Dedifferentiation-like reprogramming of degenerative nucleus pulposus cells into notochordal-like cells by defined factors.

The extensive degeneration of functional somatic cells and the depletion of endogenous stem/progenitor populations present significant challenges to tissue regeneration in degenerative diseases. Currently, a cellular reprogramming approach enabling directly generating corresponding progenitor populations from degenerative somatic cells remains elusive. The present study focused on intervertebral disc degeneration (IVDD) and identified a three-factor combination (OCT4, FOXA2, TBXT (OFT)) that could induce the dedifferentiation-like reprogramming of degenerative nucleus pulposus cells (dNPCs) toward induced notochordal-like cells (iNCs). Single-cell transcriptomics dissected the transitions of cell identity during reprogramming. Further, OCT4 was found to directly interact with bromodomain PHD-finger transcription factor (BPTF) to remodel the chromatin during the early phases, which was crucial for initiating this dedifferentiation-like reprogramming. In rat models, intradiscal injection of adeno-associated virus carrying OFT generated iNCs from in situ dNPCs and reversed IVDD. These results collectively present a proof-of-concept for dedifferentiation-like reprogramming of degenerated somatic cells into corresponding progenitors through the development of a factor-based strategy, providing a promising approach for regeneration in degenerative disc diseases.

CircEML1 facilitates the steroid synthesis in follicular granulosa cells of chicken through sponging gga-miR-449a to release IGF2BP3 expression.

Non-coding RNAs (ncRNAs) induced competing endogenous RNAs (ceRNA) play crucial roles in various biological process by regulating target gene expression. However, the studies of ceRNA networks in the regulation of ovarian ovulation processing of chicken remains deficient compared to that in mammals. Our present study revealed that circEML1 was differential expressed in hen's ovarian tissues at different ages (15 W/20 W/30 W/68 W) and identified as a loop structure from EML1 pre-mRNA, which promoted the expressions of CYP19A1/StAR and E2/P4 secretion in follicular granulosa cells (GCs). Furthermore, circEML1 could serve as a sponge of gga-miR-449a and also found that IGF2BP3 was targeted by gga-miR-449a to co-participate in the steroidogenesis, which possibly act the regulatory role via mTOR/p38MAPK pathways. Meanwhile, in the rescue experiment, gga-miR-449a could reverse the promoting role of circEML1 to IGF2BP3 and steroidogenesis. Eventually, this study suggested that circEML1/gga-miR-449a/IGF2BP3 axis exerted an important role in the steroidogenesis in GCs of chicken.

Scar Tissue-Targeting Polymer Micelle for Spinal Cord Injury Treatment.

Spinal cord injury (SCI) is a devastating disorder, leading to permanent motor and sensory deficit. Despite recent advances in neurosciences, the treatment efficacy on SCI patients remains unsatisfactory, mainly due to the poor accumulation, short retention, and lack of controlled release of therapeutics in lesion tissue. Herein, an injured spinal cord targeting prodrug polymer micelle is built. An esterase-responsive bond is used to link apocynin (APO) monomer, because of the enhanced esterase activity found in microglia cells after activation, which ensures a controlled degradation of APO prodrug (Allyloxypolyethyleneglycol-b-poly [2-(((4-acetyl-2-methoxyphenoxy)carbonyl)oxy)ethyl methacrylate], APEG-PAPO or PAPO) by activated microglia cells. A scar tissue-homing peptide (cysteine-alanine-glutamine-lysine, CAQK) is introduced to the PAPO to endow the polymer micelle the lesion tissue-targeting ability. As a result, this CAQK-modified prodrug micelle (cPAM) exhibits an improved accumulation and prolonged retention in lesion tissue compared to the control micelle. The cPAM also leads to superior tissue protection and sustained motor function recovery than the control groups in a mouse model of SCI. In conclusion, the cPAM induces an effective treatment of SCI by the lesion tissue specific delivery of the prodrug polymer via its robust scar binding effect, making the scar tissue a drug releasing platform for sustained treatment of SCI.

FoxA2 regulates the type II collagen-induced nucleus pulposus-like differentiation of adipose-derived stem cells by activation of the Shh signaling pathway.

Adipose tissue-derived stem cell (ADSC)-based therapy is promising for the treatment of intervertebral disc (IVD) degeneration, but the difficulty in inducing nucleus pulposus (NP)-like differentiation limits its clinical applications. Forkhead box (Fox)-A2 is an essential transcription factor for the formation of a normal NP. We demonstrated that type II collagen stimulates NP-like differentiation of ADSCs, partly by increasing the expression of FoxA2. We constructed FoxA2-overexpressing and -knockdown ADSCs by using lentiviral vectors. FoxA2 overexpression significantly enhanced NP-specific gene expression and the synthesis of glycosaminoglycan and collagen, whereas FoxA2 knockdown decreased NP-like differentiation and the expression of aggrecan and collagen II. The enhanced NP-like differentiation related to FoxA2 overexpression was partially rescued by an Shh signaling pathway inhibitor. In addition, FoxA2 inhibited the expression of Itg-α2 and further promoted NP-like differentiation induced by type II collagen. Furthermore, FoxA2-overexpressing ADSCs combined with type II collagen hydrogels promoted regeneration of degenerated NP in vivo. Our findings suggest that FoxA2 plays an essential role in the NP-like differentiation of ADSCs by activating the Shh signaling pathway.-Zhou, X., Ma, C., Hu, B., Tao, Y., Wang, J., Huang, X., Zhao, T., Han, B., Li, H., Liang, C., Chen, Q., Li, F. FoxA2 regulates the type II collagen-induced nucleus pulposus-like differentiation of adipose-derived stem cells by activation of the Shh signaling pathway.

Secreted klotho protein attenuates osteogenic differentiation of human bone marrow mesenchymal stem cells in vitro via inactivation of the FGFR1/ERK signaling pathway.

Increasing evidence indicates that the osteogenic differentiation of mesenchymal stem cells (MSCs) is related to bone formation, heterotopic ossification, and even vascular calcification. Therefore, it is essential to understand the microenvironment that regulates these processes. The Klotho gene plays an important role in tissue mineralization, and its secreted protein functions as a hormone. We investigated the effects of secreted Klotho protein on the osteogenesis of human bone marrow MSC (hBMSCs). To this end, the cells received osteogenic medium with or without Klotho protein. The results showed that osteoblast-specific gene expression and mineral deposition were decreased when MSCs were incubated with Klotho. Klotho reduced the expression of fibroblast growth factor receptor 1 (FGFR1) and phosphorylated extracellular signal-regulated kinase 1/2. However, both MEK and FGFR1 inhibitors delayed bone mineral formation more than Klotho. These data suggest that secreted Klotho protein attenuates the osteogenic differentiation of hBMSCs in vitro through FGFR1/ERK signaling.

Roles of FGF-2 and TGF-beta/FGF-2 on differentiation of human mesenchymal stem cells towards nucleus pulposus-like phenotype.

Human mesenchymal stem cells (MSCs) are reported to have the capability of differentiating towards nucleus pulposus (NP)-like phenotype under specific culture conditions. So far, the effects of fibroblast growth factor (FGF)-2 and the cocktail effects of transforming growth factor (TGF)-beta and FGF-2 on MSCs remain unclear. Therefore, we designed this study to clarify these effects. MSCs were cultured in conditioned medium containing FGF-2 or TGF-beta/FGF-2, and compared with basal or TGF-beta medium. The groups with FGF-2 showed the increase of cell proliferation. Functional gene markers and novel NP markers decreased in FGF-2 group, together with functional protein expression. Pho-ERK1/2 and pho-Smad3 differed significantly in the two conditioned groups. All these results suggest FGF-2 promotes MSCs' proliferation, synergistically with TGF-beta. However, FGF-2 plays a negative role in cartilage homeostasis. We also demonstrate that FGF-2 has no positive effect in differentiating MSCs into NP-like cells, but hinders the acceleration effect of TGF-beta.

TGF-ß3 and IGF-1 synergy ameliorates nucleus pulposus mesenchymal stem cell differentiation towards the nucleus pulposus cell type through MAPK/ERK signaling.

This study aimed to investigate the synergy between transforming growth factor beta 3 (TGF-ß3) and insulin-like growth factor 1 (IGF-1) on nucleus pulposus-derived mesenchymal stem cells (NP-MSCs) and the underlying mechanism using a serum-free culture system. NP-MSC proliferation and viability were measured using a CCK-8 assay and annexin V-FITC/propidium iodide, respectively. NP-MSCs in micromasses were investigated for differentiation towards nucleus pulposus cells (NPCs). SOX-9, collagen-I, collagen-II, aggrecan and decorin expressions were detected by RT-PCR and immunoblotting. Matrix deposition was assessed by sulfated glycosaminoglycan (sGAG) analysis. Novel chondrogenic and nucleus pulposus (NP) genes were detected to distinguish differentiated cell types. MAPK/ERK and TGF/Smad signaling pathways were also examined. As a result, the synergy between TGF-ß3 and IGF-1 enhanced NP-MSC viability, extracellular matrix (ECM) biosynthesis and differentiation towards NPCs, partly through the activation of the MAPK/ERK signaling pathway. Therefore, the synergy between TGF-ß3 and IGF-1 ameliorates NP-MSC viability, differentiation and promotes intervertebral disc regeneration.

BMP3 Alone and Together with TGF-ß Promote the Differentiation of Human Mesenchymal Stem Cells into a Nucleus Pulposus-Like Phenotype.

Human mesenchymal stem cells (MSCs) have the potential to differentiate into nucleus pulposus (NP)-like cells under specific stimulatory conditions. Thus far, the effects of bone morphogenetic protein 3 (BMP3) and the cocktail effects of BMP3 and transforming growth factor (TGF)-ß on MSC proliferation and differentiation remain obscure. Therefore, this study was designed to clarify these unknowns. MSCs were cultured with various gradients of BMP3 and BMP3/TGF-ß, and compared with cultures in basal and TGF-ß media. Cell proliferation, glycosaminoglycan (GAG) content, gene expression, and signaling proteins were measured to assess the effects of BMP3 and BMP3/TGF-ß on MSCs. Cell number and GAG content increased upon the addition of BMP3 in a dose-dependent manner. The expression of COL2A1, ACAN, SOX9, and KRT19 increased following induction with BMP3 and TGF-ß, in contrast to that of COL1A1, ALP, OPN, and COMP. Smad3 phosphorylation was upregulated by BMP3 and TGF-ß, but BMP3 did not affect the phosphorylation of extracellular-signal regulated kinase (ERK) 1/2 or c-Jun N-terminal kinase (JNK). Our results reveal that BMP3 enhances MSC proliferation and differentiation into NP-like cells, as indicated by increased cell numbers and specific gene expressions, and may also cooperate with TGF-ß induced positive effects. These actions are likely related to the activation of TGF-ß signaling pathway.

Nucleus pulposus mesenchymal stem cells in acidic conditions mimicking degenerative intervertebral discs give better performance than adipose tissue-derived mesenchymal stem cells.

The microenvironment of the intervertebral disc (IVD) is characterized by matrix acidity, hypoxia, hyperosmolarity and limited nutrition, which are major obstacles to stem cell-based regeneration. Our recent work showed that nucleus pulposus mesenchymal stem cells (NPMSCs) had advantages over traditional sources of cell therapy under IVD-like hypoxic and hyperosmotic conditions. Here, we examined the viability, proliferation and matrix metabolism of NPMSCs compared with adipose tissue-derived mesenchymal stem cells (ADMSCs) under IVD-like acidic conditions in vitro. ADMSCs and NPMSCs from Sprague-Dawley rats were cultured at four different pH levels representing the standard condition (pH 7.4) and the normal, mildly degenerated and severely degenerated IVD (pH 7.1, 6.8 and 6.5, respectively). Cell viability was examined by annexin-V-fluorescein isothiocyanate/propidium iodide staining. Cell proliferation was measured using a cell counting kit cell proliferation assay. The expression of aggrecan, collagen-I, collagen-II, matrix metalloproteinase-2 (MMP-2), a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS4) and the tissue inhibitor of metalloproteinase-3 (TIMP-3) was measured at mRNA and protein levels by RT-PCR and Western blotting. In both cell types, acidic pH inhibited cell viability and proliferation, downregulated the expression of aggrecan, collagen-I, collagen-II and TIMP-3, and upregulated the expression of MMP-2 and ADAMTS4. Compared with ADMSCs, NPMSCs were significantly less inhibited in viability and proliferation; they expressed significantly higher levels of aggrecan and collagen-II, and lower levels of MMP-2 and ADAMTS4. Thus, an acidic environment is a major obstacle for IVD regeneration by ADMSCs or NPMSCs. NPMSCs appeared less sensitive to inhibition by acidic pH and might be promising candidates for cell-based IVD regeneration.

A novel rat model of annulus fibrosus injury for intervertebral disc degeneration.

BACKGROUND CONTEXT: In clinical practice, acute trauma and chronic degeneration of the annulus fibrosus (AF) can promote further degeneration of the intervertebral disc (IVD). Therefore, it is critical to understand the AF repair process and its consequences on IVD. However, the lack of cost-effective and reproducible in vivo animal models of AF injury has limited research development in this field. PURPOSES: The purpose of this study was to establish and evaluate the utility of a novel animal model for full-thickness AF injury. Three foci were proposed: (1) whether this new modeling method can cause full-layer AF damage; (2) the repair processes and pathological changes in the damaged area after AF injury, and (3) the morphological and histological changes in the IVD are after AF injury. STUDY DESIGN/SETTING: In vivo rat AF injury model with characterization of AF damage repair, IVD degeneration. METHODS: A total of 72,300 g male rats were randomly assigned to one of the two groups: experimental or sham. Annulus fibrosus was separated layer by layer under the microscope with a #11 blade up to the AF- nucleus pulpous (NP) junction. The repair process of the horizontal AF and morphological changes in the sagittal IVD were evaluated with HE staining. Sirius red staining under polarized light. Immunofluorescence was conducted to analyze changes in the expression of COL1 and COL3 in the AF injury area and 8-OHdg, IL-6, MMP13, FSP1, and ACAN in the IVD. The disc height and structural changes after AF injury were measured using X-ray and contrast-enhanced micro-CT. Additionally, the resistance of the AF to stretching was analyzed using three-point bending. RESULTS: Annulus fibrosus-nucleus pulpous border was identified to stably induce the full-thickness AF injury without causing immediate NP injury. The AF repair process after injury was slow and expressed inflammation factors continuously, with abundant amounts of type III collagen appearing in the inner part of the AF. The scar at the AF lesion had decreased resistance to small molecule penetration and weakened tensile strength. Full-thickness AF injury induced disc degeneration with loss of disc height, progressive unilateral vertebral collapse, and ossification of the subchondral bone. Inflammatory-induced degeneration and extracellular matrix catabolism gradually appeared in the NP and cartilage endplate (CEP). CONCLUSIONS: We established a low-cost and reproducible small animal model of AF injury which accurately replicated the pathological state of the limited AF self-repair ability and demonstrated that injury to the AF alone could cause further degeneration of the IVD. CLINICAL RELEVANCE: This in vivo rat model can be used to study the repair process of the AF defect and pathological changes in the gradual degeneration of IVD after AF damage. In addition, the model provides an experimental platform for in vivo experimental research of potential clinical therapeutics.

Influence of hypoxia in the intervertebral disc on the biological behaviors of rat adipose- and nucleus pulposus-derived mesenchymal stem cells.

Adipose-derived mesenchymal stem cells (ADMSCs) and nucleus pulposus-derived mesenchymal stem cells (NPMSCs) are two cell candidates for cell-based therapies for intervertebral disc (IVD) regeneration. However, little work has been done to determine the influence of hypoxia in the IVD on the biological behaviors of ADMSCs and NPMSCs. This study aimed to investigate the viability, proliferation and differentiation of rat ADMSCs and NPMSCs in the hypoxic environment of IVD in vitro. ADMSCs and NPMSCs isolated from 6 SD rats were cultured under normoxia (20% O2) and hypoxia (2% O2) mimicking the standard condition and hypoxic environment of the IVD for 14 days. Cell viability was determined by the annexin-V-FITC/propidium iodide double-staining assay and cell proliferation was measured by MTT assay. The expression of hypoxia-inducible factor-1α, glucose transporter (GLUT)-1, GLUT-3 and vascular endothelial growth factor-A at the mRNA level was examined by RT-PCR. In cells cultured in three-dimensional micromass and differentiation medium, aggrecan, collagen-II and Sox-9 expression at mRNA and protein levels were examined by RT-PCR and Western blot. Hypoxia inhibited the viability and proliferation of both ADMSCs and NPMSCs, but promoted the chondrocytic differentiation of ADMSCs and NPMSCs. Compared to ADMSCs, NPMSCs showed greater viability, proliferation and chondrocytic differentiation under hypoxia. In conclusion, hypoxia in the IVD had a significant impact on the viability, proliferation and chondrocytic differentiation of ADMSCs and NPMSCs. NPMSCs exhibited more potent biological activity than ADMSCs in the hypoxic environment of the IVD and may represent another candidate for cell-based therapy for IVD regeneration.

Potential of co-culture of nucleus pulposus mesenchymal stem cells and nucleus pulposus cells in hyperosmotic microenvironment for intervertebral disc regeneration.

Nucleus pulposus mesenchymal stem cells (NPMSCs) are a potential cell source for intervertebral disc (IVD) regeneration, but little is known about their response to IVD-like high osmolarity (400 mOsm). This study was to investigate the viability, proliferation and protein biosynthesis of nucleus pulposus cells (NPCs), NPMSCs and co-cultured NPMSCs-NPCs under IVD-like high osmolarity conditions. NPCs and NPMSCs were isolated and cultured under standard and IVD-like high osmolarity conditions for 1 or 2 weeks. Cell viability was measured by annexin V-FITC and PI staining, and cell proliferation measured by MTT assay. The expression of SOX-9, aggrecan and collagen-II was measured by RT-PCR and Western blot analyses. IVD-like high osmolarity condition slightly inhibited cell viability and decreased the expression of SOX-9, aggrecan and collagen-II at the mRNA and protein levels in all groups compared with standard condition. NPMSCs could tolerate IVD-like high osmolarity, and NPCs-NPMSCs co-culture increased cell proliferation and the expression of SOX-9, aggrecan and collagen-II under both culture conditions, suggesting that co-culture of NPMSCs-NPCs has potential application for IVD regeneration.

Direct Reprogramming in Bone and Joint Degenerative Diseases: Applications, Obstacles and Directions.

With a booming aging population worldwide, bone and joint degenerative diseases have gradually become a major public health focus, attracting extensive scientific attention. However, the effective treatments of these degenerative diseases have been confined to traditional medications and surgical interventions, which easily lead to the possibility of drug abuse or loss of physiological function to varying degrees. Recently, given that the development of reprogramming has overcome shackles in the field of degenerative diseases, direct reprogramming would provide a new concept to accelerate progress in the therapy of bone and joint degenerative diseases. The process of direct reprogramming would directly induce ordinary somatic cells to the desired targeted cells without passing through pluripotent cell states. In this review, we summarize some direct reprogramming of cells that has been attempted for the repair of common bone and joint degenerative diseases, such as osteoarthritis, osteoporosis-related fracture and intervertebral disc degeneration. However, it is inevitable that some obstacles, such as accurate transcription factors, an appropriate extracellular microenvironment and efficient delivery carriers in vivo, need to be resolved. In addition, developmental and promising directions associated with direct reprogramming have attracted public attention. Investigation of the regulation of the transient genome, metabolic conversion and cellular skeleton would provide superior potential candidates for the revolution of direct reprogramming. The aim of direct reprogramming is to directly provide target cells for cell therapy and even tissue reconstruction in bone and joint degenerative diseases. Moreover, the development of direct reprogramming have potential to achieve repair and even reconstruct in situ, which would be breakthrough effect for the repair of bone and joint degenerative diseases. The advance of direct reprogramming has opened numerous opportunities for new therapeutic strategies in regenerative medicine.

Case Report: Metagenomic Next-Generation Sequencing Confirmed a Case of Spine Infection with Brucella melitensis in Non-Endemic Area.

Brucellosis is a zoonotic disease caused by Brucella spp., with the highest prevalence found in the northern cities of China. In this case report, we present an occurrence of spinal infection caused by B. melitensis in a 67-year-old man residing in a non-endemic area of southern China. The patient initially presented with chest and back pain, which was not accurately diagnosed and treated at a local hospital. Subsequently, due to worsening pain, he was admitted to our hospital. To determine the cause of the infection, we performed CT-guided aspiration biopsy and collected biopsy tissue for metagenomic next-generation sequencing (mNGS) on the second day of hospitalization. Imaging investigations revealed involvement of the thoracic vertebrae, specifically thoracic 4-7 with the main focus on 5-6, accompanied by stenosis of the intervertebral space. The mNGS results indicated that the spine infection was caused by B. melitensis. The patient's history as a shepherd and a positive Rose Bengal plate test (RBPT) further supported the diagnosis of brucella spondylitis. In order to alleviate pain and restore spinal function, the patient underwent posterior internal fixation of the thoracic spine. Treatment was initiated with cefoperazone/sulbactam, followed by doxycycline. Subsequently, the patient was switched to a combination therapy of rifampicin and doxycycline for a duration of six weeks. The patient responded well to treatment, and his condition remained stable. In conclusion, brucellosis is a common disease that can be easily misdiagnosed. This case report highlights the potential value of mNGS in early and rapid diagnosis. We believe that mNGS can serve as an effective tool to improve the diagnosis of spine infections caused by this pathogen.

Nucleus pulposus cell-derived efficient microcarrier for intervertebral disc tissue engineering.

Adipose-derived stem cells (ADSCs) show great potential for the treatment of intervertebral disc (IVD) degeneration. An ideal carrier is necessary to transplant ADSCs into degenerated IVDs without influencing cell function. Nucleus pulposus cells (NPCs) can synthesize and deposit chondroitin sulfate and type II collagen which are NP-specific extracellular matrix (ECM) and can also regulate the NP-specific differentiation of stem cells. Bioscaffolds fabricated based on the ECM synthesis functions of NPCs have possible roles in cell transplantation and differentiation induction, but it has not been studied. In this study, we first aggregated NPCs into pellets, and then, NPC-derived efficient microcarriers (NPCMs) were fabricated by pellet cultivation under specific conditions and optimized decellularization. Thirdly, we evaluated the microstructure, biochemical composition, biostability and cytotoxicity of the NPCMs. Finally, we investigated the NP-specific differentiation of ADSCs induced by the NPCMsin vitroand NP regeneration induced by the ADSC-loaded NPCMs in a rabbit model. The results indicated that the injectable NPCMs retained maximal ECM and minimal cell nucleic acid after optimized decellularization and had good biostability and no cytotoxicity. The NPCMs also promoted the NP-specific differentiation of ADSCsin vitro. In addition, the results of MRI, x-ray, and the structure and ECM content of NP showed that the ADSCs-loaded NPCMs can partly restored the degenerated NPin vivo. Our injectable NPCMs regenerated the degenerated NP and provide a simplified and efficient strategy for treating IVD degeneration.

Adiponectin inhibits GnRH secretion via activating AMPK and PI3K signaling pathways in chicken hypothalamic neuron cells.

It has been reported that adiponectin (AdipoQ), an adipokine secreted by white adipose tissue, plays an important role in the control of animal reproduction in addition to its function in energy homeostasis by binding to its receptors AdipoR1/2. However, the molecular mechanisms of AdipoQ in the regulation of animal reproduction remain elusive. In this study, we investigated the effects of AdipoQ on hypothalamic reproductive hormone (GnRH) secretion and reproduction-related receptor gene (estrogen receptor [ER] and progesterone receptor [PR]) expression in hypothalamic neuronal cells (HNCs) of chickens by using real-time fluorescent quantitative PCR (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), Western blot (WB) and cell counting kit-8 (CCK-8) assays and found that overexpression of AdipoQ could increase the expression levels of AdipoR1/2 and reproduction-related receptor genes (P < 0.05) while decreasing the expression level of GnRH. In contrast, interference with AdipoQ mRNA showed the opposite results in HNCs. Furthermore, we demonstrated that AdipoQ exerts its functions through the AMPK and PI3K signaling pathways. Finally, our in vitro experiments found that AdipoRon (a synthetic substitute for AdipoQ) treatment and AdipoR1/2 RNAi interference co-treatment resulted in no effect on GnRH secretion, suggesting that the inhibition of GnRH secretion by AdipoQ is mediated by the AdipoR1/2 signaling axis. In summary, we uncovered, for the first time, the molecular mechanism of AdipoQ in the regulation of reproductive hormone secretion in hypothalamic neurons in chickens.

Controlled extracellular vesicles release from aminoguanidine nanoparticle-loaded polylysine hydrogel for synergistic treatment of spinal cord injury.

Pharmaceutical treatments are critical for the acute and subacute phases of spinal cord injury (SCI) and significantly impact patients' prognoses. However, there is a lack of a precise, multitemporal, integrated drug delivery system for medications administered in both phases. In this study, we prepare a hybrid polylysine-based hydrogel (PBHEVs@AGN) comprising short-term release of pH-responsive aminoguanidine nanoparticles (AGN) and sustained release of extracellular vesicles (EVs) for synergistic SCI treatment. When AGN is exposed to the acidic environment at the injury site, it quickly diffuses out of the hydrogel and releases the majority of the aminoguanidine within 24 h, reducing oxidative stress in lesion tissues. Enriched EVs are gradually released from the hydrogel and remain in the tissue for weeks, providing a long-term anti-inflammatory effect and further ensuring axonal regeneration. Fast-releasing aminoguanidine can cooperate with slow-release EVs to treat SCI more effectively by reducing the production of proinflammatory cytokines and blocking the TLR4/Myd88/NF-κB inflammatory pathway, creating a sustained anti-inflammatory microenvironment for SCI recovery. Our in vivo experiments demonstrate that PBHEVs@AGN reduces the occurrence of scar tissue, encourages remyelination, and speeds up axonal regeneration. Herein, this multi-drug delivery system, which combines the acute release of aminoguanidine and the sustained release of EVs is highly effective for synergistically managing the challenging pathological processes after SCI.

Collagen Niches Affect Direct Transcriptional Conversion toward Human Nucleus Pulposus Cells via Actomyosin Contractility.

Cellular niches play fundamental roles in regulating cellular behaviors. However, the effect of niches on direct converted cells remains unexplored. In the present study, the specific combination of transcription factors is first identified to directly acquire induced nucleus pulposus-like cells (iNPLCs). Next, tunable physical properties of collagen niches are fabricated based on various crosslinking degrees. Collagen niches significantly affect actomyosin cytoskeleton and then influence the maturation of iNPLCs. Using gain- and loss of function approaches, the appropriate physical states of collagen niches are found to significantly enhance the maturation of iNPLCs through actomyosin contractility. Moreover, in a rat model of degenerative disc diseases, iNPLCs with collagen niches are transplanted into the lesion to achieve significant improvements. As a result, overexpression of transcription factors in human dermal fibroblasts are efficiently converted into iNPLCs and the optimal collagen niches affect cellular cytoskeleton and then facilitate iNPLCs maturation toward human nucleus pulposus cells. These findings encourage more in-depth studies toward the interactions of niches and direct conversion, which would contribute to the development of direct conversion.

Thermosensitive hydrogel-based GPR124 delivery strategy for rebuilding blood-spinal cord barrier.

Spinal cord injury (SCI) causes blood-spinal cord barrier (BSCB) disruption, leading to secondary damage, such as hemorrhagic infiltration, inflammatory response, and neuronal cell death. It is of great significance to rebuild the BSCB at the early stage of SCI to alleviate the secondary injury for better prognosis. Yet, current research involved in the reconstruction of BSCB is insufficient. Accordingly, we provide a thermosensitive hydrogel-based G protein-coupled receptor 124 (GPR124) delivery strategy for rebuilding BSCB. Herein, we firstly found that the expression of GPR124 decreased post-SCI and demonstrated that treatment with recombinant GPR124 could partially alleviate the disruption of BSCB post-SCI by restoring tight junctions (TJs) and promoting migration and tube formation of endothelial cells. Interestingly, GPR124 could also boost the energy metabolism of endothelial cells. However, the absence of physicochemical stability restricted the wide usage of GPR124. Hence, we fabricated a thermosensitive heparin-poloxamer (HP) hydrogel that demonstrated sustained GPR124 production and maintained the bioactivity of GPR124 (HP@124) for rebuilding the BSCB and eventually enhancing functional motor recovery post-SCI. HP@124 hydrogel can encapsulate GPR124 at the lesion site by injection, providing prolonged release, preserving wounded tissues, and filling injured tissue cavities. Consequently, it induces synergistically efficient integrated regulation by blocking BSCB rupture, decreasing fibrotic scar formation, minimizing inflammatory response, boosting remyelination, and regenerating axons. Mechanistically, giving GPR124 activates energy metabolism via elevating the expression of phosphoenolpyruvate carboxykinase 2 (PCK2), and eventually restores the poor state of endothelial cells. This research demonstrated that early intervention by combining GPR124 with bioactive multifunctional hydrogel may have tremendous promise for restoring locomotor recovery in patients with central nervous system disorders, in addition to a translational approach for the medical therapy of SCI.

Swelling-Mediated Mechanical Stimulation Regulates Differentiation of Adipose-Derived Mesenchymal Stem Cells for Intervertebral Disc Repair Using Injectable UCST Microgels.

Mechanical stimulation is an effective approach for controlling stem cell differentiation in tissue engineering. However, its realization in in vivo tissue repair remains challenging since this type of stimulation can hardly be applied to injectable seeding systems. Here, it is presented that swelling of injectable microgels can be transformed to in situ mechanical stimulation via stretching the cells adhered on their surface. Poly(acrylamide-co-acrylic acid) microgels with the upper critical solution temperature property are fabricated using inverse emulsion polymerization and further coated with polydopamine to increase cell adhesion. Adipose-derived mesenchymal stem cells (ADSCs) adhered on the microgels can be omnidirectionally stretched along with the responsive swelling of the microgels, which upregulate TRPV4 and Piezo1 channel proteins and enhance nucleus pulposus (NP)-like differentiation of ADSCs. In vivo experiments reveal that the disc height and extracellular matrix content of NP are promoted after the implantation with the microgels. The findings indicate that swelling-induced mechanical stimulation has great potential for regulating stem cell differentiation during intervertebral disc repair.