Post-Stroke Timing of ECM Hydrogel Implantation Affects Biodegradation and Tissue Restoration.

Extracellular matrix (ECM) hydrogel promotes tissue regeneration in lesion cavities after stroke. However, a bioscaffold's regenerative potential needs to be considered in the context of the evolving pathological environment caused by a stroke. To evaluate this key issue in rats, ECM hydrogel was delivered to the lesion core/cavity at 7-, 14-, 28-, and 90-days post-stroke. Due to a lack of tissue cavitation 7-days post-stroke, implantation of ECM hydrogel did not achieve a sufficient volume and distribution to warrant comparison with the other time points. Biodegradation of ECM hydrogel implanted 14- and 28-days post-stroke were efficiently (80%) degraded by 14-days post-bioscaffold implantation, whereas implantation 90-days post-stroke revealed only a 60% decrease. Macrophage invasion was robust at 14- and 28-days post-stroke but reduced in the 90-days post-stroke condition. The pro-inflammation (M1) and pro-repair (M2) phenotype ratios were equivalent at all time points, suggesting that the pathological environment determines macrophage invasion, whereas ECM hydrogel defines their polarization. Neural cells (neural progenitors, neurons, astrocytes, oligodendrocytes) were found at all time points, but a 90-days post-stroke implantation resulted in reduced densities of mature phenotypes. Brain tissue restoration is therefore dependent on an efficient delivery of a bioscaffold to a tissue cavity, with 28-days post-stroke producing the most efficient biodegradation and tissue regeneration, whereas by 90-days post-stroke, these effects are significantly reduced. Improving our understanding of how the pathological environment influences biodegradation and the tissue restoration process is hence essential to devise engineering strategies that could extend the therapeutic window for bioscaffolds to repair the damaged brain.

A systematic optimization of 19F MR image acquisition to detect macrophage invasion into an ECM hydrogel implanted in the stroke-damaged brain.

19F-MR imaging of perfluorocarbon (PFC)-labeled macrophages can provide a unique insight into their participation and spatio-temporal dynamics of inflammatory events, such as the biodegradation of an extracellular matrix (ECM) hydrogel implanted into a stroke cavity. To determine the most efficient acquisition strategy for 19F-MR imaging, five commonly used sequences were optimized using a design of experiment (DoE) approach and compared based on their signal-to-noise ratio (SNR). The fast imaging with steady-state precession (FISP) sequence produced the most efficient detection of a 19F signal followed by the rapid acquisition with relaxation enhancement (RARE) sequence. The multi-slice multi-echo (MSME), fast low angle shot (FLASH), and zero echo time (ZTE) sequences were significantly less efficient. Imaging parameters (matrix/voxel size; slice thickness, number of averages) determined the accuracy (i.e. trueness and precision) of object identification by reducing partial volume effects, as determined by analysis of the point spread function (PSF). A 96 × 96 matrix size (0.35 mm3) produced the lowest limit of detection (LOD) for RARE (2.85 mM PFPE; 119 mM 19F) and FISP (0.43 mM PFPE; 18.1 mM 19F), with an SNR of 2 as the detection threshold. Imaging of a brain phantom with PFC-labeled macrophages invading an ECM hydrogel further illustrated the impact of these parameter changes. The systematic optimization of sequence and imaging parameters provides the framework for an accurate visualization of 19F-labeled macrophage distribution and density in the brain. This will enhance our understanding of the contribution of periphery-derived macrophages in bioscaffold degradation and its role in brain tissue regeneration.

Acquisition and Analysis of Excised Neocortex from Pediatric Patients with Focal Cortical Dysplasia Using Mesoscale Diffusion MRI.

Non-invasive classification of focal cortical dysplasia (FCD) subtypes remains challenging from a radiology perspective. Quantitative imaging biomarkers (QIBs) have the potential to distinguish subtypes that lack pathognomonic features and might help in defining the extent of abnormal connectivity associated with each FCD subtype. A key motivation of diagnostic imaging is to improve the localization of a "lesion" that can guide the surgical resection of affected tissue, which is thought to cause seizures. Conversely, surgical resections to eliminate or reduce seizures provided unique opportunities to develop magnetic resonance imaging (MRI)-based QIBs by affording long scan times to evaluate multiple contrast mechanisms at the mesoscale (0.5 mm isotropic voxel dimensions). Using ex vivo hybrid diffusion tensor imaging on a 9.4 T MRI scanner, the grey to white matter ratio of scalar indices was lower in the resected middle temporal gyrus (MTG) of two neuropathologically confirmed cases of FCD compared to non-diseased control postmortem fixed temporal lobes. In contrast, fractional anisotropy was increased within FCD and also adjacent white matter tracts. Connectivity (streamlines/mm3) in the MTG was higher in FCD, suggesting that an altered connectivity at the lesion locus can potentially provide a tangible QIB to distinguish and characterize FCD abnormalities. However, as illustrated here, a major challenge for a robust tractographical comparison lies in the considerable differences in the ex vivo processing of bioptic and postmortem samples. Mesoscale diffusion MRI has the potential to better define and characterize epileptic tissues obtained from surgical resection to advance our understanding of disease etiology and treatment.

Mapping the acute time course of immune cell infiltration into an ECM hydrogel in a rat model of stroke using 19F MRI.

Extracellular matrix (ECM) hydrogel implantation into a stroke-induced tissue cavity invokes a robust cellular immune response. However, the spatio-temporal dynamics of immune cell infiltration into peri-infarct brain tissues versus the ECM-bioscaffold remain poorly understood. We here tagged peripheral immune cells using perfluorocarbon (PFC) nanoemulsions that afford their visualization by 19F magnetic resonance imaging (MRI). Prior to ECM hydrogel implantation, only blood vessels could be detected using 19F MRI. Using "time-lapse" 19F MRI, we established the infiltration of immune cells into the peri-infarct area occurs 5-6 h post-ECM implantation. Immune cells also infiltrated through the stump of the MCA, as well as a hydrogel bridge that formed between the tissue cavity and the burr hole in the skull. Tissue-based migration into the bioscaffold was observed between 9 and 12 h with a peak signal measured between 12 and 18 h post-implantation. Fluorescence-activated cell sorting of circulating immune cells revealed that 9% of cells were labeled with PFC nanoemulsions, of which the vast majority were neutrophils (40%) or monocytes (48%). Histology at 24 h post-implantation, in contrast, indicated that macrophages (35%) were more numerous in the peri-infarct area than neutrophils (11%), whereas the vast majority of immune cells within the ECM hydrogel were neutrophils (66%). Only a small fraction (12%) of immune cells did not contain PFC nanoemulsions, indicating a low type II error for 19F MRI. 19F MRI hence provides a unique tool to improve our understanding of the spatio-temporal dynamics of immune cells invading bioscaffolds and effecting biodegradation.

Physical therapy exerts sub-additive and suppressive effects on intracerebral neural stem cell implantation in a rat model of stroke.

Intracerebral cell therapy (CT) is emerging as a new therapeutic paradigm for stroke. However, the impact of physical therapy (PT) on implanted cells and their ability to promote recovery remains poorly understood. To address this translational issue, a clinical-grade neural stem cell (NSC) line was implanted into peri-infarct tissue using MRI-defined injection sites, two weeks after stroke. PT in the form of aerobic exercise (AE) was administered 5 × per week post-implantation using a paradigm commonly applied in patients with stroke. A combined AE and CT exerted sub-additive therapeutic effects on sensory neglect, whereas AE suppressed CT effects on motor integration and grip strength. Behavioral testing emerged as a potentially major component for task integration. It is expected that this study will guide and inform the incorporation of PT in the design of clinical trials evaluating intraparenchymal NSCs implantation for stroke.

ECM hydrogel improves the delivery of PEG microsphere-encapsulated neural stem cells and endothelial cells into tissue cavities caused by stroke.

Intracerebral implantation of neural stem cells (NSCs) to treat stroke remains an inefficient process with <5% of injected cells being retained. To improve the retention and distribution of NSCs after a stroke, we investigated the utility of NSCs' encapsulation in polyethylene glycol (PEG) microspheres. We first characterized the impact of the physical properties of different syringes and needles, as well as ejection speed, upon delivery of microspheres to the stroke injured rat brain. A 20 G needle size at a 10 µL/min flow rate achieved the most efficient microsphere ejection. Secondly, we optimized the delivery vehicles for in vivo implantation of PEG microspheres. The suspension of microspheres in extracellular matrix (ECM) hydrogel showed superior retention and distribution in a cortical stroke caused by photothrombosis, as well as in a striatal and cortical cavity ensuing middle cerebral artery occlusion (MCAo). Thirdly, NSCs or NSCs + endothelial cells (ECs) encapsulated into biodegradable microspheres were implanted into a large stroke cavity. Cells in microspheres exhibited a high viability, survived freezing and transport. Implantation of 110 cells/microsphere suspended in ECM hydrogel produced a highly efficient delivery that resulted in the widespread distribution of NSCs in the tissue cavity and damaged peri-infarct tissues. Co-delivery of ECs enhanced the in vivo survival and distribution of ∼1.1 million NSCs. The delivery of NSCs and ECs can be dramatically improved using microsphere encapsulation combined with suspension in ECM hydrogel. These biomaterial innovations are essential to advance clinical efforts to improve the treatment of stroke using intracerebral cell therapy.

Ex vivo biomechanical characterization of syringe-needle ejections for intracerebral cell delivery.

Intracerebral implantation of cell suspensions is finding its clinical translation with encouraging results in patients with stroke. However, the survival of cells in the brain remains poor. Although the biological potential of neural stem cells (NSCs) is widely documented, the biomechanical effects of delivering cells through a syringe-needle remain poorly understood. We here detailed the biomechanical forces (pressure, shear stress) that cells are exposed to during ejection through different sized needles (20G, 26G, 32G) and syringes (10, 50, 250 µL) at relevant flow rates (1, 5, 10 µL/min). A comparison of 3 vehicles, Phosphate Buffered Saline (PBS), Hypothermosol (HTS), and Pluronic, indicated that less viscous vehicles are favorable for suspension with a high cell volume fraction to minimize sedimentation. Higher suspension viscosity was associated with greater shear stress. Higher flow rates with viscous vehicle, such as HTS reduced viability by ~10% and also produced more apoptotic cells (28%). At 5 µL/min ejection using a 26G needle increased neuronal differentiation for PBS and HTS suspensions. These results reveal the biological impact of biomechanical forces in the cell delivery process. Appropriate engineering strategies can be considered to mitigate these effects to ensure the efficacious translation of this promising therapy.

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

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.

Diamagnetic chemical exchange saturation transfer (diaCEST) affords magnetic resonance imaging of extracellular matrix hydrogel implantation in a rat model of stroke.

Extracellular matrix (ECM) is widely used as an inductive biological scaffold to repair soft tissue after injury by promoting functional site-appropriate remodeling of the implanted material. However, there is a lack of non-invasive analysis methods to monitor the remodeling characteristics of the ECM material after implantation and its biodegradation over time. We describe the use of diamagnetic chemical exchange saturation transfer (CEST) magnetic resonance imaging to monitor the distribution of an ECM hydrogel after intracerebral implantation into a stroke cavity. In vitro imaging indicated a robust concentration-dependent detection of the ECM precursor and hydrogel at 1.8 and 3.6 ppm, which broadly corresponded to chondroitin sulfate and fibronectin. This detection was robust to changes in pH and improved at 37 °C. In vivo implantation of ECM hydrogel into the stroke cavity in a rat model corresponded macroscopically to the distribution of biomaterial as indicated by histology, but mismatches were also evident. Indeed, CEST imaging detected an endogenous "increased deposition". To account for this endogenous activity, pre-implantation images were subtracted from post-implantation images to yield a selective visualization of hydrogel distribution in the stroke cavity and its evolution over 7 days. The CEST detection of ECM returned to baseline within 3 days due to a decrease in fibronectin and chondroitin sulfate in the hydrogel. The distribution of ECM hydrogel within the stroke cavity is hence feasible in vivo, but further advances are required to warrant a selective long-term monitoring in the context of biodegradation.

Long-term retention of ECM hydrogel after implantation into a sub-acute stroke cavity reduces lesion volume.

Salvaging or functional replacement of damaged tissue caused by stroke in the brain remains a major therapeutic challenge. In situ gelation and retention of a hydrogel bioscaffold composed of 8mg/mL extracellular matrix (ECM) can induce a robust invasion of cells within 24h and potentially promote a structural remodeling to replace lost tissue. Herein, we demonstrate a long-term retention of ECM hydrogel within the lesion cavity. A decrease of approximately 32% of ECM volume is observed over 12weeks. Lesion volume, as measured by magnetic resonance imaging and histology, was reduced by 28%, but a battery of behavioral tests (bilateral asymmetry test; footfault; rotameter) did not reveal a therapeutic or detrimental effect of the hydrogel. Glial scarring and peri-infarct astrocytosis were equivalent between untreated and treated animals, potentially indicating that permeation into host tissue is required to exert therapeutic effects. These results reveal a marked difference of biodegradation of ECM hydrogel in the stroke-damaged brain compared to peripheral soft tissue repair. Further exploration of these structure-function relationships is required to achieve a structural remodeling of the implanted hydrogel, as seen in peripheral tissues, to replace lost tissue and promote behavioral recovery. STATEMENT OF SIGNIFICANCE: In situ gelation of ECM is essential for its retention within a tissue cavity. The brain is a unique environment with restricted access that necessitates image-guided delivery through a thin needle to access tissue cavities caused by stroke, as well as other conditions, such as traumatic brain injury or glioma resection. Knowledge about a brain tissue response to implanted hydrogels remains limited, especially in terms of long-term effects and potential impact on behavioral function. We here address the long-term retention of hydrogel within the brain environment, its impact on behavioral function, as well as its ability to reduce further tissue deformation caused by stroke. This study highlights considerable differences in the brain's long-term response to an ECM hydrogel compared to peripheral soft tissue. It underlines the importance of understanding the effect of the structural presence of a hydrogel within a cavity upon host brain tissue and behavioral function. As demonstrated herein, ECM hydrogel can fill a cavity long-term to reduce further progression of the cavity, while potentially serving as a reservoir for local drug or cell delivery.

Biological effects of dosing aerobic exercise and neuromuscular electrical stimulation in rats.

Aerobic exercise (AE) and non-aerobic neuromuscular electric stimulation (NMES) are common interventions used in physical therapy. We explored the dose-dependency (low, medium, high) of these interventions on biochemical factors, such as brain derived neurotrophic growth factor (BDNF), vascular endothelial growth factor-A (VEGF-A), insulin-like growth factor-1 (IGF-1) and Klotho, in the blood and brain of normal rats, as well as a treadmill-based maximum capacity test (MCT). A medium dose of AE produced the most improvement in MCT with dose-dependent changes in Klotho in the blood. A dose-dependent increase of BDNF was evident following completion of an NMES protocol, but there was no improvement in MCT performance. Gene expression in the hippocampus was increased after both AE and NMES, with IGF-1 being a signaling molecule that correlated with MCT performance in the AE conditions, but also highly correlated with VEGF-A and Klotho. Blood Klotho levels can serve as a biomarker of therapeutic dosing of AE, whereas IGF-1 is a key molecule coupled to gene expression of other molecules in the hippocampus. This approach provides a translatable paradigm to investigate the mode and mechanism of action of interventions employed in physical therapy that can improve our understanding of how these factors change under pathological conditions.

DNA-gadolinium-gold nanoparticles for in vivo T1 MR imaging of transplanted human neural stem cells.

The unambiguous imaging of transplanted cells remains a major challenge to understand their biological function and therapeutic efficacy. In vivo imaging of implanted cells is reliant on tagging these to differentiate them from host tissue, such as the brain. We here characterize a gold nanoparticle conjugate that is functionalized with modified deoxythymidine oligonucleotides bearing Gd(III) chelates and a red fluorescent Cy3 moiety to visualize in vivo transplanted human neural stem cells. This DNA-Gd@Au nanoparticle (DNA-Gd@AuNP) exhibits an improved T1 relaxivity and excellent cell uptake. No significant effects of cell uptake have been found on essential cell functions. Although T1 relaxivity is attenuated within cells, it is sufficiently preserved to afford the in vivo detection of transplanted cells using an optimized voxel size. In vivo MR images were corroborated by a post-mortem histological verification of DNA-Gd@AuNPs in transplanted cells. With 70% of cells being correctly identified using the DNA-Gd-AuNPs indicates an overall reliable detection. Less than 1% of cells were false positive for DNA-Gd@AuNPs, but a significant number of 30% false negatives reveals a dramatic underestimation of transplanted cells using this approach. DNA-Gd@AuNPs therefore offer new opportunities to visualize transplanted cells unequivocally using T1 contrast and use cellular MRI as a tool to derive biologically relevant information that allows us to understand how the survival and location of implanted cells determines therapeutic efficacy.

ECM hydrogel for the treatment of stroke: Characterization of the host cell infiltrate.

Brain tissue loss following stroke is irreversible with current treatment modalities. The use of an acellular extracellular matrix (ECM), formulated to produce a hydrogel in situ within the cavity formed by a stroke, was investigated as a method to replace necrotic debris and promote the infiltration of host brain cells. Based on magnetic resonance imaging measurements of lesion location and volume, different concentrations of ECM (0, 1, 2, 3, 4, 8 mg/mL) were injected at a volume equal to that of the cavity (14 days post-stroke). Retention of ECM within the cavity occurred at concentrations >3 mg/mL. A significant cell infiltration into the ECM material in the lesion cavity occurred with an average of ∼36,000 cells in the 8 mg/mL concentration within 24 h. An infiltration of cells with distances of >1500 µm into the ECM hydrogel was observed, but the majority of cells were at the tissue/hydrogel boundary. Cells were typically of a microglia, macrophage, or neural and oligodendrocyte progenitor phenotype. At the 8 mg/mL concentration, ∼60% of infiltrating cells were brain-derived phenotypes and 30% being infiltrating peripheral macrophages, polarizing toward an M2-like anti-inflammatory phenotype. These results suggest that an 8 mg/mL ECM concentration promotes a significant acute endogenous repair response that could potentially be exploited to treat stroke.

Concentration-dependent rheological properties of ECM hydrogel for intracerebral delivery to a stroke cavity.

Biomaterials composed of mammalian extracellular matrix (ECM) promote constructive tissue remodeling with minimal scar tissue formation in many anatomical sites. However, the optimal shape and form of ECM scaffold for each clinical application can vary markedly. ECM hydrogels have been shown to promote chemotaxis and differentiation of neuronal stem cells, but minimally invasive delivery of such scaffold materials to the central nervous system (CNS) would require an injectable form. These ECM materials can be manufactured to exist in fluid phase at room temperature, while forming hydrogels at body temperature in a concentration-dependent fashion. Implantation into the lesion cavity after a stroke could hence provide a means to support endogenous repair mechanisms. Herein, we characterize the rheological properties of an ECM hydrogel composed of urinary bladder matrix (UBM) that influence its delivery and in vivo interaction with host tissue. There was a notable concentration-dependence in viscosity, stiffness, and elasticity; all characteristics important for minimally invasive intracerebral delivery. An efficient MRI-guided injection with drainage of fluid from the cavity is described to assess in situ hydrogel formation and ECM retention at different concentrations (0, 1, 2, 3, 4, and 8mg/mL). Only ECM concentrations >3mg/mL gelled within the stroke cavity. Lower concentrations were not retained within the cavity, but extensive permeation of the liquid phase ECM into the peri-infarct area was evident. The concentration of ECM hydrogel is hence an important factor affecting gelation, host-biomaterial interface, as well intra-lesion distribution. STATEMENT OF SIGNIFICANCE: Extracellular matrix (ECM) hydrogel promotes constructive tissue remodeling in many tissues. Minimally invasive delivery of such scaffold materials to the central nervous system (CNS) would require an injectable form that exists in fluid phase at room temperature, while forming hydrogels at body temperature in a concentration-dependent fashion. We here report the rheological characterization of an injectable ECM hydrogel and its concentration-dependent delivery into a lesion cavity formed after a stroke based on MRI-guidance. The concentration of ECM determined its retention within the cavity or permeation into tissue and hence influenced its interaction with the host brain. This study demonstrates the importance of understanding the structure-function relationship of biomaterials to guide particular clinical applications.