Biodegradation of ECM hydrogel promotes endogenous brain tissue restoration in a rat model of stroke

Abstract

The brain is considered to have a limited capacity to repair damaged tissue and no regenerative capacity following injury. Tissue lost after a stroke is therefore not spontaneously replaced. Extracellular matrix (ECM)-based hydrogels implanted into the stroke cavity can attract endogenous cells. These hydrogels can be formulated at different protein concentrations that govern their rheological and inductive properties. We evaluated histologically 0, 3, 4 and 8 mg/mL of porcine-derived urinary bladder matrix (UBM)-ECM hydrogel concentrations implanted in a 14-day old stroke cavity. Less concentrated hydrogels (3 and 4 mg/mL) were efficiently degraded with a 95% decrease in volume by 90 days, whereas only 32% of the more concentrated and stiffer hydrogel (8 mg/mL) was resorbed. Macrophage infiltration and density within the bioscaffold progressively increased in the less concentrated hydrogels and decreased in the 8 mg/mL hydrogels. The less concentrated hydrogels showed a robust invasion of endothelial cells with neovascularization. No neovascularization occurred with the stiffer hydrogel. Invasion of neural cells increased with time in all hydrogel concentrations. Differentiation of neural progenitors into mature neurons with axonal projections was evident, as well as a robust invasion of oligodendrocytes. However, relatively few astrocytes were present in the ECM hydrogel, although some were present in the newly forming tissue between degrading scaffold patches. Implantation of an ECM hydrogel partially induced neural tissue restoration, but a more complete understanding is required to evaluate its potential therapeutic application. STATEMENT OF SIGNIFICANCE: Extracellular matrix hydrogel promotes tissue regeneration in many peripheral soft tissues. However, the brain has generally been considered to lack the potential for tissue regeneration. We here demonstrate that tissue regeneration in the brain can be achieved using implantation of ECM hydrogel into a tissue cavity. A structure-function relationship is key to promote tissue regeneration in the brain. Specifically, weaker hydrogels that were retained in the cavity underwent an efficient biodegradation within 14 days post-implantation to promote a tissue restoration within the lesion cavity. In contrast, stiffer ECM hydrogel only underwent minor biodegradation and did not lead to a tissue restoration. Inductive hydrogels weaker than brain tissue provide the appropriate condition to promote an endogenous regenerative response that restores tissue in a cavity. This approach offers new avenues for the future treatment of chronic tissue damage caused by stroke and other acute brain injuries.

Keywords: Biodegradation; Biomaterial; Cell invasion; Extracellular matrix; Hydrogel; Magnetic resonance imaging; Regeneration; Scaffold; Stroke; Tissue repair.

Figure 1.. Macroscopic distribution of ECM hydrogel in the stroke cavity.

A. Pre-implantation T₂-weighted magnetic resonance images (MRI) were used to define stereotactic coordinates and calculate volumes of ECM hydrogel precursor for injection. A complete coverage of the lesion cavity with an ECM bioscaffold (Collagen I in green, DAPI in Blue) was achieved with this approach. A concentration of 8 mg/mL ECM shows limited degradation over 90 days, whereas 3 and 4 mg/mL show a very efficient structural remodeling, with only a small amount of hydrogel being present at the final time point. B. Anterior-Posterior pre-implantation MRI scans revealed the location and volume of the lesion cavity for comparison with 4 mg/mL ECM hydrogel at 90 days post-implantation. Active tissue remodeling inside the ECM bioscaffold and around the lesion cavity is evident (DAPI in Blue, Collagen I in green, GFAP in red). C. At the lesion-tissue boundary, astrocytes (GFAP+ cells) cross the glial scar and invade the bioscaffold that is replacing the stroke cavity. Capillary-like structures were also apparent in ECM remodeling regions.

Figure 2.. ECM biodegradation and tissue deformation.

A. Lesion volumes calculated from T₂-weighted MR images acquired 4 days before ECM injection (10 days post-stroke) were used to assign rats to groups with equivalent lesion volumes. B. Remaining volume of ECM hydrogel was quantified to determine biodegradation at 1, 14 and 90 days post-implantation. At 90 days post-implantation a decrease in ECM volume of 94.1%, 95%, and 32% was recorded in the 3, 4, and 8 mg/mL. A locally weighted scatter plot smoother (LOWESS) fitted curve visualize the anticipated degradation pattern with a 3 mg/mL concentration providing a half-life of 4.5 days, the 4 mg/mL 4.9 days and 8 mg/mL >90 days. A biodegradation plateau is reached for the 3 and 4 mg/mL concentration around 28 days, whereas the 8 mg/mL concentration plateaued at 66 days. C. Less concentrated 3 and 4 mg/mL ECM concentrations halted lesion progression, whereas the 8 mg/mL had a more limited impact on the evolution of the cavity compared to no treatment (0 mg/mL). D. Midline shift was calculated by a ratio between distance of the ipsi- and contralateral hemisphere midpoints. A gradual shift of the midline was evident in all groups. The 4 mg/mL condition exhibited the most promise to reduce tissue deformation. E. Hydrogel implantation did not affect the imbalance in ipsi-and contralateral parenchymal volumes that follows a stroke. F. There was also no effect on the hydrocephalus ex vacuo that ensues as a result of stroke damage, as evidence by the ratio of the ipsi- and contralateral lateral ventricles. (** p<0.01)

Figure 3.. Glial scarring and tissue astrocytosis.

A. To evaluate the impact of ECM hydrogel on glial scarring at the tissue interface, whole brain slices covering the lesion cavity were acquired to measure the level of astrocytic (GFAP) reactivity in the striatal and cortical tissues. A 4 mg/mL concentration condition is shown here. B. However, it is important to note a clear morphological difference in astrocytic activity at the different time points, with the 14 day time point showing the sharpest interface border, whereas by 90 days post-implantation a complex mesh of astrocytic processes blurring the line between established and regenerating tissue. C. A quantitative comparison indicated a marked increase in GFAP intensity at the border of the cavity 1 day post-implantation that was equivalent for all groups. A gradual decrease of reactivity away from the cavity border was evident. The highest increase in astrocyte reactivity was observed at 14 days post-implantation with a surge in intensity reaching further into both striatum and cortex. By 90 days, the extent and intensity of glial reactivity was reducing, but not back to the level present on day 1. D. Peri-infarct astrocytosis was extensive in areas surrounding the ECM hydrogel implantation at all time points. E. A quantitative comparison mirrored the results of glial scarring, where an increase occurred in the 14 days post-implantation. This was nevertheless equivalent between all groups, including the 0 mg/mL condition indicating that this astrocytosis is not related to the ECM hydrogel, but either due to lesion progression or the implantation procedure. The 3 mg/mL condition exhibited the lowest level of astrocytosis, potentially revealing a minor effect of ECM permeating into peri-infarct tissue.

Figure 4.. Biodegradation of the material is crucial for supporting cell infiltration and tissue remodeling.

Biodegradation of ECM hydrogel is concentration dependent with less concentrated 3 and 4 mg/mL bioscaffolds getting efficiently degraded, whereas the 8 mg/mL persists longer. A. At Day 90, a very small amount (5.9 %) of the 3 mg/mL ECM was present with host cells showing an excellent invasion and structural remodeling. An even distribution of the invading GFAP+ cells is seen throughout the remaining hydrogel. B. With 4 mg/mL, chain cell invasion can be seen with GFAP+ cells filling the space in between patches of ECM hydrogel, as identified by collagen I staining. C. In contrast with these less concentrated hydrogels, a sharp boundary between the biomaterial and host was evident in animals injected with 8 mg/mL. Density of cells in the hydrogel at 90 days was much lower compared to the less concentrated gels. These observations highlight key differences in biodegradation and cell infiltration between different concentrations of ECM hydrogel.

Figure 5.. Presence of host cells in ECM hydrogel.

A. Using collagen I staining, a region of interest (ROI) was defined around the edges of the biomaterial (8 mg/mL shown) and applied to the DAPI image to provide a quantification of the number of cells present within the hydrogel. B. Total cell infiltration indicated that the 8 mg/mL hydrogel consistently contained the highest number of cells. In all conditions, a gradual decrease in total number of cells is seen that is related to the biodegradation of the scaffold. C. To account for ECM hydrogel volume changes due to biodegradation, cell density was calculated. The 4 mg/mL hydrogel concentration provided a very consistent density of approximately 4000 cells/μL. Cell density for the 8 mg/mL decreased from a 4 mg/mL comparable level, whereas 3 mg/mL increased to a comparable level at 90 days. These cell density dynamics reveal key differences in the inductive potential of ECM hydrogel concentrations. D. Cell infiltration and density here focus on the bioscaffold content (4 mg/mL shown). However, a significant number of cells are evident within the previous cavity in between patches of ECM hydrogel. E. Iba-1+ macrophages and GFAP+ astrocytes are common phenotypes, but no scar or foreign body response was evident. (* p<0.05)

Figure 6.. Phenotypic characterization of invading immune cells in ECM hydrogel.

A. Invasion of Iba-1+ macrophage is evident at the tissue/hydrogel interface (4 mg/mL). Collagen I staining of the ECM hydrogel defined the region of analysis of macrophage invasion. Individual leader cells spread through the material, typically with an amoeboid shape, 1 day post-implantation. B. At 14 days post-implantation, clusters of Iba-1 positive cells were increasingly common, with some macrophages exhibiting an activated and ramified morphology. C. M1-like (CD86) and M2-like (CD206) polarization of macrophages was also evident with some cells expressing both markers, especially at 90 days. D. The 8 mg/mL ECM concentration invoked the highest proportion of macrophage and this increased with time. However, the total number of macrophage gradually decreased in all conditions. Density of macrophages within efficiently degrading hydrogel was high and persisted at approximately 700–800 cells/ μL. Only in the 8 mg/mL ECM hydrogel was there a decrease in macrophage density. E. Analysis of polarization of macrophages indicated that M1-like phenotypes were predominant 1 day post-implantation, but that M2-like cells were common 14 days post-implantation. By 90 days, both M1 and M2 were commonly found in the same macrophage cell. (* p<0.05; ** p<0.01)

Figure 7.. Vascularization of the ECM hydrogel.

A. Neovascularization inside the hydrogel was evident at 14 days if hydrogel underwent an efficient biodegradation, as illustrated here after implantation of a 3 mg/mL ECM bioscaffold. B. However, in some cases very tortuous vessels can be seen. C. Preceding the formation of vasculature is the infiltration of endothelial cells. In the 8 mg/mL condition, infiltration of endothelial cells is seen, but there is a lack of vascular formation. A higher magnification of RECA-1+ cells highlights the early stages of alignment of individual cells that invaded the hydrogel. D. A quantification of endothelial cell infiltration indicated a higher infiltration in the 8 mg/mL condition 1 day post-implantation, but a turning point is reached at 14 days where there is decrease of endothelial cells at this concentration. Endothelial cell infiltration was linearly increased in the 3 and 4 mg/mL concentrations, constituting almost 30% of all cells in the hydrogel at 90 days. (*** p<0.001)

Figure 8.. Neuronal and glial cell invasion into the ECM hydrogel.

A. While most of the migrating neural progenitors (doublecortin, DCX) were seen at the host-biomaterial interface, a small number of DCX+ cells could be seen inside the material (4 mg/mL shown). B. Immunostaining with beta III-tubulin (Tuj) neuron marker revealed further differentiation of these progenitors inside, as well as in between the remnant of ECM hydrogel. Occasionally GFAP+ astrocytes were adjacent to these neurons, but often these neurons were not paired with astrocytes. C. To verify if mature neurons were being generated in this de novo tissue, NeuN staining was performed to target post-mitotic neurons that typically extend processes for tissue integration. Fewer of these were evident, mostly in between ECM hydrogel patches, rather than within the scaffold per se. D. Occasional clusters containing NeuN+ cells in between ECM hydrogel were also found, potentially illustrating different stages of development within newly forming tissue. E. Neuron and tissue maturation were evident at 90 days with some neurons extending neurofilament (NF) containing axons. F. Glia lineage cells also invaded the ECM hydrogel. There were surprisingly fewer astrocytes inside the hydrogel, whereas oligodendrocytes efficiently colonized the weaker 3 and 4 mg/mL scaffolds by 90 days post-injection.

Figure 9.. Phenotypic characterization and quantification of invading neuronal cells.

The proportion of neural cells 1 day post-implantation was approximately 30% with neural progenitors and oligodendrocytes being the predominant phenotypes (images show 4 mg/mL condition). The 8 mg/mL ECM hydrogel was especially efficient in attracting neural progenitors at this time point. However, the proportion of neural progenitor content reduced by 14 days, as mature phenotypes became more prominent, consistent with differentiation of cells and maturation of tissue. At 90 days, the less concentrated 3 and 4 mg/mL hydrogel contained a higher density of neurons, astrocytes and oligodendrocytes compared to the 8 mg/mL concentration. The 3 and 4 mg/mL ECM concentrations therefore provide favorable conditions for neural tissue formation. (* p<0.05; ** p<0.01)

Figure 10.. Correlations of ECM hydrogel volume and cellular content.

To evaluate the relationship between ECM hydrogel degradation and cellular content, a correlational analysis at each time point was performed for lesion volume (A), all invading cells (B), macrophages (C), endothelial cells (D), neural progenitors (E), neurons (F), astrocytes (G) and oligodendrocytes (H). A dramatic inversion of the relationship between ECM volume at day 1 and 90 occurred. At day 1 all measures, apart of astrocytes, revealed a positive correlation with ECM volume. On day 14, the association between ECM volume and cell content was weak, but by 90 days the relationship inverted with biodegradation of ECM (i.e. lower volume) producing greater cell content and differentiation.