Bioscaffold-Induced Brain Tissue Regeneration

Abstract

Brain tissue lost after a stroke is not regenerated, although a repair response associated with neurogenesis does occur. A failure to regenerate functional brain tissue is not caused by the lack of available neural cells, but rather the absence of structural support to permit a repopulation of the lesion cavity. Inductive bioscaffolds can provide this support and promote the invasion of host cells into the tissue void. The putative mechanisms of bioscaffold degradation and its pivotal role to permit invasion of neural cells are reviewed and discussed in comparison to peripheral wound healing. Key differences between regenerating and non-regenerating tissues are contrasted in an evolutionary context, with a special focus on the neurogenic response as a conditio sine qua non for brain regeneration. The pivotal role of the immune system in biodegradation and the formation of a neovasculature are contextualized with regeneration of peripheral soft tissues. The application of rehabilitation to integrate newly forming brain tissue is suggested as necessary to develop functional tissue that can alleviate behavioral impairments. Pertinent aspects of brain tissue development are considered to provide guidance to produce a metabolically and functionally integrated de novo tissue. Although little is currently known about mechanisms involved in brain tissue regeneration, this review outlines the various components and their interplay to provide a framework for ongoing and future studies. It is envisaged that a better understanding of the mechanisms involved in brain tissue regeneration will improve the design of biomaterials and the methods used for implantation, as well as rehabilitation strategies that support the restoration of behavioral functions.

Keywords: biodegradation; biomaterial; extracellular matrix; physical therapy; regeneration; scaffold; stroke; tissue repair.

FIGURE 1

Tissue response, repair, and regenerative response. (A) After acute brain injuries, such as stroke, astrocytes and microglia get activated to respond to tissue damage that at its core eventually produce a loss of cells, as well as the extracellular matrix, leaving behind a cavity. The glial scar is aimed at providing structural support but also preventing neurotoxic fluid to permeate into the peri-infarct tissue. In addition to this tissue response, the brain mounts reparative activities, which include an upregulation of neurogenesis in the subventricular zone. (B) To supplement the endogenous repair response, neural stem cells (NSCs) can be transplanted into damaged tissue where these differentiate and integrate with host cells, but they also increase local angiogenesis providing a better blood supply to poorly perfused tissues (Smith et al., 2012). (C) In contrast to cell transplantation, implantation of inductive extracellular matrix (ECM) bioscaffolds promotes a restoration of tissue inside the lesion cavity from endogenous cells that invade the material (Ghuman et al., 2018). (D) An alternative approach to tissue regeneration using an inductive bioscaffolds is to implant a mixture of NSCs and ECs that spontaneously organize into a vascular and neuropil compartment, potentially accelerating the restoration process and overcoming the potential limitation of the reservoir of endogenous cells (Nicholls et al., 2015).

FIGURE 2

Evolutionary aspects of tissue regeneration. (A) The capacity of different species to regenerate different organ tissue over their lifespan widely varies. Anatomical complexity of the species, as well as tissues, is a major factor in their ability to regenerate. Lower species more readily regenerate more complex tissue, whereas more complex species, such as mammals, are only able to regenerate very few organs. It is pertinent to contrast a replenishing of blood and immune system from the bone marrow, which essentially restitutes single cells, from constructing a tissue that involves multiple cell lineages and the deposition of ECM to create a geometric arrangement of cells. (B) In wound healing, where a cut in a tissue, such as the skin, restores tissue integrity, typically follows 4 phases. These phases are characterized by a major difference in inflammation, which peaks in phase 2, but also by changes in tissue characteristics, with the deposition of a transient granulation tissue that is being remodeled by the infiltration of host tissue cells. The formation of blood vessels and epithelization of the tissue are further key events required to ensure that a seamless wound closure occurs. Depending on which process is interrupted, a wound breakdown can occur or scarring occurs that limits tissue function. This process can take months to complete.

FIGURE 3

Brain development and tissue repair. During brain development, the pallio-subpallial boundary defines the divide between regions that mature into cortical and striatal tissues. The subventricular zone (SVZ) lies beneath the ventricular zone (VZ) and during development is the birthplace for cells that colonize tissue by migrating along radial glia. Within these tissues, particular gene expressions define the positional specifications of cells to become neurons that characterize the functions of individual regions. The medial ganglionic eminence (MGE) produces striatal interneurons (i.e., calretinin, paravalbumin, calbindin, cholinergic positive), whereas the lateral ganglionic eminence (LGE) is the main source of striatal projection neurons, which constitute 90% of neurons in the striatum. Of these 98% are DARPP-32 positive neurons. A further sub-division of the LGE into the ventral (vLGE) and dorsal (dLGE) has emerged, with vLGE producing a subset of projection neurons. The dLGE is thought to be the main source of interneurons in the olfactory bulb. In contrast, the pallium (a.k.a. telencephalon) is giving rise to the cortex with subdivisions of the ventral pallium (VP), lateral pallium (LP), deep pallium (DP) and medial pallium (MP). These regions produce different subdivision of the cortex, such as the motor cortex and somatosensory cortex. In the adult brain, this positional specification is retained within the subventricular zone (SVZ), the site of adult endogenous neurogenesis. Neurons born along different segments of the SVZ therefore contain a certain positional specification to produce region-specific cells. In response to acute brain injury. These cells respond and migrate through damaged tissue. In the context of tissue regeneration, the SVZ is the main source of cells to replenish lost cells. It remains currently unclear if these cells will cross the pallio-subpallial division that is defined by the lateral corpus callosum in adults. It also remains unknown if cells can change their positional specification and what functional consequence ensue if cells are not expressing a site-appropriate neuron differentiation.

FIGURE 4

The immune response in tissue regeneration. (A) The inflammatory response is driven by the immune system. Although the immune system is a complex network of circulating and tissue-resident cells, these originate from a hematopoietic stem cell. Traditionally the immune system was characterized in studies of infection and cells have therefore been divided into those that contribute to an innate rapid response versus those that produce an adaptive slow response. Only a few types of cells, such as natural killer cells and γδT cells, have been thought to contribute to both. However, more recently the importance of inflammation in tissue repair and regeneration has revealed distinct phenotypic changes in cells, such as macrophages, that questions the traditional division into an innate and adaptive immune response to recognize a pro-repair response of the immune system. It is likely that a range of immune cells are involved in this pro-repair response, but that the function of cells might be different to their role in response to an infection. (B) A characterization of cells infiltrating an ECM bioscaffold implanted into a stroke cavity revealed that >75% of cells (mean – standard deviation) at 1 day post-implantation are not of a brain origin (Ghuman et al., 2018). Although the majority of these are currently unidentified phenotypes, it is likely that these are of an immune origin, such as neutrophils and eosinophils, which respond rapidly, but also transiently, to tissue changes. (C) Inflammation in wound healing is thought to be initiated by resident macrophages that release inflammatory cytokines (IL-1; IL-6, TNF-; IFN-) in response to detection of an injury. The chemokine CXCL8 is released and drives the rapid invasion of neutrophils into the damaged tissue from the blood. A secondary invasion response is recruiting inflammatory macrophages, as well as lymphocytes, such as helper (T_h), regulatory (T_reg) and γδT cells. T cells are thought to play a key role in modulating macrophage activity and promoting a pro-fibrotic response that results in tissue scarring or a pro-repair response that leads to tissue regeneration.

FIGURE 5

Evaluating brain tissue regeneration. (A) A bioscaffold is implanted into the lesion cavity through a narrow bore needle that produces an injection tract and defines the trajectory of the injection. The aim of the procedure is to fill the tissue cavity and to produce a close-fitting interface with host tissue that affords invasion of cells into the scaffold and produces a seamless integration between veterate and de novo brain tissue (Ghuman et al., 2018). De novo tissue growing inside the cavity can be identified based on collagen I staining of the ECM hydrogel and the neovasculature, surrounded by veterate tissue, as indicated by the peri-infarct area and a dense presence of microglia (Iba1 + cells). (B) At 90 days post-implantation, the bioscaffold is almost completely degraded. In this case, two small remnants of the ECM hydrogel, characterized by a dense collagen I content are still undergoing cell invasion and degradation (yellow box). However, the rest of the scaffold is degraded and replaced with de novo tissue, in which blood vessels contain a higher level of collagen I compared to host tissue. Regenerated tissue (orange box) has blood vessels high in collagen I, but a dense tissue structure is evident with a reduced number of Iba-1 positive microglia/macrophages. It was also noted that in de novo tissue, some particulates were present that were not evident in veterate brain (white box). (C) In regenerating tissue, the bioscaffold is degraded, but there is still a higher collagen I background compared to host brain. Morphologically it is also distinct with a higher content of microglia/macrophages and strongly collagen I positive blood vessels. Nevertheless, more robust unique identifiable markers are desirable to contrast these different microenvironments. (D) In between patches of ECM bioscaffold, tissue is developing that contains neurons (Tuj) at a higher density than within the bioscaffold. However, at present there are no robust markers that allow a reliable identification of this as de novo tissue, complicating the quantification of the regenerative process.

FIGURE 6

Four phase of brain tissue regeneration. Phase 1: The repose phase is characterized by tissue loss being mostly complete and a repair response having been instigated. Although gliosis is ongoing, no defining scar along the tissue cavitation has emerged. During this phase, a bioscaffold can be implanted to initiate the tissue regeneration process. In the absence of an introduction of a bioscaffold a scar is forming around the cavity. Phase 2: During the repopulation phase, host cells are invading the bioscaffold. Even during the acute invasion phase, brain derived cells, such as neural progenitors and astrocytes, are infiltrating the biomaterial, but immune cells are more rapidly invading and provide additional soluble and juxtracrine signaling to recruit host brain cells to repopulate the tissue cavity. Phase 3: Invading cells deposit transient matrix molecules and take-up positions inside the bioscaffold that leads to a gradual degradation of the scaffold. Blood vessel formation during this phase plays a key role to promote biodegradation, but also to remodel individual compartments that will develop into neuropil. Within the vascular compartment, cells are depositing appropriate matrix molecules, such as vitronectin, laminin and collagen. In the neuropil, neural cells deposit matrix molecules, such as laminin, aggrecan, decorin, thrombospondin that are involved in maintaining structure and juxtracrine signaling. At the end of this phase, the bioscaffold is completed degraded and replaced with host matrix. Phase 2 and Phase 3 overlap within different parts of the cavity. Phase 4: Once host brain cells are in position and formed a neuropil in between blood vessels, tissue maturation is occurring with terminal differentiation of neurons through interaction with astrocytes, oligodendrocytes and ECM molecules. It can be anticipated that de novo tissue formation in phase 3 and maturation processes, such as axonal and dendritic branching, can occur side-by-side. Axonal and dendritic processes are required to form a functional neuronal circuitry.