Background

Neurovascular unit restoration is crucial for nerve regeneration, especially in critical gaps of injured peripheral nerve. Multipotent vascular stem cells (MVSCs) harvested from an adult blood vessel are involved in vascular remodeling; however, the therapeutic benefit for nerve regeneration is not clear.

Methods

MVSCs were isolated from rats expressing green fluorescence protein (GFP), expanded, mixed with Matrigel matrix, and loaded into the nerve conduits. A nerve autograft or a nerve conduit (with acellular matrigel or MVSCs in matrigel) was used to bridge a transected sciatic nerve (10-mm critical gap) in rats. The functional motor recovery and cell fate in the regenerated nerve were investigated to understand the therapeutic benefit.

Results

MVSCs expressed markers such as Sox 17 and Sox10 and could differentiate into neural cells in vitro. One month following MVSC transplantation, the compound muscle action potential (CMAP) significantly increased as compared to the acellular group. MVSCs facilitated the recruitment of Schwann cell to regenerated axons. The transplanted cells, traced by GFP, differentiated into perineurial cells around the bundles of regenerated myelinated axons. In addition, MVSCs enhanced tight junction formation as a part of the blood-nerve barrier (BNB). Furthermore, MVSCs differentiated into perivascular cells and enhanced microvessel formation within regenerated neurovascular bundles.

Conclusions

In rats with peripheral nerve injuries, the transplantation of MVSCs into the nerve conduits improved the recovery of neuromuscular function; MVSCs differentiated into perineural cells and perivascular cells and enhanced the formation of tight junctions in perineural BNB. This study demonstrates the in vivo therapeutic benefit of adult MVSCs for peripheral nerve regeneration and provides insight into the role of MVSCs in BNB regeneration.

Electroceuticals provide promising opportunities for peripheral nerve regeneration, in terms of modulating the extensive endogenous tissue repair mechanisms between neural cell body, axons and target muscles. However, great challenges remain to deliver effective and controllable electroceuticals via bioelectronic implantable device. In this review, the modern fabrication methods of bioelectronic conduit for bridging critical nerve gaps after nerve injury are summarized, with regard to conductive materials and core manufacturing process. In addition, to deliver versatile electrical stimulation, the integration of implantable bioelectronic device is discussed, including wireless energy harvesters, actuators and sensors. Moreover, a comprehensive insight of beneficial mechanisms is presented, including up-to-datein vitro, in vivoand clinical evidence. By integrating conductive biomaterials, 3D engineering manufacturing process and bioelectronic platform to deliver versatile electroceuticals, the modern biofabrication enables comprehensive biomimetic therapies for neural tissue engineering and regeneration in the new era.

Cell reprogramming is a revolutionized biotechnology that offers a powerful tool to engineer cell fate and function for regenerative medicine, disease modeling, drug discovery, and beyond. Leveraging advances in biomaterials and micro/nanotechnologies can enhance the reprogramming performance in vitro and in vivo through the development of delivery strategies and the control of biophysical and biochemical cues. In this review, we present an overview of the state-of-the-art technologies for cell reprogramming and highlight the recent breakthroughs in engineering biomaterials with micro/nanotechnologies to improve reprogramming efficiency and quality. Finally, we discuss future directions and challenges for reprogramming technologies and clinical translation.

Muscle loss and impairment resulting from traumatic injury can be alleviated by therapies using muscle stem cells. However, collecting sufficient numbers of autologous myogenic stem cells and expanding them efficiently has been challenging. Here we show that myogenic stem cells (predominantly Pax7⁺ cells)-which were selectively expanded from readily obtainable dermal fibroblasts or skeletal muscle stem cells using a specific cocktail of small molecules and transplanted into muscle injuries in adult, aged or dystrophic mice-led to functional muscle regeneration in the three animal models. We also show that sustained release of the small-molecule cocktail in situ through polymer nanoparticles led to muscle repair by inducing robust activation and expansion of resident satellite cells. Chemically induced stem cell expansion in vitro and in situ may prove to be advantageous for stem cell therapies that aim to regenerate skeletal muscle and other tissues.

Background

Muscle denervation from trauma and motor neuron disease causes disabling morbidities. A limiting step in functional recovery is the regeneration of neuromuscular junctions (NMJs) for reinnervation. Stem cells have the potential to promote these regenerative processes, but current approaches have limited success, and the optimal types of stem cells remain to be determined. Neural crest stem cells (NCSCs), as the developmental precursors of the peripheral nervous system, are uniquely advantageous, but the role of NCSCs in neuromuscular regeneration is not clear. Furthermore, a cell delivery approach that can maintain NCSC survival upon transplantation is critical.

Methods

We established a streamlined protocol to derive, isolate, and characterize functional p75⁺ NCSCs from human iPSCs without genome integration of reprogramming factors. To enhance survival rate upon delivery in vivo, NCSCs were centrifuged in microwell plates to form spheroids of desirable size by controlling suspension cell density. Human bone marrow mesenchymal stem cells (MSCs) were also studied for comparison. NCSC or MSC spheroids were injected into the gastrocnemius muscle with denervation injury, and the effects on NMJ formation and functional recovery were investigated. The spheroids were also co-cultured with engineered neuromuscular tissue to assess effects on NMJ formation in vitro.

Results

NCSCs cultured in spheroids displayed enhanced secretion of soluble factors involved in neuromuscular regeneration. Intramuscular transplantation of spheroids enabled long-term survival and retention of NCSCs, in contrast to the transplantation of single-cell suspensions. Furthermore, NCSC spheroids significantly improved functional recovery after four weeks as shown by gait analysis, electrophysiology, and the rate of NMJ innervation. MSC spheroids, on the other hand, had insignificant effect. In vitro co-culture of NCSC or MSC spheroids with engineered myotubes and motor neurons further evidenced improved innervated NMJ formation with NCSC spheroids.

Conclusions

We demonstrate that stem cell type is critical for neuromuscular regeneration and that NCSCs have a distinct advantage and therapeutic potential to promote reinnervation following peripheral nerve injury. Biophysical effects of spheroidal culture, in particular, enable long-term NCSC survival following in vivo delivery. Furthermore, synthetic neuromuscular tissue, or "tissues-on-a-chip," may offer a platform to evaluate stem cells for neuromuscular regeneration.

Little is known regarding the interplays between the mechanical and molecular bases for vein graft restenosis. We elucidated the stenosis initiation using a high-frequency ultrasonic (HFU) echogenicity platform and estimated the endothelium yield stress from von-Mises stress computation to predict the damage locations in living rats over time. The venous-arterial transition induced the molecular cascades for autophagy and apoptosis in venous endothelial cells (ECs) to cause neointimal hyperplasia, which correlated with the high echogenicity in HFU images and the large mechanical stress that exceeded the yield strength. The ex vivo perfusion of arterial laminar shear stress to isolated veins further confirmed the correlation. EC damage can be rescued by inhibiting autophagy formation using 3-methyladenine (3-MA). Pretreatment of veins with 3-MA prior to grafting reduced the pathological increases of echogenicity and neointima formation in rats. Therefore, this platform provides non-invasive temporal spatial measurement and prediction of restenosis after venous-arterial transition as well as monitoring the progression of the treatments.

Bioresorbable electronic stimulators are of rapidly growing interest as unusual therapeutic platforms, i.e., bioelectronic medicines, for treating disease states, accelerating wound healing processes and eliminating infections. Here, we present advanced materials that support operation in these systems over clinically relevant timeframes, ultimately bioresorbing harmlessly to benign products without residues, to eliminate the need for surgical extraction. Our findings overcome key challenges of bioresorbable electronic devices by realizing lifetimes that match clinical needs. The devices exploit a bioresorbable dynamic covalent polymer that facilitates tight bonding to itself and other surfaces, as a soft, elastic substrate and encapsulation coating for wireless electronic components. We describe the underlying features and chemical design considerations for this polymer, and the biocompatibility of its constituent materials. In devices with optimized, wireless designs, these polymers enable stable, long-lived operation as distal stimulators in a rat model of peripheral nerve injuries, thereby demonstrating the potential of programmable long-term electrical stimulation for maintaining muscle receptivity and enhancing functional recovery.