Cortical Reshaping and Functional Recovery Induced by Silk Fibroin Hydrogels-Encapsulated Stem Cells Implanted in Stroke Animals

Far into the future with our non-existent stroke leadership.
Cortical Reshaping and Functional Recovery Induced by Silk Fibroin Hydrogels-Encapsulated Stem Cells Implanted in Stroke Animals 

Laura Fernández-García¹, José Pérez-Rigueiro^1,2,3, Ricardo Martinez-Murillo⁴, Fivos Panetsos^5,6, Milagros Ramos^1,3,7, Gustavo V. Guinea^1,2,3 and Daniel González-Nieto^1,3,7*

• ¹Center for Biomedical Technology, Universidad Politécnica de Madrid, Madrid, Spain
• ²Departamento de Ciencia de Materiales, Escuela Técnica Superior de Ingenieros de Caminos, Canales y Puertos, Universidad Politécnica de Madrid, Madrid, Spain
• ³Biomedical Research Networking Center in Bioengineering Biomaterials and Nanomedicine, Madrid, Spain
• ⁴Department of Translational Neuroscience, Instituto Cajal – Consejo Superior de Investigaciones Científicas, Madrid, Spain
• ⁵Neurocomputing and Neurorobotics Research Group, Faculty of Biology and Faculty of Optics, Universidad Complutense de Madrid, Madrid, Spain
• ⁶Neural Plasticity Research Group, Health Research Institute of the Hospital Clínico San Carlos, Madrid, Spain
• ⁷Departamento de Tecnología Fotónica y Bioingeniería, Escuela Técnica Superior de Ingenieros de Telecomunicación, Universidad Politécnica de Madrid, Madrid, Spain

The restitution of damaged circuitry and functional remodeling of peri-injured areas constitute two main mechanisms for sustaining recovery of the brain after stroke. In this study, a silk fibroin-based biomaterial efficiently supports the survival of intracerebrally implanted mesenchymal stem cells (mSCs) and increases functional outcomes over time in a model of cortical stroke that affects the forepaw sensory and motor representations. We show that the functional mechanisms underlying recovery are related to a substantial preservation of cortical tissue in the first days after mSCs-polymer implantation, followed by delayed cortical plasticity that involved a progressive functional disconnection between the forepaw sensory (FLs₁) and caudal motor (cFLm₁) representations and an emergent sensory activity in peri-lesional areas belonging to cFLm₁. Our results provide evidence that mSCs integrated into silk fibroin hydrogels attenuate the cerebral damage after brain infarction inducing a delayed cortical plasticity in the peri-lesional tissue, this later a functional change described during spontaneous or training rehabilitation-induced recovery. This study shows that brain remapping and sustained recovery were experimentally favored using a stem cell-biomaterial-based approach.

Introduction

Stroke represents the leading cause of disability and a main reason for premature mortality worldwide (Benjamin et al., 2017). The early recognition of symptoms and the rapidity of medical intervention influence the clinical evolution of each patient. In ischemic stroke, the most frequent form of stroke, the intravenous injection of the tissue plasminogen activator (tPA) and surgical procedures such as endovascular thrombectomy are currently the main advanced treatments for the early reestablishment of blood flow in the occluded vessel (Saver et al., 2015). However, a minority of stroke patients can really get benefits from these treatments due to the narrow time window for administration after the onset of symptoms and the risks of complications such as intracranial hemorrhage, major systemic hemorrhage, and angioedema. Thus, most of the patients who do not receive acute reperfusion therapies show long-term disabilities. Unfortunately, we do not have therapies to target the subacute and chronic phases of ischemic stroke and efficiently repair the damaged brain promoting a satisfactory degree of functional recovery in most patients (George and Steinberg, 2015).

A well-established observation in different species, including humans, is that the brain reorganizes itself under physiological and pathological conditions (Merzenich et al., 1984; Jenkins et al., 1990; Panetsos et al., 1995; Nudo et al., 1996b; Elbert et al., 1997; Traversa et al., 1997; Jaillard et al., 2005; Schaechter et al., 2006; Brown et al., 2009). During normal brain development, the emerging circuitry is organized to represent sensory, motor and variably distributed cognitive abilities. However, the initially established topographic and functional representations are not static and change dynamically in size and location throughout adult life in response to sensory input, experience and learning (Jenkins et al., 1990; Elbert et al., 1995; Nudo et al., 1996a). Brain reorganization also occurs after central or periphery injury (Merzenich et al., 1984; Calford and Tweedale, 1988; Elbert et al., 1997; Simoes et al., 2012). Reciprocal GABA-mediated inhibitory signals between distinct representations (Jacobs and Donoghue, 1991; Jones, 1993) and the regular activity of sensory/motor fields together with the maintenance of axonal pathways (Graziano and Jones, 2009) have been proposed to contribute to the mechanism defining topographic areas and preventing the functional invasion between surrounding representations (Calford and Tweedale, 1988; Jacobs and Donoghue, 1991).

The general principles that orchestrate the nascent brain circuitry during development are believed to be similar to those forming the compensatory circuits underlying functional recovery after brain damage. Based on accumulating evidence, motor and sensory representations are modified in the affected and non-affected hemispheres after unilateral brain damage (Nudo et al., 1996b; Brown et al., 2009; Harrison et al., 2013; Tennant et al., 2015). An evolutionarily conserved program exists in mammals to sustain the reorganization of non-affected areas surrounding the damaged regions that then perform the specific functions that were lost and initially depended of the injured areas. However, under spontaneous recovery, brain remapping in the undamaged surrounding tissue does not always correlate with functional outcomes (Nudo and Milliken, 1996; Xerri et al., 1998; Schaechter et al., 2006; Nishibe et al., 2015). A direct link between the post-stroke cortical changes and the temporal pattern of functional recovery has not been directly obtained in the majority of the studies, making a cause-effect relationship difficult to establish.

Stem cell therapy constitutes a promising approach to stimulate functional recovery after stroke (Chen et al., 2001a,b; Trounson and McDonald, 2015; Wang et al., 2016). Different types of stem cells have been used as a potential source of both replacement cells and neurotrophic factors although the precise mechanisms of action and the optimal administration route are unclear (Hermann et al., 2014; Gervois et al., 2016). Compared with the systemic delivery (Prasad et al., 2014; Hess et al., 2017), cerebral implantation requires fewer cells and provides a precision graft (Yang et al., 2011; Du G. et al., 2014; Du S. et al., 2014; Borlongan, 2016). However, the cerebral route has also reached relatively modest levels of post-stroke functional recovery, which has been associated with the severe loss of grafted cells that are generally not observed in the brain for more than 1–3 weeks after transplantation, as reported in several preclinical models (Kelly et al., 2004; Bliss et al., 2010; Mora-Lee et al., 2012; George and Steinberg, 2015; Wang et al., 2016). In patients, the cerebral implantation of stem cells has been reported to be safe and clinical improvements were observed, but the small sample size and heterogeneity between subjects currently preclude the use of this specific approach in clinical practice (Chen et al., 2014; Borlongan, 2016; Steinberg et al., 2016).

The use of biomaterials in tissue engineering is booming and has provided examples of how the integration of neurotrophic cells and factors in biomaterial-based polymers results in better post-stroke functional recovery compared to the implantation of therapeutic cells or factors alone (Guan et al., 2013; Jendelova et al., 2016; Nih et al., 2016). Different natural and synthetic polymers have been used to support stem cell engraftment including hyaluronic acid, collagen, hyaluronan-methylcellulose, polyethylene glycol, PLGA, alginate and matrigel among others (González-Nieto et al., 2018).

Although improved functional outcomes have been achieved in the majority of stroke models after the implantation of stem cells or stem cells plus different biomaterials, the mechanisms of recovery are in most cases unclear, but they might be associated with the reestablishment of the destroyed circuitry in damaged regions (Taguchi et al., 2004; Ramos-Cabrer et al., 2010; Wang et al., 2016). However, other tentative benefits of stem cell therapy could be related to the reorganization of the pre-existing circuitry in peri-lesional areas of the affected hemisphere, involving the reorganization of cortical maps (Dijkhuizen et al., 2001; Brown et al., 2009; Andres et al., 2011).

In the present study, we examined the ability of bone marrow mesenchymal stem cells (mSCs) to enhance functional outcomes after cortical stroke in mice. The mSCs were intracerebrally implanted together with a silk fibroin (SF)-based hydrogel (Fernandez-Garcia et al., 2016). We report evidence of cortical plasticity linked to functional recovery occurring late after treatment. To our knowledge, this study is unique because it shows that brain remapping and sustained recovery were experimentally favored using a stem cell-biomaterial-based approach.

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