimic and modulate native tissue microenvironments, to replace form and to elucidate structure–function relationships of cell–material interactions. The therapeutic relevance of these biomaterial properties can only be studied after clinical translation, whereby key parameters for efficacy can be defined and then used for future design. In this Review, we present the development and translation of biomaterials for two tissue engineering targets, cartilage and cornea, both of which lack the ability to self-repair. Finally, looking to the future, we discuss the role of the immune system in regeneration and the potential for biomaterial scaffolds to modulate immune signalling to create a pro-regenerative environment. The clinical translation of biomaterials for tissue engineering reveals their therapeutic performance and relevance, and thus enables the improvement of biomaterials design. In this Review, the design and translation of biomaterials, particularly cartilage and cornea repair, and a new understanding of the interaction between biomaterials and the host immune system are discussed.

Foxn1 Transcription Factor Regulates Wound Healing of Skin through Promoting Epithelial-Mesenchymal Transition.

Transcription factors are key molecules that finely tune gene expression in response to injury. We focused on the role of a transcription factor, Foxn1, whose expression is limited to the skin and thymus epithelium. Our previous studies showed that Foxn1 inactivity in nude mice creates a pro-regenerative environment during skin wound healing. To explore the mechanistic role of Foxn1 in the skin wound healing process, we analyzed post-injured skin tissues from Foxn1::Egfp transgenic and C57BL/6 mice with Western Blotting, qRT-PCR, immunofluorescence and flow cytometric assays. Foxn1 expression in non-injured skin localized to the epidermis and hair follicles. Post-injured skin tissues showed an intense Foxn1-eGFP signal at the wound margin and in leading epithelial tongue, where it co-localized with keratin 16, a marker of activated keratinocytes. This data support the concept that suprabasal keratinocytes, expressing Foxn1, are key cells in the process of re-epithelialization. The occurrence of an epithelial-mesenchymal transition (EMT) was confirmed by high levels of Snail1 and Mmp-9 expression as well as through co-localization of vimentin/E-cadherin-positive cells in dermis tissue at four days post-wounding. Involvement of Foxn1 in the EMT process was verified by co-localization of Foxn1-eGFP cells with Snail1 in histological sections. Flow cytometric analysis showed the increase of double positive E-cadherin/N-cadherin cells within Foxn1-eGFP population of post-wounded skin cells isolates, which corroborated histological and gene expression analyses. Together, our findings indicate that Foxn1 acts as regulator of the skin wound healing process through engagement in re-epithelization and possible involvement in scar formation due to Foxn1 activity during the EMT process.

Evidence for Mechanisms Underlying the Functional Benefits of a Myocardial Matrix Hydrogel for Post-MI Treatment

BackgroundThere is increasing need for better therapies to prevent the development of heart failure after myocardial infarction (MI). An injectable hydrogel derived from decellularized porcine ventricular myocardium has been shown to halt the post-infarction progression of negative left ventricular remodeling and decline in cardiac function in both small and large animal models. ObjectivesThis study sought to elucidate the tissue-level mechanisms underlying the therapeutic benefits of myocardial matrix injection. MethodsMyocardial matrix or saline was injected into infarcted myocardium 1 week after ischemia–reperfusion in Sprague–Dawley rats. Cardiac function was evaluated by magnetic resonance imaging and hemodynamic measurements at 5 weeks after injection. Whole transcriptome microarrays were performed on RNA isolated from the infarct at 3 days and 1 week after injection. Quantitative polymerase chain reaction and histologic quantification confirmed expression of key genes and their activation in altered pathways. ResultsPrincipal component analysis of the transcriptomes showed that samples collected from myocardial matrix-injected infarcts are distinct and cluster separately from saline-injected control subjects. Pathway analysis indicated that these differences are due to changes in several tissue processes that may contribute to improved cardiac healing after MI. Matrix-injected infarcted myocardium exhibits an altered inflammatory response, reduced cardiomyocyte apoptosis, enhanced infarct neovascularization, diminished cardiac hypertrophy and fibrosis, altered metabolic enzyme expression, increased cardiac transcription factor expression, and progenitor cell recruitment, along with improvements in global cardiac function and hemodynamics. ConclusionsThese results indicate that the myocardial matrix alters several key pathways after MI creating a pro-regenerative environment, further demonstrating its promise as a potential post-MI therapy.

A new three dimensional biomimetic hydrogel to deliver factors secreted by human mesenchymal stem cells in spinal cord injury

Stem cell therapy with human mesenchymal stem cells (hMSCs) represents a promising strategy in spinal cord injury (SCI). However, both systemic and parenchymal hMSCs administrations show significant drawbacks as a limited number and viability of stem cells in situ. Biomaterials able to encapsulate and sustain hMSCs represent a viable approach to overcome these limitations potentially improving the stem cell therapy. In this study, we evaluate a new agarose/carbomer based hydrogel which combines different strategies to optimize hMSCs viability, density and delivery of paracrine factors. Specifically, we evaluate a new loading procedure on a lyophilized scaffold (soaked up effect) that reduces mechanical stress in encapsulating hMSCs into the hydrogel. In addition, we combine arginine–glycine–aspartic acid (RGD) tripeptide and 3D extracellular matrix deposition to increase the capacity to attach and maintain healthy hMSCs within the hydrogel over time. Furthermore, the fluidic diffusion from the hydrogel toward the injury site is improved by using a cling film that oriented efficaciously the delivery of paracrine factors in vivo. Finally, we demonstrate that an improved combination as here proposed of hMSCs and biomimetic hydrogel is able to immunomodulate significantly the pro-inflammatory environment in a SCI mouse model, increasing M2 macrophagic population and promoting a pro-regenerative environment in situ.

Improvement of diaphragmatic performance through orthotopic application of decellularized extracellular matrix patch

Muscle tissue engineering can provide support to large congenital skeletal muscle defects using scaffolds able to allow cell migration, proliferation and differentiation. Acellular extracellular matrix (ECM) scaffold can generate a positive inflammatory response through the activation of anti-inflammatory T-cell populations and M2 polarized macrophages that together lead to a local pro-regenerative environment. This immunoregulatory effect is maintained when acellular matrices are transplanted in a xenogeneic setting, but it remains unclear whether it can be therapeutic in a model of muscle diseases. We demonstrated here for the first time that orthotopic transplantation of a decellularized diaphragmatic muscle from wild animals promoted tissue functional recovery in an established atrophic mouse model. In particular, ECM supported a local immunoresponse activating a pro-regenerative environment and stimulating host muscle progenitor cell activation and migration. These results indicate that acellular scaffolds may represent a suitable regenerative medicine option for improving performance of diseased muscles.

Surgical Sutures Filled with Adipose-Derived Stem Cells Promote Wound Healing

Delayed wound healing and scar formation are among the most frequent complications after surgical interventions. Although biodegradable surgical sutures present an excellent drug delivery opportunity, their primary function is tissue fixation. Mesenchymal stem cells (MSC) act as trophic mediators and are successful in activating biomaterials. Here biodegradable sutures were filled with adipose-derived mesenchymal stem cells (ASC) to provide a pro-regenerative environment at the injured site. Results showed that after filling, ASCs attach to the suture material, distribute equally throughout the filaments, and remain viable in the suture. Among a broad panel of cytokines, cell-filled sutures constantly release vascular endothelial growth factor to supernatants. Such conditioned media was evaluated in an in vitro wound healing assay and showed a significant decrease in the open wound area compared to controls. After suturing in an ex vivo wound model, cells remained in the suture and maintained their metabolic activity. Furthermore, cell-filled sutures can be cryopreserved without losing their viability. This study presents an innovative approach to equip surgical sutures with pro-regenerative features and allows the treatment and fixation of wounds in one step, therefore representing a promising tool to promote wound healing after injury.

Fcγ receptor-mediated inflammation inhibits axon regeneration.

Anti-glycan/ganglioside antibodies are the most common immune effectors found in patients with Guillain-Barré Syndrome, which is a peripheral autoimmune neuropathy. We previously reported that disease-relevant anti-glycan autoantibodies inhibited axon regeneration, which echo the clinical association of these antibodies and poor recovery in Guillain-Barré Syndrome. However, the specific molecular and cellular elements involved in this antibody-mediated inhibition of axon regeneration are not previously defined. This study examined the role of Fcγ receptors and macrophages in the antibody-mediated inhibition of axon regeneration. A well characterized antibody passive transfer sciatic nerve crush and transplant models were used to study the anti-ganglioside antibody-mediated inhibition of axon regeneration in wild type and various mutant and transgenic mice with altered expression of specific Fcγ receptors and macrophage/microglia populations. Outcome measures included behavior, electrophysiology, morphometry, immunocytochemistry, quantitative real-time PCR, and western blotting. We demonstrate that the presence of autoantibodies, directed against neuronal/axonal cell surface gangliosides, in the injured mammalian peripheral nerves switch the proregenerative inflammatory environment to growth inhibitory milieu by engaging specific activating Fcγ receptors on recruited monocyte-derived macrophages to cause severe inhibition of axon regeneration. Our data demonstrate that the antibody orchestrated Fcγ receptor-mediated switch in inflammation is one mechanism underlying inhibition of axon regeneration. These findings have clinical implications for nerve repair and recovery in antibody-mediated immune neuropathies. Our results add to the complexity of axon regeneration in injured peripheral and central nervous systems as adverse effects of B cells and autoantibodies on neural injury and repair are increasingly recognized.

Fgf2 improves functional recovery—decreasing gliosis and increasing radial glia and neural progenitor cells after spinal cord injury

ObjectivesA major impediment for recovery after mammalian spinal cord injury (SCI) is the glial scar formed by proliferating reactive astrocytes. Finding factors that may reduce glial scarring, increase neuronal survival, and promote neurite outgrowth are of major importance for improving the outcome after SCI. Exogenous fibroblast growth factor (Fgf) has been shown to decrease injury volume and improve functional outcome; however, the mechanisms by which this is mediated are still largely unknown.MethodsIn this study, Fgf2 was administered for 2 weeks in mice subcutaneously, starting 30 min after spinal cord hemisection.ResultsFgf2 treatment decreased the expression of TNF-a at the lesion site, decreased monocyte/macrophage infiltration, and decreased gliosis. Fgf2 induced astrocytes to adopt a polarized morphology and increased expression of radial markers such as Pax6 and nestin. In addition, the levels of chondroitin sulfate proteoglycans (CSPGs), expressed by glia, were markedly decreased. Furthermore, Fgf2 treatment promotes the formation of parallel glial processes, “bridges,” at the lesion site that enable regenerating axons through the injury site. Additionally, Fgf2 treatment increased Sox2-expressing cells in the gray matter and neurogenesis around and at the lesion site. Importantly, these effects were correlated with enhanced functional recovery of the left paretic hind limb.ConclusionsThus, early pharmacological intervention with Fgf2 following SCI is neuroprotective and creates a proregenerative environment by the modulation of the glia response.

Skin, heal thyself

Simply grabbing an African spiny mouse can cause it to lose up to 60% of the skin on its back. Analysis of the mouse's astounding shedding and healing capacity provides insight into the biomechanics of tissue regeneration. See Letter p.561 Many animals can regenerate tissues, organs and even whole limbs after injury. In mammals, this capability is believed to be at best extremely limited, but these authors report that the African spiny mice (Acomys) are potentially important exceptions. Live-trapped A. kempi and A. percivali from Kenya are shown to share with certain lizards the ability to shed and then rapidly regenerate areas of skin. This skin autotomy is an effective defence against predators, who may gain a mouthful of easily-torn skin but miss out on the main prize as the spiny mouse scuttles away. Quite large, deep lesions can be regenerated, complete with hair follicles and cartilage in damaged ears, and with no scarring. The authors propose that these mice produce a pro-regenerative environment similar to that seen in limb-regenerating salamanders. This study suggests that mammals may retain a higher capacity for regeneration than previously recognized, and introduces spiny mice as a novel model organism for study of this subject.

Basic fibroblast growth factor (bFGF) enhances tissue sparing and functional recovery following moderate spinal cord injury.

The rapid increase in basic fibroblast growth factor (bFGF) production following spinal cord injury (SCI) in rats is thought to serve a role in the cellular processes responsible for the functional recovery often observed. In this study, bFGF was intrathecally administered continuously for 1 week beginning 30 min after a moderate (12.5 mm) spinal cord contusion in adult rats using the New York University impactor device. Osmotic minipumps were implanted into the lateral ventricle and lumbar thecal sac to deliver bFGF at a rate of 3 microg or 6 microg per day versus control vehicle. Animals were behaviorally tested for 6 weeks using the Basso, Beattie, Bresnahan locomotor rating scale and histologically assessed for both tissue sparing and glial reactivity rostral and caudal to the lesion. Rats treated with bFGF regained coordinated hindlimb movements earlier than controls and demonstrated consistent coordination from 4 to 6 weeks. Vehicle-treated rats showed only modest improvements in hindlimb function. The amount of spared tissue was significantly higher in bFGF-treated rats than in controls. Astrocyte and microglial reactivity was more pronounced in bFGF-treated animals versus controls. In summary, intrathecal infusion of exogenous bFGF following SCI significantly reduces tissue damage and enhances functional recovery. Early pharmacological intervention with bFGF following SCI may serve a neuroprotective role and/or create a proregenerative environment, possibly by modulating the neuroglial response.