Engineering self-assembling silk hydrogels for the delivery of stem cells

Advanced cell therapies require robust delivery materials and silk is a promising contender with a long clinical track record. Our aim was to optimise self-assembling silk hydrogels as a mesenchymal stem cell (MSC)-support matrix that would allow future minimally invasive brain application.

Context of the research: Stroke is the number one cause of disability in the UK, with an estimated 150,000 new cases annually. Following the acute onset of stroke, lack of oxygen leads to massive neuronal cell death within minutes and progressive brain damage over the following hours and days. A therapeutic intervention that would retard or even halt stroke progression would benefit stroke survivors. In this context, stem cell-based therapies are currently being assessed in patients. However, delivering those cells safely and effectively to the area in the brain where they are needed most is challenging. There is the need to develop delivery systems that can place, retain, support and protect applied stem cells to maximise their therapeutic potential.

Aims and objectives: Ensuring that a delivery system can fulfil all these needs is not trivial and requires careful selection of the most suitable strategy. The overall aim is to develop gels that can be loaded with stem cells and injected into the stroked brain, supports and protects the applied stem cell load at the target site.

Outcomes: We used energy to programme the transition of liquid silk to a stable gel. This method allowed us to fine tune the self-assembly process of silk gels to achieve space conformity in the absence of any silk gel swelling while supporting healthy cell growth. Embedded stem cells showed excellent health even after injection through a 30G needle, especially when the gels were in the pre-gelled state. Silk gels with physical characteristics matching brain tissue exhibited good space conformity in stroke brains. Next, the impact on stroke symptoms, interaction with the stroke scar, interference with the normal inflammatory response and cell growth in the lesion cavity were examined for several weeks. Self-assembling gels presented neither an overt stroke scar response nor adverse side effects. This study informs on an optimal stem cell gel matrix for minimally invasive application as a platform for targeting brain repair. The stroke model confirmed that self-assembling silk gels provide a favorable microenvironment as a future support matrix in the stroke cavity.

We used sonication energy to programme the transition of silk (1–5% w/v) secondary structure from a random coil to a stable β-sheet configuration. This allowed fine tuning of self-assembling silk hydrogels to achieve space conformity in the absence of any silk hydrogel swelling and to support uniform cell distribution as well as cell viability. Embedded cells underwent significant proliferation over 14 days in vitro, with the best proliferation achieved with 2% w/v hydrogels. Embedded MSCs showed significantly better viability in vitro after injection through a 30G needle when the gels were in the pre-gelled versus post-gelled state. Silk hydrogels (4% w/v) with physical characteristics matching brain tissue were visualised in preliminary in vivo experiments to exhibit good space conformity in an ischemic cavity (intraluminal thread middle cerebral artery occlusion model) in adult male Sprague-Dawley rats (n = 3). This study informs on optimal MSC-hydrogel matrix conditions for minimally invasive application as a platform for future experiments targeting brain repair.

Next, we assessed the performance of these silk hydrogels in the stroke setting. Targeting the brain cavity formed by an ischemic stroke is
appealing for many regenerative treatment strategies but requires a robust delivery technology. We hypothesised that self-assembling silk fibroin hydrogels could serve as a reliable support matrix for regeneration in the stroke cavity. We therefore performed in vivo evaluation studies of self-assembling silk fibroin hydrogels after intracerebral injection in a rat stroke model. Adult male Sprague−Dawley rats (n = 24) underwent transient middle cerebral artery occlusion (MCAo) 2 weeks before random
assignment to either no stereotaxic injection or a stereotaxic injection of
either self-assembling silk fibroin hydrogels (4% w/v) or PBS into the
lesion cavity. The impact on morbidity and mortality, space conformity,
interaction with glial scar, interference with inflammatory response, and
cell proliferation in the lesion cavity were examined for up to 7 weeks by a
blinded investigator. Self-assembling hydrogels filled the stroke cavity with excellent space conformity and presented neither an overt microglial/macrophage response nor an adverse morbidity or mortality. The relationship between the number of proliferating cells and lesion volume was significantly changed by injection of self-assembling silk hydrogels. This in vivo stroke model confirmed that self-assembling silk fibroin hydrogels provide a favorable microenvironment as a future support matrix in the stroke cavity.

Engineering self-assembling silk hydrogels for the delivery of stem cells