Biologic-free mechanically induced muscle regeneration.

Severe skeletal muscle injuries are common and can lead to extensive fibrosis, scarring, and loss of function. Clinically, no therapeutic intervention exists that allows for a full functional restoration. As a result, both drug and cellular therapies are being widely investigated for treatment of muscle injury. Because muscle is known to respond to mechanical loading, we investigated instead whether a material system capable of massage-like compressions could promote regeneration. Magnetic actuation of biphasic ferrogel scaffolds implanted at the site of muscle injury resulted in uniform cyclic compressions that led to reduced fibrous capsule formation around the implant, as well as reduced fibrosis and inflammation in the injured muscle. In contrast, no significant effect of ferrogel actuation on muscle vascularization or perfusion was found. Strikingly, ferrogel-driven mechanical compressions led to enhanced muscle regeneration and a ∼threefold increase in maximum contractile force of the treated muscle at 2 wk compared with no-treatment controls. Although this study focuses on the repair of severely injured skeletal muscle, magnetically stimulated bioagent-free ferrogels may find broad utility in the field of regenerative medicine.

Ultrasound-triggered disruption and self-healing of reversibly cross-linked hydrogels for drug delivery and enhanced chemotherapy.

Biological systems are exquisitely sensitive to the location and timing of physiologic cues and drugs. This spatiotemporal sensitivity presents opportunities for developing new therapeutic approaches. Polymer-based delivery systems are used extensively for attaining localized, sustained release of bioactive molecules. However, these devices typically are designed to achieve a constant rate of release. We hypothesized that it would be possible to create digital drug release, which could be accelerated and then switched back off, on demand, by applying ultrasound to disrupt ionically cross-linked hydrogels. We demonstrated that ultrasound does not permanently damage these materials but enables nearly digital release of small molecules, proteins, and condensed oligonucleotides. Parallel in vitro studies demonstrated that the concept of applying temporally short, high-dose "bursts" of drug exposure could be applied to enhance the toxicity of mitoxantrone toward breast cancer cells. We thus used the hydrogel system in vivo to treat xenograft tumors with mitoxantrone, and found that daily ultrasound-stimulated drug release substantially reduced tumor growth compared with sustained drug release alone. This approach of digital drug release likely will be applicable to a broad variety of polymers and bioactive molecules, and is a potentially useful tool for studying how the timing of factor delivery controls cell fate in vivo.

Sustained delivery of VEGF maintains innervation and promotes reperfusion in ischemic skeletal muscles via NGF/GDNF signaling.

Tissue reinnervation following trauma, disease, or transplantation often presents a significant challenge. Here, we show that the delivery of vascular endothelial growth factor (VEGF) from alginate hydrogels ameliorates loss of skeletal muscle innervation after ischemic injury by promoting both maintenance and regrowth of damaged axons in mice. Nerve growth factor (NGF) and glial-derived neurotrophic factor (GDNF) mediated VEGF-induced axonal regeneration, and the expression of both is induced by VEGF presentation. Using both in vitro and in vivo modeling approaches, we demonstrate that the activity of NGF and GDNF regulates VEGF-driven angiogenesis, controlling endothelial cell sprouting and blood vessel maturation. Altogether, these studies produce evidence of new mechanisms of VEGF action, further broaden the understanding of the roles of NGF and GDNF in angiogenesis and axonal regeneration, and suggest approaches to improve axonal and ischemic tissue repair therapies.

Active scaffolds for on-demand drug and cell delivery.

Porous biomaterials have been widely used as scaffolds in tissue engineering and cell-based therapies. The release of biological agents from conventional porous scaffolds is typically governed by molecular diffusion, material degradation, and cell migration, which do not allow for dynamic external regulation. We present a new active porous scaffold that can be remotely controlled by a magnetic field to deliver various biological agents on demand. The active porous scaffold, in the form of a macroporous ferrogel, gives a large deformation and volume change of over 70% under a moderate magnetic field. The deformation and volume variation allows a new mechanism to trigger and enhance the release of various drugs including mitoxantrone, plasmid DNA, and a chemokine from the scaffold. The porous scaffold can also act as a depot of various cells, whose release can be controlled by external magnetic fields.

Functional muscle regeneration with combined delivery of angiogenesis and myogenesis factors.

Regenerative efforts typically focus on the delivery of single factors, but it is likely that multiple factors regulating distinct aspects of the regenerative process (e.g., vascularization and stem cell activation) can be used in parallel to affect regeneration of functional tissues. This possibility was addressed in the context of ischemic muscle injury, which typically leads to necrosis and loss of tissue and function. The role of sustained delivery, via injectable gel, of a combination of VEGF to promote angiogenesis and insulin-like growth factor-1 (IGF1) to directly promote muscle regeneration and the return of muscle function in ischemic rodent hindlimbs was investigated. Sustained VEGF delivery alone led to neoangiogenesis in ischemic limbs, with complete return of tissue perfusion to normal levels by 3 weeks, as well as protection from hypoxia and tissue necrosis, leading to an improvement in muscle contractility. Sustained IGF1 delivery alone was found to enhance muscle fiber regeneration and protected cells from apoptosis. However, the combined delivery of VEGF and IGF1 led to parallel angiogenesis, reinnervation, and myogenesis; as satellite cell activation and proliferation was stimulated, cells were protected from apoptosis, the inflammatory response was muted, and highly functional muscle tissue was formed. In contrast, bolus delivery of factors did not have any benefit in terms of neoangiogenesis and perfusion and had minimal effect on muscle regeneration. These results support the utility of simultaneously targeting distinct aspects of the regenerative process.

Improved magnetic regulation of delivery profiles from ferrogels.

While providing the ability to magnetically enhance delivery rates, ferrogels have not been able to produce the various types of regulated delivery profiles likely needed to direct complex biological processes. For example, magnetically triggered release after prolonged periods of payload retention have not been demonstrated and little has been accomplished towards remotely controlling release rate through alterations in the magnetic signal. Also, strategies do not exist for magnetically coordinating multi-drug sequences. The purpose of this study was to develop these capabilities through improved ferrogel design and investigating how alterations in the magnetic signal impact release characteristics. Results show that delivery rate can be remotely regulated using the frequency of magnetic stimulation. When using an optimized biphasic ferrogel design, stimulation at optimized frequencies enabled magnetically triggered deliveries after a delay of 5 days that were 690- to 1950-fold higher than unstimulated baseline values. Also, a sequence of two payloads was produced by allowing one payload to initially diffuse out of the ferrogel, followed by magnetically triggered release of a different payload on day 5. Finally, it was demonstrated that two payloads could be sequentially triggered for release by first stimulating at a frequency tuned to preferentially release one payload (after 24 h), followed by stimulation at a different frequency tuned to preferentially release the other payload (After 4 days). The strategies developed here may expand the utility of ferrogels in clinical scenarios where the timing and sequence of biological events can be tuned to optimize therapeutic outcome.

Timed Delivery of Therapy Enhances Functional Muscle Regeneration.

Cell transplantation is a promising therapeutic strategy for the treatment of traumatic muscle injury in humans. Previous investigations have typically focused on the identification of potent cell and growth factor treatments and optimization of spatial control over delivery. However, the optimal time point for cell transplantation remains unclear. Here, this study reports how myoblast and morphogen delivery timed to coincide with specific phases of the inflammatory response affects donor cell engraftment and the functional repair of severely injured muscle. Delivery of a biomaterial-based therapy timed with the peak of injury-induced inflammation leads to potent early and long-term regenerative benefits. Diminished inflammation and fibrosis, enhanced angiogenesis, and increased cell engraftment are seen during the acute stage following optimally timed treatment. Over the long term, treatment during peak inflammation leads to enhanced functional regeneration, as indicated by reduced chronic inflammation and fibrosis along with increased tissue perfusion and muscle contractile force. Treatments initiated immediately after injury or after inflammation had largely resolved provided more limited benefits. These results demonstrate the importance of appropriately timing the delivery of biologic therapy in the context of muscle regeneration. Biomaterial-based timed delivery can likely be applied to other tissues and is of potential wide utility in regenerative medicine.

Biomaterial-based delivery for skeletal muscle repair.

Skeletal muscle possesses a remarkable capacity for regeneration in response to minor damage, but severe injury resulting in a volumetric muscle loss can lead to extensive and irreversible fibrosis, scarring, and loss of muscle function. In early clinical trials, the intramuscular injection of cultured myoblasts was proven to be a safe but ineffective cell therapy, likely due to rapid death, poor migration, and immune rejection of the injected cells. In recent years, appropriate therapeutic cell types and culturing techniques have improved progenitor cell engraftment upon transplantation. Importantly, the identification of several key biophysical and biochemical cues that synergistically regulate satellite cell fate has paved the way for the development of cell-instructive biomaterials that serve as delivery vehicles for cells to promote in vivo regeneration. Material carriers designed to spatially and temporally mimic the satellite cell niche may be of particular importance for the complete regeneration of severely damaged skeletal muscle.

Biphasic ferrogels for triggered drug and cell delivery.

Ferrogels are an attractive material for many biomedical applications due to their ability to deliver a wide variety of therapeutic drugs on-demand. However, typical ferrogels have yet to be optimized for use in cell-based therapies, as they possess limited ability to harbor and release viable cells. Previously, an active porous scaffold that exhibits large deformations and enhanced biological agent release under moderate magnetic fields has been demonstrated. Unfortunately, at small device sizes optimal for implantation (e.g., 2 mm thickness), these monophasic ferrogels no longer achieve significant deformation due to a reduced body force. A new biphasic ferrogel, containing an iron oxide gradient, capable of large deformations and triggered release even at small gel dimensions, is presented in this study. Biphasic ferrogels demonstrate increased porosity, enhanced mechanical properties, and potentially increased biocompatibility due to their reduced iron oxide content. With their ability to deliver drugs and cells on-demand, it is expected that these ferrogels will have wide utility in the fields of tissue engineering and regenerative medicine.

The role of multifunctional delivery scaffold in the ability of cultured myoblasts to promote muscle regeneration.

Many cell types of therapeutic interest, including myoblasts, exhibit reduced engraftment if cultured prior to transplantation. This study investigated whether polymeric scaffolds that direct cultured myoblasts to migrate outwards and repopulate the host damaged tissue, in concert with release of angiogenic factors designed to enhance revascularizaton of the regenerating tissue, would enhance the efficacy of this cell therapy and lead to functional muscle regeneration. This was investigated in the context of a severe injury to skeletal muscle tissue involving both myotoxin-mediated direct damage and induction of regional ischemia. Local and sustained release of VEGF and IGF-1 from macroporous scaffolds used to transplant and disperse cultured myogenic cells significantly enhanced their engraftment, limited fibrosis, and accelerated the regenerative process. This resulted in increased muscle mass and, improved contractile function. These results demonstrate the importance of finely controlling the microenvironment of transplanted cells in the treatment of severe muscle damage.