In situ bone regeneration with sequential delivery of aptamer and BMP2 from an ECM-based scaffold fabricated by cryogenic free-form extrusion

In situ tissue engineering is a powerful strategy for the treatment of bone defects.It could overcome the limitations of traditional bone tissue engineering,which typically involves extensive cell expansion steps,low cell survival rates upon transplantation,and a risk of immuno-rejection.Here,a porous scaffold polycaprolactone(PCL)/decellularized small intestine submucosa(SIS)was fabricated via cryogenic free-form extrusion,followed by surface modification with aptamer and PlGF-2_(123-144)*-fused BMP2(pBMP2).The two bioactive molecules were delivered sequentially.The aptamer Apt19s,which exhibited binding affinity to bone marrow-derived mesenchymal stem cells(BMSCs),was quickly released,facilitating the mobilization and recruitment of host BMSCs.BMP2 fused with a PlGF-2_(123-144)peptide,which showed“super-affinity”to the ECM matrix,was released in a slow and sustained manner,inducing BMSC osteogenic differentiation.In vitro results showed that the sequential release of PCL/SIS-pBMP2-Apt19s promoted cell migration,proliferation,alkaline phosphatase activity,and mRNA expression of osteogenesis-related genes.The in vivo results demonstrated that the sequential release system of PCL/SIS-pBMP2-Apt19s evidently increased bone formation in rat calvarial critical-sized defects compared to the sequential release system of PCL/SIS-BMP2-Apt19s.Thus,the novel delivery system shows potential as an ideal alternative for achieving cell-free scaffold-based bone regeneration in situ.

Alkaline activation of endogenous latent TGFβ1 by an injectable hydrogel directs cell homing for in situ complex tissue regeneration

Utilization of the body’s regenerative potential for tissue repair is known as in situ tissue regeneration.However,the use of exogenous growth factors requires delicate control of the dose and delivery strategies and may be accompanied by safety,efficacy and cost concerns.In this study,we developed,for the first time,a biomaterial-based strategy to activate endogenous transforming growth factor beta 1(TGFβ1)under alkaline conditions for effective in situ tissue regeneration.We demonstrated that alkaline-activated TGFβ1 from blood serum,bone marrow fluids and soaking solutions of meniscus and tooth dentin was capable of increasing cell recruitment and early differentiation,implying its broad practicability.Furthermore,we engineered an injectable hydrogel(MS-Gel)consisting of gelatin microspheres for loading strong alkaline substances and a modified gelatin matrix for hydrogel click crosslinking.In vitro models showed that alkaline MS-Gel controllably and sustainably activated endogenous TGFβ1 from tooth dentin for robust bone marrow stem cell migration.More importantly,infusion of in vivo porcine prepared root canals with alkaline MS-Gel promoted significant pulp-dentin regeneration with neurovascular stroma and mineralized tissue by endogenous proliferative cells.Therefore,this work offers a new bench-to-beside translation strategy using biomaterial-activated endogenous biomolecules to achieve in situ tissue regeneration without the need for cell or protein delivery.

Effect of electroacupuncture on the mRNA and protein expression of Rho-A and Rho-associated kinase Ⅱ in spinal cord injury rats

Electroacupuncture is beneficial for the recovery of spinal cord injury, but the underlying mechanism is unclear. The Rho/Rho-associated kinase（ROCK） signaling pathway regulates the actin cytoskeleton by controlling the adhesive and migratory behaviors of cells that could inhibit neurite regrowth after neural injury and consequently hinder the recovery from spinal cord injury. Therefore, we hypothesized electroacupuncture could affect the Rho/ROCK signaling pathway to promote the recovery of spinal cord injury. In our experiments, the spinal cord injury in adult Sprague-Dawley rats was caused by an impact device. Those rats were subjected to electroacupuncture at Yaoyangguan（GV3）, Dazhui（GV14）, Zusanli（ST36） and Ciliao（BL32） and/or monosialoganglioside treatment. Behavioral scores revealed that the hindlimb motor functions improved with those treatments. Real-time quantitative polymerase chain reaction, fluorescence in situ hybridization and western blot assay showed that electroacupuncture suppressed the m RNA and protein expression of Rho-A and Rho-associated kinase Ⅱ（ROCKⅡ） of injured spinal cord. Although monosialoganglioside promoted the recovery of hindlimb motor function, monosialoganglioside did not affect the expression of Rho-A and ROCKⅡ. However, electroacupuncture combined with monosialoganglioside did not further improve the motor function or suppress the expression of Rho-A and ROCKⅡ. Our data suggested that the electroacupuncture could specifically inhibit the activation of the Rho/ROCK signaling pathway thus partially contributing to the repair of injured spinal cord. Monosialoganglioside could promote the motor function but did not suppress expression of Rho A and ROCKⅡ. There was no synergistic effect of electroacupuncture combined with monosialoganglioside.

Loss of micro RNA-124 expression in neurons in the peri-lesion area in mice with spinal cord injury

Micro RNA-124（mi R-124） is abundantly expressed in neurons in the mammalian central nervous system, and plays critical roles in the regulation of gene expression during embryonic neurogenesis and postnatal neural differentiation. However, the expression profile of mi R-124 after spinal cord injury and the underlying regulatory mechanisms are not well understood. In the present study, we examined the expression of mi R-124 in mouse brain and spinal cord after spinal cord injury using in situ hybridization. Furthermore, the expression of mi R-124 was examined with quantitative RT-PCR at 1, 3 and 7 days after spinal cord injury. The mi R-124 expression in neurons at the site of injury was evaluated by in situ hybridization combined with Neu N immunohistochemical staining. The mi R-124 was mainly expressed in neurons throughout the brain and spinal cord. The expression of mi R-124 in neurons significantly decreased within 7 days after spinal cord injury. Some of the neurons in the peri-lesion area were Neu N＋/mi R-124-. Moreover, the neurons distal to the peri-lesion site were Neu N＋/mi R-124＋. These findings indicate that mi R-124 expression in neurons is reduced after spinal cord injury, and may reflect the severity of spinal cord injury.

A novel therapeutic target for peripheral nerve injury-related diseases: aminoacyl-tRNA synthetases

Aminoacyl-t RNA synthetases（Amino ARSs） are essential enzymes that perform the first step of protein synthesis. Beyond their original roles, Amino ARSs possess non-canonical functions, such as cell cycle regulation and signal transduction. Therefore, Amino ARSs represent a powerful pharmaceutical target if their non-canonical functions can be controlled. Using Amino ARSs-specific primers, we screened m RNA expression in the spinal cord dorsal horn of rats with peripheral nerve injury created by sciatic nerve axotomy. Of 20 Amino ARSs, we found that phenylalanyl-t RNA synthetase beta chain（FARSB）, isoleucyl-t RNA synthetase（IARS） and methionyl-t RNA synthetase（MARS） m RNA expression was increased in spinal dorsal horn neurons on the injured side, but not in glial cells. These findings suggest the possibility that FARSB, IARS and MARS, as a neurotransmitter, may transfer abnormal sensory signals after peripheral nerve damage and become a new target for drug treatment.