Periodic Reporting for period 1 - Multiple Sclerosis (Development of a Functionalised Biomaterial Scaffold to Treat Multiple Sclerosis)
Periodo di rendicontazione: 2018-08-01 al 2020-07-31
Multiple sclerosis (MS) is a chronic debilitating demyelinating disease of the central nervous system (CNS) which affects over 700,000 people in Europe with a total annual health care cost of €27.3 billion (Ernstsson et al., 2016). Current therapeutic strategies offer modest or ephemeral relieve of MS symptoms. However, they fail to modify disease progression. As a result, there is currently no licensed disease-modifying therapy for MS (Hauser & Cree, 2020). Inflammation is a common pathological feature of MS (Calabrese et al., 2015; Milstein, Barbour, Jackson, Kosa, & Bielekova, 2019). In particular, the inflammatory insult that drives the pathology becomes condoned off behind an intact blood-brain barrier (BBB) during MS progression. This leads to a clinical scenario where the delivery of antibody-based therapeutics is extraordinarily challenging and ineffective due to the inability to cross the non-compromised BBB (Matthews, 2019).
In the last 15 years, some studies showed the critical role of extracellular matrix (ECM) in the development and homeostasis of the CNS (Aleithe et al., 2019; Back et al., 2005; Crapser et al., 2020). The main components of neural ECM include chondroitin sulphate proteoglycans, tenascin-R and hyaluronic acid, which are synthesized by both neurons and glial cells (Srivastava, Sherman, & Back, 2020). These molecules are regulated in physiological conditions, shaping both neuronal and glial functions through a multitude of molecular mechanisms. The upregulation of some particular ECM molecules by reactive astrocytes during neuroinflammation and neurodegeneration results in the formation of growth-impermissive environment and impaired synaptic plasticity. Some authors also described the fact that ECM molecules have a neuroprotective effect, at least in the form of perineuronal nets. All this data suggests that targeting expression of specific ECM molecules may lead to the development of new therapeutic strategies promoting regeneration and synaptic plasticity (Song & Dityatev, 2018)
Changes in ECM content and metabolism have been related to several pathological conditions, including stroke, traumatic injury and MS, and these changes disrupt repair by endogenous neural stem cells.
Herein, we set out for the first time to test address local delivery of an ECM scaffold into the CNS that could downregulate neuroinflammation associated to the disease. We hypothesized that an external ECM source would lead to a downregulating of the astrogliosis generated after MS-like induction reducing its severity and decreasing the incidence of the disease. This will also avoid the ECM deposition and scar generation in the demyelinated axons, allowing oligodendrocytes to remyelinate the injured axons after the MS inflammatory insult.
In the last 15 years, some studies showed the critical role of extracellular matrix (ECM) in the development and homeostasis of the CNS (Aleithe et al., 2019; Back et al., 2005; Crapser et al., 2020). The main components of neural ECM include chondroitin sulphate proteoglycans, tenascin-R and hyaluronic acid, which are synthesized by both neurons and glial cells (Srivastava, Sherman, & Back, 2020). These molecules are regulated in physiological conditions, shaping both neuronal and glial functions through a multitude of molecular mechanisms. The upregulation of some particular ECM molecules by reactive astrocytes during neuroinflammation and neurodegeneration results in the formation of growth-impermissive environment and impaired synaptic plasticity. Some authors also described the fact that ECM molecules have a neuroprotective effect, at least in the form of perineuronal nets. All this data suggests that targeting expression of specific ECM molecules may lead to the development of new therapeutic strategies promoting regeneration and synaptic plasticity (Song & Dityatev, 2018)
Changes in ECM content and metabolism have been related to several pathological conditions, including stroke, traumatic injury and MS, and these changes disrupt repair by endogenous neural stem cells.
Herein, we set out for the first time to test address local delivery of an ECM scaffold into the CNS that could downregulate neuroinflammation associated to the disease. We hypothesized that an external ECM source would lead to a downregulating of the astrogliosis generated after MS-like induction reducing its severity and decreasing the incidence of the disease. This will also avoid the ECM deposition and scar generation in the demyelinated axons, allowing oligodendrocytes to remyelinate the injured axons after the MS inflammatory insult.
Up to our knowledge, this is the first time that an ECM based hydrogel has been tested in an in vitro model of MS-like neuroinflammation. As we mentioned before, in MS, ECM deposits are present in areas of demyelination. Reactive astrocytes produce high levels of hyaluronic acid and chondroitin sulphate after an inflammatory insult, which will accumulate in the damaged areas. Considering this, we aimed to test the effect of an exogenous ECM hydrogel in a model mimicking neuroinflammation associated with MS and characterize how the material can affect the physiological changes related to the inflammatory insult.
Different ECM scaffolds formulations were prepared and characterized in terms of their physicochemical properties exhibiting suitable mechanical properties to be use in the CNS.
In vitro studies with primary mixed cerebellar cells and primary mixed cortical cells were performed to assess the cytotoxicity and compatibility of the HA based scaffold. None of the tested formulations had any adverse effect in terms of metabolic activity and dsDNA content up to seven days in vitro in the presence of the materials. Also, the inflammatory profile of the mixed glial cells after the treatment has been asset by nitrite concentration in the media, ELISA and immunocytochemistry of the most relevant markers, showing significant improvement when compared with controls. Changes in relevant ECM biomarkers after inflammation and treatment with the scaffold were determined by immunocytochemistry. All these data together suggested a potential modulatory effect on ECM intrinsic metabolism that could lead to a downregulation in inflammation to be confirmed in vivo.
Different ECM scaffolds formulations were prepared and characterized in terms of their physicochemical properties exhibiting suitable mechanical properties to be use in the CNS.
In vitro studies with primary mixed cerebellar cells and primary mixed cortical cells were performed to assess the cytotoxicity and compatibility of the HA based scaffold. None of the tested formulations had any adverse effect in terms of metabolic activity and dsDNA content up to seven days in vitro in the presence of the materials. Also, the inflammatory profile of the mixed glial cells after the treatment has been asset by nitrite concentration in the media, ELISA and immunocytochemistry of the most relevant markers, showing significant improvement when compared with controls. Changes in relevant ECM biomarkers after inflammation and treatment with the scaffold were determined by immunocytochemistry. All these data together suggested a potential modulatory effect on ECM intrinsic metabolism that could lead to a downregulation in inflammation to be confirmed in vivo.
The suitability of the formulations was tested in an in vitro screening to reduce the number of animals needed and based on previous work on the group with similar devices in CNS for other applications. The performance of the tested ECM scaffolds is promising for their potential use in vivo. Besides, the effect of the degradation products of the proposed scaffolds was tested in the same models to avoid any potential undesired effect in animal studies, showing no impact on metabolic activity nor dsDNA content.
The most suitable system has been selected for further studies and to be tested in vivo in a model of cortical neuroinflammation that would potentially confirm the promising outcome shown in vitro. However, due to the global pandemic, the tasks related to this particular study are still ongoing
The most suitable system has been selected for further studies and to be tested in vivo in a model of cortical neuroinflammation that would potentially confirm the promising outcome shown in vitro. However, due to the global pandemic, the tasks related to this particular study are still ongoing