This study aimed to assess the feasibility and safety of the novel hydrogel Alpha2 for application in CNS regeneration, and to determine any potential therapeutic effects. Overall, when injected 7 days after ICH, the unfunctionalised Alpha2 hydrogel had neutral effects on functional and histological outcomes and did not alter the gene expression profile in the affected tissues but did lead to a greater number of proliferating cells in the lesion.
The Alpha2 hydrogel was found to be injectable through clinically-relevant gauge needles, which is essential for minimally-invasive delivery to deep brain regions [19], was able to fill the irregular shape of the cavity formed after ICH, becoming contiguous with the surrounding tissue and, estimations of brain tissue elasticity (stiffness) showed the hydrogel to be softer than that of brain tissue [37–39]. The hydrogel remained in situ for at least 2 weeks after injection but appeared to be degraded by 8 weeks. Although the exact timeline for regenerative events after ICH is unclear, angiogenesis and neurogenesis are thought to commence in the sub-acute to chronic phase after an acute brain injury and continue for several weeks or months thereafter [40–42]. Therefore the ideal hydrogel should remain in situ for at least several weeks to facilitate these processes but should also eventually be degraded, to be replaced by native tissue [19]. The hydrogel also appeared to provide a permissive microenvironment for host cell infiltration, which started as early as 24 h post-injection and continued for over 2 weeks. Several previous reports using unfunctionalised hydrogels in ICH models found very little material-tissue interaction and host cell infiltration, even at much later time points after injection [14, 15]. A key concern in the design of biomaterials for application in the brain, is matrix elasticity mismatch, which can exacerbate glial scarring [6, 43]. In this work, Alpha2 hydrogel administration did not affect glial scarring or cause a change in expression of the macrophage/microglia marker Iba1, which together indicate that the material was well-tolerated and perhaps non-immunogenic. The potential non-immunogenicity of Alpha2 is supported by in vitro evidence, in which the inflammatory profile of mouse monocytes encapsulated within the hydrogel, was studied. The authors reported no significant increase in the production of several pro-inflammatory cytokines, after 48 or 72 h of culture, indicating that the hydrogel did not elicit a pro-inflammatory response [20]. Overall, these data indicate that the Alpha2 hydrogel appeared to fulfil several of the features of an ideal hydrogel for brain repair.
Prior to hydrogel administration, there was no difference in haematoma volume between groups and 8 weeks later the hydrogel had no influence on the lesion volume or tissue atrophy. This is in contrast to a previous study that found reduced atrophy when a hyaluronic acid-based hydrogel was administered one week after ICH, which may represent a regenerative effect [44]. A keratin-based hydrogel encapsulated with an iron chelator administered acutely after ICH, also led to reduced tissue atrophy [45], although this may be attributable to the protective effect of the iron chelator. However, data in this study indicates that the Alpha2 hydrogel did not exacerbate or improve the amount of tissue lost after ICH.
To confirm whether the hydrogel had any effect on functional outcomes, body weight monitoring and functional tests covering different domains of post-ICH deficits were performed. No effect on body weight, neuroscore, or performance in the corner and cylinder tests, were detected over 8 weeks after ICH. The lack of an effect on body weight, an indicator of welfare and ICH severity, suggests there were no major adverse effects or acute phase response to hydrogel injection [46, 47]. Similarly, a study by Ghuman and colleagues found that an ECM-based hydrogel administered at a chronic time point after ischaemic stroke, was retained for several weeks and allowed host cell infiltration but had neutral effects on a battery of functional tests [48]. However, some studies using alternative hydrogels, have reported a beneficial effect on the behavioral impairments seen in the neuroscore and corner test after ICH [12, 45, 49–51], although functional benefits are sometimes seen only when the hydrogel is combined with other factors, such as growth factors ([51]). Although this study found no detrimental or beneficial effect of hydrogel administration on functional outcomes, it is important to note that there may be other domains of impairment, such as fine motor dexterity or sickness behaviour that are not captured by the tests selected.
A key aim of regenerative medicine is to facilitate and enhance endogenous regenerative processes, such as angiogenesis and neurogenesis, that are typically halted by the biochemical and biophysical conditions in the damaged tissue. Using collagen IV as a marker of the basement membrane of blood vessels, there appeared to be an upregulation of collagen IV labelling within the core of the lesion, both with and without hydrogel treatment. However, quantification of blood vessel parameters at the lesion rim revealed no effect of the hydrogel on vessel area, density or number of branchpoints compared to the contralateral hemisphere, at 8 weeks after ICH. These data suggest that the hydrogel had no effect on the extent of vascularisation after ICH, although collagen IV may only label mature vessels and may not have detected nascent, actively developing blood vessels. However, injection of the Alpha2 hydrogel led to significantly more proliferative cells (positive for Ki67) within the lesion core. Ki67-expressing cells appeared to be positive for DCX, indicating the presence of proliferating neural progenitors in the lesion. Several reports using hydrogels in ICH have studied the expression of neurogenic markers [15, 49, 51, 52]. Interestingly, Lim and colleagues found a significant increase in the number of nestin-positive neural stem cells, after delivery of a EGF and gelatin-hydrogel combination therapy, but no increase with the hydrogel scaffold alone [51]. Similarly, another report found a significant increase in BrdU-positive cells with a hydrogel encapsulated with mesenchymal stem cells but not with the hydrogel alone [45]. Consequently, the significant increase in proliferative cells after administration of the Alpha2 hydrogel alone in the current work suggests that the biomaterial can support the proliferation of new cells. Labelling of β-III tubulin, a marker of immature neurones, uncovered elaborate networks of neuronal microtubules within the lesion itself in both experimental groups. Overall, these data suggest that the chronic ICH lesion is richly populated with not only Iba1-positive macrophages, but a range of other cell types such as astrocytes, neuroblasts and immature neurones, together with the basement membrane protein, collagen IV. This is promising for the regenerative medicine approach, as it demonstrates that a number of cell types important for regeneration can enter the damaged tissue – a response which could be amplified with a targeted adjunct therapy.
RNA-seq was performed to study whether the hydrogel had an effect on the transcriptional profile after ICH. Several of up- and downregulated genes were identified 8 weeks after ICH in both hydrogel and vehicle-injected tissue, but there were no changes in the DEGs between groups, supporting the idea that the material is safe but perhaps, largely functionally inert. Gene set enrichment and pathway analysis found that in both the hydrogel and vehicle-injected groups, DEGs were involved in pathways related predominantly to inflammation, such as neutrophil mediated immunity, macrophage activation and cytokine mediated signalling pathways. The top upregulated cellular and molecular functions identified in both experimental groups in the current work suggest that even as late as 8 weeks, inflammation is the dominant process occurring in the ICH lesion. However, the expression of some myeloid-related genes, such as Cd68 and Clec7a, could be associated with a reparative immune cell phenotype. In addition, the canonical inflammatory interleukins are conspicuously absent from the list of DEGs in both groups, which is not typical of acutely inflamed tissue. Nevertheless, despite the presence of some DEGs associated with tissue repair and regeneration (Hmox1, Dcx, Gfap) in both groups, gene enrichment and pathway analysis did not identify pathways related to the either angiogenesis or neurogenesis, indicating that active repair is not a dominant process in lesioned tissue at 8 weeks.
Overall, this study is the first characterisation of the effects of the novel SAPH (Alpha2) hydrogel in the CNS. In testing in an experimental rat model of ICH, the hydrogel had several suitable properties for application in the brain and when injected in vivo, the material was found to be safe, well-tolerated and could support the infiltration of multiple cell types. Furthermore, hydrogel injection had no effect on functional outcomes but did lead to an increase in the number of proliferating cells in the ICH lesion. These results indicate that the Alpha2 hydrogel may have potential as a delivery platform for other pro-regenerative therapies, as the retention and degradation rate could allow for sustained and targeted release of the therapy over time to enable regeneration after ICH.