Sufang Han

The linear-ordered collagen scaffold-BDNF complex significantly promotes functional recovery after completely transected spinal cord injury in canine.

Spinal cord injury (SCI) is still a worldwide clinical challenge for which there is no viable therapeutic method. We focused on developing combinatorial methods targeting the complex pathological process of SCI. In this study, we implanted linear-ordered collagen scaffold (LOCS) fibers with collagen binding brain-derived neurotrophic factor (BDNF) by tagging a collagen-binding domain (CBD) (LOCS + CBD-BDNF) in completely transected canine SCI with multisystem rehabilitation to validate its potential therapeutic effect through a long-term (38 weeks) observation. We found that LOCS + CBD-BDNF implants strikingly promoted locomotion and functional sensory recovery, with some dogs standing unassisted and transiently moving. Further histological analysis showed that administration of LOCS + CBD-BDNF reduced lesion volume, decreased collagen deposits, promoted axon regeneration and improved myelination, leading to functional recovery. Collectively, LOCS + CBD-BDNF showed striking therapeutic effect on completely transected canine SCI model and it is the first time to report such breakthrough in the war with SCI. Undoubtedly, it is a potentially promising therapeutic method for SCI paralysis or other movement disorders caused by neurological diseases in the future.

The three-dimensional collagen scaffold improves the stemness of rat bone marrow mesenchymal stem cells.

Mesenchymal stem cells (MSCs) show the great promise for the treatment of a variety of diseases because of their self-renewal and multipotential abilities. MSCs are generally cultured on two-dimensional (2D) substrate in vitro. There are indications that they may simultaneously lose their stemness and multipotentiality as the result of prolonged 2D culture. In this study, we used three-dimensional (3D) collagen scaffolds as rat MSCs carrier and compared the properties of MSCs on 3D collagen scaffolds with monolayer cultured MSCs. The results demonstrated that collagen scaffolds were suitable for rat MSCs adherence and proliferation. More importantly, compared to MSCs under 2D culture, 3D MSCs significantly maintained higher expression levels of stemness genes (Oct4, Sox2, Rex-1 and Nanog), yielded high frequencies of colony-forming units-fibroblastic (CFU-F) and showed enhanced osteogenic and adipogenic differentiation efficiency upon induction. Thus, 3D collagen scaffolds may be beneficial for expanding rat MSCs while maintaining the stem cell properties in vitro.

Functionalized Collagen Scaffold Neutralizing the Myelin-Inhibitory Molecules Promoted Neurites Outgrowth in Vitro and Facilitated Spinal Cord Regeneration in Vivo.

Research has demonstrated that many myelin-associated inhibitory molecules jointly contribute to the failure of adult spinal cord regeneration. Therapies comprehensively targeting the multiple inhibitory nature of the injured spinal cord are being concerned. Here, two collagen-binding proteins, CBD-EphA4LBD and CBD-PlexinB1LBD, were constructed, respectively, to neutralize the axon guidance molecules ephrinB3 and sema4D that inhibit the regeneration of nerve fibers. The two neutralizing proteins have proven their ability to specifically bind collagen and to continuously release from collagen scaffolds. They could also promote neurites outgrowth of cerebellar granular neurons and dorsal root ganglion neurons in vitro. Subsequently, the functionalized collagen scaffolds by physically absorbing NEP1-40 and immobilizing CBD-EphA4LBD and CBD-PlexinB1LBD were transplanted into a rat T10 complete spinal cord transection model. Our results showed that rats that received the treatment of transplanting the functionalized collagen scaffold exhibited great advantage on axonal regeneration and locomotion recovery after spinal cord injury.

Functionalized collagen scaffold implantation and cAMP administration collectively facilitate spinal cord regeneration

UNLABELLED Previous studies have demonstrated that several mechanisms, including numerous inhibitory molecules, weak neurotrophic stimulation and deficient intrinsic regenerative responses, collectively contribute to the failure of mature spinal cord axon regeneration. Thus, combinatorial therapies targeting multiple mechanisms have attracted much attention. In the present study, a porous collagen scaffold was used to support neuronal attachment and bridge axonal regeneration. The scaffold was specifically functionalized using neutralizing proteins (CBD-EphA4LBD, CBD-PlexinB1LBD and NEP1-40) and collagen-binding neurotrophic factors (CBD-BDNF and CBD-NT3) to simultaneously antagonize myelin inhibitory molecules (ephrinB3, Sema4D and Nogo) and exert neurotrophic protection and stimulation. Cerebellar granular neurons cultured on the functionalized collagen scaffold promoted neurite outgrowth in the presence of myelin. Furthermore, a full combinatorial treatment comprising functionalized scaffold implantation and cAMP administration was developed to evaluate the synergistic repair ability in a rat T10 complete removal spinal cord injury model. The results showed that full combinatorial therapy exhibited the greatest advantage in reducing the volume of cavitation, facilitating axonal regeneration, and promoting neuronal generation. The newborn neurons generated in the lesion area could form the neuronal relay and enhance the locomotion recovery after severe spinal cord injury. STATEMENT OF SIGNIFICANCE A porous collagen scaffold was specifically functionalized with neutralizing proteins and neurotrophic factors to antagonize the myelin inhibitory molecules and exert neurotrophic protection and stimulation for spinal cord regeneration. Cerebellar granular neurons seeded on the functionalized collagen scaffold showed enhanced neurite outgrowth ability in vitro. The functionalized scaffold implantation combined with cAMP administration exhibited synergistic repair ability for rat T10 complete spinal cord transection injury.

One-year clinical study of NeuroRegen scaffold implantation following scar resection in complete chronic spinal cord injury patients

The objective of this clinical study was to assess the safety and feasibility of the collagen scaffold, NeuroRegen scaffold, one year after scar tissue resection and implantation. Scar tissue is a physical and chemical barrier that prevents neural regeneration. However, identification of scar tissue is still a major challenge. In this study, the nerve electrophysiology method was used to distinguish scar tissue from normal neural tissue, and then different lengths of scars ranging from 0.5–4.5 cm were surgically resected in five complete chronic spinal cord injury (SCI) patients. The NeuroRegen scaffold along with autologous bone marrow mononuclear cells (BMMCs), which have been proven to promote neural regeneration and SCI recovery in animal models, were transplanted into the gap in the spinal cord following scar tissue resection. No obvious adverse effects related to scar resection or NeuroRegen scaffold transplantation were observed immediately after surgery or at the 12-month follow-up. In addition, patients showed partially autonomic nervous function improvement, and the recovery of somatosensory evoked potentials (SSEP) from the lower limbs was also detected. The results indicate that scar resection and NeuroRegen scaffold transplantation could be a promising clinical approach to treating SCI.

Maintenance of the self-renewal properties of neural progenitor cells cultured in three-dimensional collagen scaffolds by the REDD1-mTOR signal pathway

Three-dimensional (3-D) culture, compared with traditional two-dimensional (2-D) cell culture, can provide physical signals and 3-D matrix close to the in vivo microenvironments. Here, sponge-like collagen scaffolds were used to assess how 3-D culture would affect the differentiation and self-renewal of neural progenitor cells (NPCs). Cultured in differentiation medium without growth factors, cells in 3-D collagen scaffolds yielded much higher clone formation efficiency and expressed less neuron marker, TUJ1, compared with cells cultured on 2-D plates. mTOR inactivation was identified and showed to supported the self-renewal of NPCs in 3-D culture. At the same time, REDD1 was highly expressed in cells cultured in 3-D conditions, which blocks the activity of mTOR. Moreover, knocking-down REDD1 induced the differentiation of NPCs in 3-D collagen scaffolds. These results indicated that mTOR inactivation by REDD1 mediated the self-renewal regulation of NPCs in 3-D cultures. Thus, 3-D collagen scaffolds maintained self-renewal properties of NPCs, and the inhibitory regulator of mTOR (such as REDD1) played an important role in the regulation of self-renewal and differentiation of NPCs.

Cetuximab modified collagen scaffold directs neurogenesis of injury-activated endogenous neural stem cells for acute spinal cord injury repair

Studies have shown that endogenous neural stem cells (NSCs) activated by spinal cord injury (SCI) primarily generate astrocytes to form glial scar. The NSCs do not differentiate into neurons because of the adverse microenvironment. In this study, we defined the activation timeline of endogenous NSCs in rats with severe SCI. These injury-activated NSCs then migrated into the lesion site. Cetuximab, an EGFR signaling antagonist, significantly increased neurogenesis in the lesion site. Meanwhile, implanting cetuximab modified linear ordered collagen scaffolds (LOCS) into SCI lesion sites in dogs resulted in neuronal regeneration, including neuronal differentiation, maturation, myelination, and synapse formation. The neuronal regeneration eventually led to a significant locomotion recovery. Furthermore, LOCS implantation could also greatly decrease chondroitin sulfate proteoglycan (CSPG) deposition at the lesion site. These findings suggest that endogenous neurogenesis following acute complete SCI is achievable in species ranging from rodents to large animals via functional scaffold implantation. LOCS-based Cetuximab delivery system has a promising therapeutic effect on activating endogenous neurogenesis, reducing CSPGs deposition and improving motor function recovery.

A modified collagen scaffold facilitates endogenous neurogenesis for acute spinal cord injury repair

Due to irreversible neuronal loss and glial scar deposition, spinal cord injury (SCI) ultimately results in permanent neurological dysfunction. Neuronal regeneration of neural stem cells (NSCs) residing in the spinal cord could be an ideal strategy for replenishing the lost neurons and restore function. However, many myelin-associated inhibitors in the SCI microenvironment limit the ability of spinal cord NSCs to regenerate into neurons. Here, a linearly ordered collagen scaffold was used to prevent scar deposition, guide nerve regeneration and carry drugs to neutralize the inhibitory molecules. A collagen-binding EGFR antibody Fab fragment, CBD-Fab, was constructed to neutralize the myelin inhibitory molecules, which was demonstrated to promote neuronal differentiation and neurite outgrowth under myelin in vitro. This fragment could also specifically bind to the collagen and undergo sustained release from collagen scaffold. Then, the scaffolds modified with CBD-Fab were transplanted into an acute rat SCI model. The robust neurogenesis of endogenous injury-activated NSCs was observed, and these NSCs could not only differentiate into neurons but further mature into functional neurons to reconnect the injured gap. The results indicated that the modified collagen scaffold could be an ideal candidate for spinal cord regeneration after acute SCI. STATEMENTS OF SIGNIFICANCE A linearly ordered collagen scaffold was specifically modified with collagen-binding EGFR antibody, allowed for sustained release of this EGFR neutralizing factor, to block the myelin associated inhibitory molecules and guide spinal cord regeneration along its linear fibers. Dorsal root ganglion neurons and neural stem cells induced by CBD-Fab exhibited enhanced neurite outgrowth and neuronal differentiation rate under myelin in vitro. Transplantation of the modified collagen scaffold with moderate EGFR neutralizing proteins showed greatest advantage on endogenous neurogenesis of injury-activated neural stem cells for acute spinal cord injury repair.

The miR-20-Rest-Wnt signaling axis regulates neural progenitor cell differentiation

Increasing evidence suggests that three dimensional (3-D) cell cultures are an improvement over traditional two dimensional (2-D) cell cultures. Current researches have extensively focused on the study of utilizing biomaterial-based 3-D culture systems to study and direct stem-cell fate both in vitro and in vivo. Here in our study, we screened the differential expression patterns of miRNAs between 2-D cultured and 3-D cultured NPCs using microarray analysis. Among these differentially expressed miRNAs, miR-20 was found to increase during differentiation of NPCs. Specifically, the facilitative effect on neural differentiation of miR-20 is mediated, at least in part by directly target the Rest gene, which is essential for preventing neural differentiation and maintaining NPCs self-renewal. Furthermore, the expression of miR-20 was decreased when the WNT pathway was inhibited by knock down of β-catenin or by exogenous Dkk protein, whereas it increased when the WNT pathway was activated by exogenous Wnt3a protein. Overall, miR-20, Rest and Wnt signaling are suggested to be involved in a regulatory circuit that can modulate the neural differention of NPCs. This novel regulatory circuit provides additional insight into how microRNAs interact with signaling molecules during neural differentiation of NPCs, allowing for fine-tuning of intricate cellular processes.

Transplantation of hUC-MSCs seeded collagen scaffolds reduces scar formation and promotes functional recovery in canines with chronic spinal cord injury.

Spinal cord injury (SCI) can lead to locomotor deficits, and the repair of chronic SCI is considered one of the most challenging clinical problems. Although extensive studies have evaluated treatments for acute SCI in small animals, comparatively fewer studies have been conducted on large-animal SCI in the chronic phase, which is more clinically relevant. Here, we used a collagen-based biomaterial, named the NeuroRegen scaffold, loaded with human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) in a canine chronic SCI model. To generate chronic SCI, the T8 spinal cord segment was removed by complete transection of the spinal cord. Two months later, glial scar tissue was removed and a NeuroRegen scaffold was transplanted into the lesion area. Functionalized NeuroRegen scaffold implantation promoted both locomotor recovery and endogenous neurogenesis in the lesion area. Moreover, some newly generated neurons successfully matured into 5-HT-positive neurons at 1 year post-injury. In addition, many regenerated axon fibers in the lesion area exhibited remyelination and synapse formation at 1 year post-injury in the functionalized NeuroRegen scaffold group. In conclusion, the NeuroRegen scaffold functionalized with hUC-MSCs is a promising potential therapeutic approach to chronic SCI that promotes neuronal regeneration, reduces glial scar formation, and ultimately improves locomotor recovery.