Injectable new design Schwann Cell Loss Hydrogel material for preventing transplant treatment of spinal cord injury

Spinal cord injury (SCI) is a serious disease affecting the body function, there is no current clinical treatments based on the damaged spinal cord regeneration. Spinal cord injury patients and their families to bring huge economic, physical and emotional burden. Cell-based therapies have become the method to encourage regeneration and functional recovery of spinal cord injury promising. Patients Currently, the US Food and Drug Administration for breast and cervical level spinal cord injury are studying autologous human Schwann cells (SCs) transplantation (Figure 1). Human Schwann cells are found in the peripheral nervous system glial cells, which promote axonal regeneration following peripheral nerve injury. In the past two decades, several preclinical studies indicate that the lesion cavity in the human Schwann cells delivered directly to the site of injury formed may promote regeneration after spinal cord injury. Compared to the potential of pluripotent stem cells regenerative therapy of spinal cord injury, the Schwann cells with high purity, well-characterized, and is relatively easily separated from the patient and amplified sural nerve. Unfortunately, the SCs local injection directly into spinal cord injury can cause a lot of transplanted cell loss and death. During the injection, typically up to 60% of the cells can not even reach the target site. This may be due to the result of many factors, including reflux during injection membrane damage and spinal cord cells. In addition, studies have shown that 10 minutes after injection of the cells transplanted cell death will occur quickly, leading to poor SC survival. Typically, only about 20% of the cells survived after one week, but not to 5% graft survival SC 1 month after transplantation, but due to the limited volume of transplanted spinal cord injury lesions, only increase the number of injected cells is not feasible of.

新型设计可注射高分子水凝胶材料用于防止脊髓损伤治疗过程中移植的施旺细胞流失
Figure 1. A schematic diagram of cells to treat spinal cord injury

In response to these cells for the treatment of spinal cord injury problems encountered, School of Materials Science and Engineering at Stanford University [123 ] Professor Sarah C. Heilshorn research group at Science Advance published an article proposes the use of the injectable hydrogel polymer material strategies to solve SC hinder spinal cord injury during transplantation three key challenges of survival. In this study, the authors use the use of injectable hydrogel polymer material solutions during spinal cord injury hampered SC transplant survival 新型设计可注射高分子水凝胶材料用于防止脊髓损伤治疗过程中移植的施旺细胞流失 threeThe key challenge (FIG. 2). In the process of cell delivery, cell membrane damage by cell loss during tensile force (i) injection of cells and induced leakage reflux and (ii) the injection site due to the loss of cells. Shortly after injection of the lack of (iii) the diseased tissue extracellular matrix (the ECM), further reducing the viability of injected cells, resulting in apoptosis is dependent on the growth of adherent SCs. Therefore, if the transplanted cells can interact with endogenous surrounding tissue and promote long-term repair, cell therapy must first overcome the three key challenges.

新型设计可注射高分子水凝胶材料用于防止脊髓损伤治疗过程中移植的施旺细胞流失 FIG. 2. SC transplantation challenge
The system described herein illustrates the hydrogel material designed three different characteristics, and to propose new strategies to solve the transplanted cell survival three key challenges: (i) shear rheology to protect cell membranes from damage during the injection process; (ii) in situ to repair itself quickly and to stabilize the hardness lift injected into the cells in the spinal cord injury lesions, and (iii ) and added to promote adhesion SC promote cell adhesion ligands.

Specific steps:

1, injectable hydrogel design The first phase occurs hydrogel recombinant protein (C7) and a proline-rich peptide bound multi-arm polyethylene glycol (PEG) – physical crosslinking (FIG. 3, B and C) of poly (N- isopropyl acrylamide) (of PNIPAM) is formed between the copolymer. Two components by two peptide domains (CC43 WW domain and proline-rich peptide) reversibly binding heterodimeric ectopic assembled to form a weak gel with SC encapsulated. When the gel subjected to a force, a peptide – peptide bond cleavage, shearing the material in liquid form to flow and thin. After the elimination of the force, peptide – peptide bond rapid restructuring, the rapid self-healing gel. The second stage crosslinked hydrogel occurs in situ after injection, to stabilize and enhance the gel firmness and in order to prevent loss of cells from the lesion extruded site. PNIPAM at body temperature above the lower critical solution temperature 32 ° C, the polymer will intramolecular crosslinking hydrogen bonds, thereby providing the secondary physically crosslinked hydrogels to enhance and increase the network (FIG. 3B). Since it is not clear whether the lesions in spinal cord injury feasible over the gel firmness retention ported SC, and thus the thermally responsive article prepared having a range of poly PNIPAMMaterial composition, were obtained soft gel, a gel medium hardness and high hardness gel (Figure 3D). These materials cover the approximate range of tissue stiffness in injured spinal cord lesions it reported.

新型设计可注射高分子水凝胶材料用于防止脊髓损伤治疗过程中移植的施旺细胞流失 Figure 2. Challenge SC transplantation

2, an injectable hydrogel membrane provides protection for SC injection. When subjected to tensile force during the injection needle, can be damaged cell membranes (FIG. 2A). Suppose even when the injection flow rate is very slow (500 nl / min) also occurs such a case, because the size of the linear velocity variation was determined by the geometry of the syringe needle device. When the cells from the syringe barrel [inner diameter (ID) = 0.485 mm] needle into the ultra-small (No. 33, ID = 0.11 mm), the linear velocity of the fluid 20-fold increase. Transplantation in the same injection parameters (materials and methods) In order to assess the ability of the body against cell membrane damage designs SC hydrogel materials protect the transplant process, used herein. When the cell delivery in physiological saline or a viscous polymer solution C7 RGD (5 wt%) observed obviously damage the membrane (25% and 20%, respectively). The C7 RGD polymer and PEG-P-PNIPAM polymers (both final concentration of 5 wt% mixed) may produce shear thinning rapid self-healing hydrogel (FIG. 4C), changes in cell adhesion gel formulation PNIPAM or amount of the ligand does not change the shear thinning behavior of the gel (FIG. 4C). These pre-packaged in SC These hydrogels variants, can provide significant protection of cell membrane (FIG. 4, A and D) during flow injection needle. Cells as compared to saline delivery, soft, medium hard hydrogel cells with higher levels of protection. Whether there RGD cell adhesion domain material does not affect cell membrane protection and the individual was observed not protect C7 RGD polymer. These data indicate that the shear thinning gel / self-healing properties (adhesive properties of the gel rather than a cell) is the cause of an injectable hydrogel capable of providing protection to the cell membrane.

新型设计可注射高分子水凝胶材料用于防止脊髓损伤治疗过程中移植的施旺细胞流失 FIG. 4. injectable hydrogel injection syringe needle is prevented during cell membrane damage.

3, injectable hydrogel increasesSC diffusion and retention of morphology transplantation. we selected unilateral neck contusion model of spinal cord injury in female Fischer 344 rats, to represent patients with the most common spinal cord injury. Performing backside laminectomy was 75-kdyne contusion right side (FIG. 5A) in the fifth cervical vertebra (C5) level. 48 hours after transplantation (N = 10) and 4 weeks (N = 16) Evaluation retention SC transplantation. Consistent with the in vitro transplantation model data, when delivered in the hydrogel, the lesion was observed compared to saline P75 + cell number was significantly increased (FIG. 5D). At 48 hours, animals were observed at the hydrogel injected into 32,000 ± 7700 cells surviving transplanted animals received saline injections found 4300 ± 2500 survival of transplanted cells. At four weeks, may be observed injectable hydrogel delivered 29,000 ± 8000 viable cells, saline and counted delivered animals 2400 ± 800 viable cells, compared to saline, local delivery of viable cells increased about 10-fold . In the spinal cord cross section of two points in time it was observed compared to saline delivered SC to deliver more (FIG. 5E). In vitro binding data herein, these observations suggest that the use of shear-thinning hydrogel may be self-healing by providing a cell membrane protection during the injection process and limiting the exudation cells from the injection site to significantly improve SC injury to the spinal cord lesion deliver.

新型设计可注射高分子水凝胶材料用于防止脊髓损伤治疗过程中移植的施旺细胞流失 FIG. 5, the injectable hydrogel deliver improved cervical spinal cord injury in a rat model of short-term and long-term retention SC

4, hydrogel-mediated increased intracellular delivery functional recovery As used herein evaluated the effect of a combination of injectable hydrogel and cellular therapy forelimb on functional recovery. Motion and sensory-motor test can be a more correct view of the right forelimb function, since contusion of the spinal cord injury can cause motor and sensory defects. In order to assess forelimb movement recovery, we measured the forelimb grip strength assessment, for example, a combination of the right arm and left arm strength to compensate for the assessment of whether the impact of the behavior of the right arm. The results showed that while unilateral C5 would cause contusions combined and right forelimb grip strength decreased, and only the right forearm test This decline significantly greater (FIG. 6, A and B). Merging the right forelimb and evaluation forelimb, with only injury was observed in the control group compared to hydrogel injectable aqueousAnimals treated with SC grip significantly increased (FIG. 6, A and B). Further, the injectable hydrogel animals received treatment, the right forelimb grip strength was significantly higher than that of physiological saline SC of SC (FIG. 6, A and B), but in the animals treated with saline SCs significant improvement was not observed . This article also go through tests to assess the level of animal-step ladder sensorimotor recovery. The method of calculating the number of steps by an animal unevenly spaced across the cross ladder forearm miss measured forelimb coordination observed after cervical spinal cord contusion injury in step missing significantly increased (FIG. 6C).

新型设计可注射高分子水凝胶材料用于防止脊髓损伤治疗过程中移植的施旺细胞流失 Figure 6. injectable hydrogel injected by SC transplantation after spinal cord injury can significantly restore animal forelimb.

Summary and Outlook: This paper systematically explores new design injectable hydrogel compared to the saline delivery of clinical criteria, so that the injected cells have the ability to retain the lesion improved significantly. The characterization results indicate that a large amount, by using a modular design hydrogel system, such as a hydrogel composition of the cell adhesion polypeptide thereby further optimized by exploring the complete system to further improve cell viability. In addition, in this study we reported using cell plus hydrogel combination therapy for spinal regeneration and functional recovery. The next opportunity to continue the experiment injectable hydrogel optimized highlights the potential applications of new injectable hydrogel, not only for the treatment of spinal cord injury, but also for many other clinical indications have similar limitations. Finally, although the study focused on the use of hydrogels for SC injection designed transplant, but this study has broader implications for the use of other cell types to treat spinal cord injuries. References Marquardt LM, Doulames VM, Wang AT, et al Designer, injectable gels to prevent transplanted Schwann cell loss during spinal cord injury therapy [J] Science Advances, 2020, 6 (14):. Eaaz1039 [123. ] The full text link: https: //advances.sciencemag.org/content/6/14/eaaz1039

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