A roadmap for promoting endogenous in situ tissue restoration using inductive bioscaffolds after acute brain injury

Abstract

The regeneration of brain tissue remains one of the greatest unsolved challenges in medicine and by many is considered unfeasible. Indeed, the adult mammalian brain does not regenerate tissue, but there is ongoing endogenous neurogenesis, which is upregulated after injury and contributes to tissue repair. This endogenous repair response is a conditio sine que non for tissue regeneration. However, scarring around the lesion core and cavitation provide unfavorable conditions for tissue regeneration in the brain. Based on the success of using extracellular matrix (ECM)-based bioscaffolds in peripheral soft tissue regeneration, it is plausible that the provision of an inductive ECM-based hydrogel inside the volumetric tissue loss can attract neural cells and create a de novo viable tissue. Following perturbation theory of these successes in peripheral tissues, we here propose 9 perturbation parts (i.e. requirements) that can be solved independently to create an integrated series to build a functional and integrated de novo neural tissue. Necessities for tissue formation, anatomical and functional connectivity are further discussed to provide a new substrate to support the improvement of behavioral impairments after acute brain injury. We also consider potential parallel developments of this tissue engineering effort that can support therapeutic benefits in the absence of de novo tissue formation (e.g. structural support to veterate brain tissue). It is envisaged that eventually top-down inductive "natural" bioscaffolds composed of decellularized tissues (i.e. ECM) will be replaced by bottom-up synthetic designer hydrogels that will provide very defined structural and signaling properties, potentially even opening up opportunities we currently do not envisage using natural materials.

Keywords: Biodegradation; Bioscaffold; Brain; Hydrogel; Magnetic resonance imaging; Regeneration; Stroke; Tissue repair.

Figure 1.. Pathophysiology of acute brain injury and therapeutic interventions.

An acute insult to the brain through brute force or ischemia will lead to the rapid loss of neurons at the core of the infarct, this area will eventually cavitate and be sealed off by a glial scar. Apoptosis will further lead to neuronal death, as well as other cells, such as astrocytes, oligodendrocytes and endothelial cells (not illustrated here). Cell stress and cell death will provoke microglia to invoke an immune response that recruits neutrophils into brain tissue and produce a disruption in the blood brain barrier (BBB). The BBB will close again, but a second disruption will occur transitioning between the acute and subacute phase, again characterized by a major influx of peripheral immune cells, such as macrophages and lymphocytes. Most infiltrating macrophages are of a M1-type (i.e. pro-inflammatory) during the cell death and ECM clearance phase. However, as tissue is cleared these macrophages tend to polarize towards an M2-like (i.e. pro-repair) phenotype. It currently remains unclear if these are newly infiltrating cells or if ECM clearance in the brain promotes a shift towards a pro-repair phenotype. The implantation of inductive bioscaffolds needs to be considered against the backdrop of these pathophysiological events, but also in the context of other therapeutics and their time window. Endogenous in situ tissue engineering at present is best considered for treatment after a cavity formed in the sub-chronic phase. However, the use of bioscaffolds to change inflammatory events, promote neuronal survival, or neovascularization might be suitable for earlier interventions.

Figure 2.. Overview of materials used for bioscaffolds.

A variety of natural and synthetic materials have been used to create bioscaffolds. Natural material can typically be distinguished based on their provenance from mammalian or non-mammalian species. Of the mammalian sources, extracellular matrix (ECM) produces an inductive response, i.e. it invokes a host cells response that leads to its degradation and replacement with new tissue. Other materials, such as hyaluronic acid (HA) do not by themselves produces these types of response, but do influence cell behavior based on their affinity to its juxtacrine signaling. Combinations of the materials in hybrid designs is possible and further functionalization by altering composition or structure are also undertaken based on specific design criteria (e.g. anisotropic structure for axonal growth requiring spatial patterning). Bioscaffolds can also carry cargo (e.g. cells, growth factors) that is thought to promote specific therapeutic effects. Most commonly cells are integrated into bioscaffolds with an eye towards cell/tissue replacement. Bioscaffolds/biomaterials can improve the survival of delivered cells, but also provide a structural support to promote de novo tissue formation.

Figure 3.. Perturbation series defined requirements for endogenous in situ brain tissue engineering.

Based on endogenous tissue regeneration is peripheral soft tissue defects, 9 perturbation parts can be defined and arranged in a series to solve the same problem in the brain. For each biological challenge, an engineering solution can be envisaged. To ensure that these concerted parts unfold in vivo, ideally non-invasive monitoring is used to visualize and check that an appropriate biology continues to develop. Magnetic resonance imaging techniques, such as T₂-weighted (T2w) images, T2 maps, Apparent diffusion coefficient (ADC) maps, diffusion tensor imaging (DTI), chemical exchange saturation transfer (CEST), magnetic resonance spectroscopy (MRS), as well as functional brain imaging techniques, such as manganese-enhance MRI (MEMRI), functional MRI (fMRI), resting-state fMRI (rs-fMRI) and pharmacological MRI (phMRI) can be employed. To define the integrity of the blood brain barrier (BBB), gadoterate (Gd-DOTA) can be used to visualize leakage of molecules from the vascular compartment into the neuropil. A leaky BBB would occur during angiogenesis, when the barrier has not sufficiently matured and could hence indicate neovascularization.

Figure 4.. ECM hydrogel implantation for endogenous in situ brain tissue engineering.

A. Magnetic resonance imaging (MRI)-based image guidance afforded an injection-drainage delivery of ECM hydrogel into a tissue cavity caused by a stroke. This afforded a complete coverage of the cavity with ECM bioscaffold, as evidence by the immunohistochemistry. An overlay of the pre-implantation MR image with 1-day post-mortem histology verifies the accuracy and efficiency of ECM bioscaffold delivery. B. Implantation of an ECM bioscaffold produced host cell invasion following the pattern of chain cell migration. C. The interface between the biomaterial and host tissue is essential to ensure an efficient invasion. D. A poor tissue-biomaterial interface or limited permeation of ECM derived signals into host tissue create invasion blind spots where no cell invasion occurs. Typically, invasion follows an outside-in migration pattern from all sides of the scaffold. E. Invading cells participate in constructive remodeling of the ECM hydrogel. In many cases, small pockets of tissue are forming inside the hydrogel, which gradually is being degraded by phagocytes. F. De novo tissue formed inside the cavity caused by a stroke after implantation of an ECM hydrogel (4 mg/mL). G. Blood vessels are being formed in the de novo tissue, with some vessels presented in the newly formed tissue and penetrating/passing through remnants of the ECM bioscaffold. H. In some cases, neovascularization passed in between ECM patches. The patches reflect a common pattern observed in degrading ECM bioscaffolds. Blood vessels supported newly forming tissue in between these patches that were remodeling the cavity and creating novel tissue.