The high risks of traumatic cataract treatments promoted the development of the concept of autologous lens regeneration. Biochemical cues can influence the cellular behavior of stem cells, and in this case, biophysical cues may be the important factors in producing rapid activation of cellular behavior. Here we bio-printed mesenchymal stem cells (MSCs) using a commonly used bioink sodium alginate-gelatin blends, and investigated the induction effect of MSC differentiation towards lens epithelial stem cells (LESCs) under a combination of biochemical cues and biophysical cues. It was found that biochemical cues in the porous three-dimensional (3D) matrix constructed using bioink sodium alginate-gelatin blends for bio-printing did not reduce the cell viability of loaded MSCs in the matrix by scanning electron microscope (SEM) observation and cell viability detection. Loaded MSCs in the matrix were consistently upregulated in the expression of proteins and genes involved in phenotypes and development signaling pathways of LESCs, as detected by polymerase chain reaction (PCR) with the support of biochemical cues. These results indicated that biophysical cues could rapidly activate the cellular behavior of MSCs differentiation, and biochemical cues could continuously induce MSCs differentiation towards LESCs.

INTRODUCTION:Acute kidney injury (AKI) is a severe condition with high morbidity and mortality. Innovative biomarkers and treatments are essential for improving patient outcomes. This study aims to investigate the role of ferroptosis-related genes (FRGs) in AKI for identifying potential biomarkers and therapeutic targets. METHODS:We analyzed mRNA expression profiles from the Gene Expression Omnibus (GEO: GSE139061) dataset, comparing 36 AKI samples with 9 normal samples. Differentially expressed genes (DEGs) were identified using the R software package limma. Functional enrichment analyses were conducted using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Key biomarkers were validated through area under the curve (AUC) values, and immune cell infiltration was analyzed using CIBERSORT. RESULTS:We identified 78 differentially expressed FRGs, with 27 up-regulated and 51 down-regulated genes. Key signaling pathways included MAPK, ferroptosis, and p53. Five genes-NR4A1, GLRX5, USP35, AEBP2, and MDM4-were identified as potential biomarkers, each demonstrating AUC values greater than 0.800. Specifically, MDM4 showed significant potential by promoting the phosphorylation of p53 at Ser46, enhancing mitochondrial apoptotic activity. Immune analysis revealed a significant elevation of M0 macrophages in AKI samples compared to normal samples (P < 0.01). CONCLUSION:Our findings highlight the critical role of ferroptosis-related genes in AKI, identifying NR4A1, GLRX5, USP35, AEBP2, and MDM4 as key biomarkers with high diagnostic potential. These results provide novel insights into the molecular mechanisms of AKI.

Given afferent functions, sensory nerves have recently been found to exert efferent effects and directly alter organ physiology. Additionally, several studies have highlighted the indirect but crucial role of sensory nerves in the regulation of the physiological function of osteoclasts. Nonetheless, evidence regarding the direct sensory nerve efferent influence on osteoclasts is lacking. In the current study, we found that high levels of efferent signals were transported directly from the sensory nerves into osteoclasts. Furthermore, sensory hypersensitivity significantly increased osteoclastic bone resorption, and sensory neurons (SNs) directly promoted osteoclastogenesis in an in vitro coculture system. Moreover, we screened a novel neuropeptide, Cyp40, using an isobaric tag for relative and absolute quantitation (iTRAQ). We observed that Cyp40 is the efferent signal from sensory nerves, and it plays a critical role in osteoclastogenesis via the aryl hydrocarbon receptor (AhR)-Ras/Raf-p-Erk-NFATc1 pathway. These findings revealed a novel mechanism regarding the influence of sensory nerves on bone regulation, i.e., a direct promoting effect on osteoclastogenesis by the secretion of Cyp40. Therefore, inhibiting Cyp40 could serve as a strategy to improve bone quality in osteoporosis and promote bone repair after bone injury.

Groove patterns are widely used in material surface modifications. However, the independent role of ditches/ridges in regulating fibrosis of soft tissues is not well-understood, especially the lack of linkage evidence in vitro and in vivo. Herein, two kinds of combinational microgroove chips with the gradient ditch/ridge width were fabricated by photolithography technology, termed R and G groups, respectively. In group R, the ridge width was 1, 5, 10, and 30 μm, with a ditch width of 30 μm; in group G, the groove width was 5, 10, 20, and 30 μm, and the ridge width was 5 μm. The effect of microgrooves on the morphology, proliferation, and expression of fibrous markers of stem cells was systematically investigated in vitro. Moreover, thicknesses of fibrous capsules were evaluated after chips were implanted into the muscular pouches of rats for 5 months. The results show that microgrooves have almost no effect on cell proliferation but significantly modulate the morphology of cells and focal adhesions (FAs) in vitro, as well as fibrosis differentiation. In particular, the differentiation of stem cells is attenuated after the intracellular force caused by stress fibers and FAs is interfered by drugs, such as rotenone and blebbistatin. Histological analysis shows that patterns of high intracellular force can apparently stimulate soft tissue fibrosis in vivo. This study not only reveals the specific rules and mechanisms of ditch/ridge regulating stem cell behaviors but also offers insight into tailoring implant surface patterns to induce controlled soft tissue fibrosis.

Background Bone defects remain an unsolved clinical problem due to the lack of effective osteogenic induction protocols. Nanomaterials play an important role in bone defect repair by stimulating osteogenesis. However, constructing an effective bioactive nanomaterial remains a substantial challenge. Methods In this study, mesoporous silica nanoparticles (MSNs) were prepared and used as nanocarriers for basic fibroblast growth factor (bFGF). The characteristics and biological properties of the synthetic bFGF@MSNs were tested. The osteogenic effects of the particles on the behavior of MC3T3-E1 cells were investigated in vitro. In addition, the differentially expressed genes during induction of osteogenesis were analyzed by transcriptomic sequencing. Radiological and histological observations were carried out to determine bone regeneration capability in a distal femur defect model. Results Achieving bFGF sustained release, bFGF@MSNs had uniform spherical morphology and good biocompatibility. In vitro osteogenesis induction experiments showed that bFGF@MSNs exhibited excellent osteogenesis performance, with upregulation of osteogenesis-related genes (RUNX2, OCN, Osterix, ALP). Transcriptomic sequencing revealed that the Wnt/β-catenin signalling pathway could be activated in regulation of biological processes. In vivo, bone defect repair experiments showed enhanced bone regeneration, as indicated by radiological and histological analysis, after the application of bFGF@MSNs. Conclusion bFGF@MSNs can promote bone regeneration by activating the Wnt/β-catenin signalling pathway. These particles are expected to become a potential therapeutic bioactive material for clinical application in repairing bone defects in the future.

Large bone defects remain an unsolved clinical challenge because of the lack of effective vascularization in newly formed bone tissue. 3D bioprinting is a fabrication technology with the potential to create vascularized bone grafts with biological activity for repairing bone defects. In this study, vascular endothelial cells laden with thermosensitive bio-ink were bioprinted in situ on the inner surfaces of interconnected tubular channels of bone mesenchymal stem cell-laden 3D-bioprinted scaffolds. Endothelial cells exhibited a more uniform distribution and greater seeding efficiency throughout the channels. In vitro, the in situ bioprinted endothelial cells can form a vascular network through proliferation and migration. The in situ vascularized tissue-engineered bone also resulted in a coupling effect between angiogenesis and osteogenesis. Moreover, RNA sequencing analysis revealed that the expression of genes related to osteogenesis and angiogenesis is upregulated in biological processes. The in vivo 3D-bioprinted in situ vascularized scaffolds exhibited excellent performance in promoting new bone formation in rat calvarial critical-sized defect models. Consequently, in situ vascularized tissue-engineered bones constructed using 3D bioprinting technology have a potential of being used as bone grafts for repairing large bone defects, with a possible clinical application in the future.

The technique bottleneck of repairing large bone defects with tissue engineered bone is the vascularization of tissue engineered grafts. Although some studies have shown that extracellular vesicles (EVs) derived from bone marrow mesenchymal stem cells (BMSCs) promote bone healing and repair by accelerating angiogenesis, the effector molecules and the mechanism remain unclear, which fail to provide ideas for the future research and development of cell-free interventions. Here, we found that Nidogen1-enriched EV (EV-NID1) derived from BMSCs interferes with the formation and assembly of focal adhesions (FAs) by targeting myosin-10, thereby reducing the adhesion strength of rat arterial endothelial cells (RAECs) to the extracellular matrix (ECM), and enhancing the migration and angiogenesis potential of RAECs. Moreover, by delivery with composite hydrogel, EV-NID1 is demonstrated to promote angiogenesis and bone regeneration in rat femoral defects. This study identifies the intracellular binding target of EV-NID1 and further elucidates a novel approach and mechanism, thereby providing a cell-free construction strategy with precise targets for the development of vascularized tissue engineering products.

Injectable hydrogels have long been gaining attention in the bone tissue engineering field owing to their ability to mix homogeneously with cells and therapeutic agents, minimally invasive administration, and seamless defect filling. Despite the advantages, the use of injectable hydrogels as cell delivery carriers is currently limited by the challenge of mimicking the natural microenvironment of the loaded cells, promoting cell proliferation, and enhancing bone regeneration. To overcome these problems, we aimed to develop an injectable and in situ-forming nanocomposite hydrogel composed of gelatin, alginate, and LAPONITE® to mimic the architecture and composition of the extracellular matrix. The encapsulated rat bone marrow mesenchymal stem cells (rBMSCs) survived in the nanocomposite hydrogel, and the gel promoted cell proliferation in vitro. Systematic in vivo research of the biomimetic hydrogel with or without cells was conducted in a critical-size (8 mm) rat bone defect model. The in vivo results proved that the hydrogel loaded with rBMSCs significantly promoted bone healing in rat calvarial defects, compared to the hydrogel without cells, and that the hydrogel did not provoked side effects on the recipients. Given these advantageous properties, the developed cell-loaded injectable nanocomposite hydrogel can greatly accelerate the bone healing in critical bone defects, thus providing a clinical potential candidate for orthopedic applications.

Background Induced pluripotent stem cells (iPSCs) exhibit limitless pluripotent plasticity and proliferation capability to provide an abundant cell source for tissue regenerative medicine. Thus, inducing iPSCs toward a specific differentiation direction is an important scientific question. Traditionally, iPSCs have been induced to chondrocytes with the help of some small molecules within 21–36 days. To speed up the differentiation of iPSCs, we supposed to utilize bioactive ceramics to assist chondrogenic-induction process. Methods In this study, we applied ionic products (3.125~12.5 mg/mL) of the lithium-containing bioceramic (Li 2 Ca 4 Si 4 O 13 , L2C4S4) and individual Li + (5.78~23.73 mg/L) in the direct chondrogenic differentiation of human iPSCs. Results Compared to pure chondrogenic medium and extracts of tricalcium phosphate (TCP), the extracts of L2C4S4 at a certain concentration range (3.125~12.5 mg/mL) significantly enhanced chondrogenic proteins Type II Collagen (COL II)/Aggrecan/ SRY-Box 9 (SOX9) synthesis and reduced hypertrophic protein type X collagen (COL X)/matrix metallopeptidase 13 (MMP13) production in iPSCs-derived chondrocytes within 14 days, suggesting that these newly generated chondrocytes exhibited favorable chondrocytes characteristics and maintained a low-hypertrophy state. Further studies demonstrated that the individual Li + ions at the concentration range of 5.78~23.73 mg/L also accelerated the chondrogenic differentiation of iPSCs, indicating that Li + ions played a pivotal role in chondrogenic differentiation process. Conclusions These findings indicated that lithium-containing bioceramic with bioactive specific ionic components may be used for a promising platform for inducing iPSCs toward chondrogenic differentiation and cartilage regeneration.

Recent studies show that biomaterials are capable of regulating immune responses to induce a favorable osteogenic microenvironment and promote osteogenesis and angiogenesis. In this study, we investigated the effects of zinc silicate/nano-hydroxyapatite/collagen (ZS/HA/Col) scaffolds on bone regeneration and angiogenesis and explored the related mechanism. We demonstrate that 10ZS/HA/Col scaffolds significantly enhanced bone regeneration and angiogenesis in vivo compared with HA/Col scaffolds. ZS/HA/Col scaffolds increased tartrate-resistant acid phosphatase (TRAP)-positive cells, nestin-positive bone marrow stromal cells (BMSCs) and CD31-positive neovessels, and expression of osteogenesis (Bmp-2 and Osterix) and angiogenesis-related (Vegf-α and Cd31) genes increased in nascent bone. ZS/HA/Col scaffolds with 10 wt. % ZS activated the p38 signaling pathway in monocytes. The monocytes subsequently differentiated into TRAP+ cells and expressed higher levels of the cytokines SDF-1, TGF-β1, VEGF-α, and PDGF-BB, which recruited BMSCs and endothelial cells (ECs) to the defect areas. Blocking the p38 pathway in monocytes reduced TRAP+ differentiation and cytokine secretion, and resulted in a decrease in BMSC and EC homing and angiogenesis. Overall, these findings demonstrate that 10ZS/HA/Col scaffolds modulate monocytes, and thereby create a favorable osteogenic microenvironment that promotes BMSC migration and differentiation and vessel formation by activating the p38 signaling pathway.

Three-dimensional (3D) bioprinting is an extremely convenient biofabrication technique for creating biomimetic tissue-engineered bone constructs and has promising applications in regenerative medicine. However, existing bioinks have shown low mechanical strength, poor osteoinductive ability, and lacking a suitable microenvironment for laden cells. Nanosilicate (nSi) has shown to be a promising biomaterial, due to its unique properties such as excellent biocompatibility, degrade into nontoxic products, and with osteoinductive properties, which has been used in bone bioprinting. However, the long term bone healing effects and associating risks, if any, of using nSi in tissue engineering bone scaffolds in vivo are unclear and require a more thorough assessment prior to practical use. Hence, a functional and biomimetic nanocomposite bioink composed of rat bone marrow mesenchymal stem cells (rBMSCs), nSi, gelatin and alginate for the 3D bioprinting of tissue-engineered bone constructs is firstly demonstrated, mimicking the structure of extracellular matrix, to create a conducive microenvironment for encapsulated cells. It is shown that the addition of nSi significantly increases the printability and mechanical strength of fabricated human-scale tissue or organ structures (up to 15 mm height) and induces osteogenic differentiation of the encapsulated rBMSCs in the absence of in vitro osteoinductive factors. A systematic in vivo research of the biomimetic nanocomposite bioink scaffolds is further demonstrated in a rat critical-size (8 mm) bone defect-repair model. The in vivo results demonstrate that the 3D bioprinted nanocomposite scaffolds can significantly promote the bone healing of the rat calvarial defects compared to other scaffolds without nSi or cells, and show rarely side effects on the recipients. Given the above advantageous properties, the 3D bioprinted nanocomposite scaffolds can greatly accelerate the bone healing in critical bone defects, thus providing a clinical potential candidate for orthopedic applications.

OBJECTIVES:Activation of the sympathetic system and adrenergic β-receptors following traumatic bone defects negatively impairs bone regeneration. Whether preventing β-receptor activation could potentially improve bone defect repair is unknown. In this study, we investigated the effect of systematic administration and local delivery of propranolol through composite scaffolds on bone healing.MATERIALS AND METHODS:Collagen/PVA/propranolol/hydroxyapatite（CPPH）composite scaffolds were fabricated with 3D printing technique and characterized by scanning electron microscope (SEM). Micro-CT analysis and bone formation histology were performed to detect new bone formation. Osteogenic differentiation of bone marrow stromal cells (BMSCs) and osteoclastogenesis of bone marrow monocytes cultured with scaffolds extract were performed for further verification.RESULTS:Intraperitoneal injection of propranolol did not significantly improve bone repair, as indicated by micro-CT analysis and bone formation histology. However, CPPH scaffolds exhibited sustained release of propranolol in vitro and significantly enhanced bone regeneration compared with vehicle collagen/PVA/hydroxyapatite (CPH) scaffolds in vivo. Moreover, in vitro experiments indicated the scaffolds containing propranolol promoted the osteogenic differentiation and migration of rat BMSCs and inhibited osteoclastogenesis by preventing β-receptor activation.CONCLUSIONS:This study demonstrates that local adrenergic β-receptor blockade can effectively enhance the treatment of bone defects by stimulating osteogenic differentiation, inhibiting osteoclastogenesis and enhancing BMSCs migration.

The prevascularization of tissue-engineered bone grafts (TEBGs) has been shown to accelerate capillary vessel ingrowth in bone defect remodeling and to enhance new bone formation. However, the exact mechanisms behind this positive effect remain unknown. Here, we report that basic fibroblast growth factor (FGF2)-Ras homolog gene family member A (RhoA)/Rho-associated protein kinase (ROCK) signaling functions as a molecular switch to regulate the lineage fate of bone mesenchymal stem cells (BMSCs) and that prevascularization promotes the cell fate switch, which contributes to increased bone regeneration with the use of prevascularized TEBGs compared with control TEBGs. Prevascularized TEBGs enhanced the in vivo endothelial differentiation of BMSCs by inhibiting RhoA/ROCK signaling. In vitro data more clearly showed that BMSCs differentiated into von Willebrand factor (vWF)-positive endothelial cells, and FGF2-induced inhibition of RhoA/ROCK signaling played a key role. Our novel findings uncovered a new mechanism that stimulates the increased vascularization of engineered bone and enhanced regeneration by promoting the endothelial differentiation of BMSCs implanted in TEBGs. These results offer a new molecular target to regulate TEBG-induced bone regeneration.

Vascularization is one of the most important processes in tissue-engineered bone graft (TEBG)-mediated regeneration of large segmental bone defects. We previously showed that prevascularization of TEBGs promoted capillary vessel formation within the defected site and accelerated new bone formation. However, the precise mechanisms and contribution of endogenous cells were not explored.

Objective To establish specific induced pluripotent stem cells (iPSCs) fromthe patient with congenitalclubfoot by taking exfoliated cells in urine as source.Methods 180 ml of urine from clubfoot patient were collected,and the exfoliated cells in urine were isolated and cultured by centrifugation.Then,the cells were reprogrammed with virus system and identified by pluripotency gene detection,alkaline phosphatase (ALP) staining,karyotype identification,Fluorescein activated cell sorter (FACS) surface markers detection,embryoid body (EB) suspension ball assay and methylation assay.Results The expression of pluripotentgenes between obtained cellsand human embryonic stem cells H9 was similar,ALP staining showed strong positive.The cells possessed normal karyotype and 84.1％ positive rate of pluripotency marker Tra-1-60,they could form a large number of EB suspension balls and almost lost methylation of the promoter regionsin Nanog homeobox (NANOG) and organic cation/carnitine transporter4 (Oct4) genes.Therefore,werevealed that the obtained cells owned all of the characteristics of pluripotent cells,and wecan identified them as iPSCs.Conclusion iPSCs were successfully constructed in this study,which provided cell materials for the clarification of evolution characteristics of congenital clubfootdisease.

Massive bone defects are a challenge in orthopaedic research. Defective regeneration leads to bone atrophy, non-union of bone, and physical morbidity. Large animals are important models, however, production costs are high, nursing is complex, and evaluation methods are limited. A suitable laboratory animal model is required to explore the underlying molecular mechanism and cellular process of bone tissue engineering. We designed a stainless steel plate with 8 holes; the middle 2 holes were used as a guide to create a standardized critical size defect in the femur of anaesthetized rats. The plate was fixed to the bone using 6 screws, serving as an inner fixed bracket to secure a tricalcium phosphate implant seeded with green fluorescent protein-positive rat bone marrow mesenchymal stem cells within the defect. In some animals, we also grafted a vessel bundle into the lateral side of the implant, to promote vascularized bone tissue engineering. X-ray, microcomputed tomography, and histological analyses demonstrated the stainless steel plate resulted in a stable large segmental defect model in the rat femur. Vascularization significantly increased bone formation and implant degradation. Moreover, survival and expansion of green fluorescent protein-positive seeded cells could be clearly monitored in vivo at 1, 4, and 8 weeks postoperation via fluorescent microscopy. This standardized large segmental defect model in a small animal may help to advance the study of bone tissue engineering. Furthermore, availability of antibodies and genetically modified rats could help to dissect the precise cellular and molecular mechanisms of bone repair.

Our previous studies found that sensory nerve tracts implanted in tissue-engineered bone (TEB) could result in better osteogenesis. To explore the mechanism of the sensory nerve promoting osteogenesis in TEB in vitro, a transwell coculture experiment was designed between dorsal root ganglion (DRG) cells and bone marrow mesenchymal stem cells (BMSCs). BMSC proliferation was determined by CCK8 assay, and osteo-, chondro-, and adipogenic differentiation were assessed by alizarin red, alcian blue, and oil red staining. We found that the proliferation and multipotent differentiation of BMSCs were all enhanced in the coculture group compared to the BMSCs group. Crystal violet staining showed that the clone-forming ability of BMSCs in the coculture group was also enhanced and mRNA levels of Sox2, Nanog, and Oct4 were significantly upregulated in the coculture group. Moreover, the autophagy level of BMSCs, regulating their stemness, was promoted in the coculture group, mediated by the AMPK/mTOR pathway. In addition, AMPK inhibitor compound C could significantly downregulate the protein expression of LC3 and the mRNA level of stemness genes in the coculture group. Finally, we found that the NK1 receptor antagonist, aprepitant, could partly block this effect, which indicated that substance P played an important role in the effect. Together, we conclude that DRG could maintain the stemness of BMSCs by enhancing autophagy through the AMPK/mTOR pathway in a transwell coculture system, which may help explain the better osteogenesis after implantation of the sensory nerve into TEB.

CD31hiEmcnhi vessels were a subtype of vessels in the murine skeletal system, with high levels of platelet and endothelial cell adhesion molecule-1 (PECAM-1/CD31) and endomucin (Emcn). They were reported coupling angiogenesis and osteogenesis during bone development. We investigated the distribution of these vessels in rat tibiae and their temporal and spatial distribution during the bone defect repair process to improve our understanding of the importance of these vessels. We confirmed that CD31hiEmcnhi vessels were specially distributed around the trabecular bones near metaphysis and endosteum in rat tibiae. At 3 days post bone injury, CD31hiEmcnhi vessels proliferated and were extensively distributed across the entire repair area. At 7 and 14 days post-injury, these vessels decreased but were specially distributed around the growing trabecular bones near the frontier growth area, suggesting that these vessels support new bone formation. The distribution of CD31hiEmcnhi vessels and the transcriptions of Hif - 1α and VEGFA , as well as BMP2 and Osterix decreased at 7 and 14 days post-injury under osteoporotic conditions, in combination with insufficient osteogenesis. Our research is of great significance to help understand the important role of CD31hiEmcnhi vessels in supporting new trabecular bones formation during bone defect repair process.

Objective To investigate the effect of sensory and motor nerve homogenates at different concentrations on the proliferation and osteogenesis differentiation of bone marrow mesenchymal stem cells (BMSCs) in rats.Methods The saphenous nerve and the muscle branch of the sciatic nerve in rats were extracted surgically as sensory and motor nerve tissues,respectively.The primary nerve homogenates (10 mg/mL) were prepared as per 10 mg tissue with 1 mL osteoblast inducing conditional media,and 10 times diluted after filtration purification to prepare sensory and motor nerve homogenates at concentration gradients of 1.0,0.1,0.01,0.001 and 0.0001 mg/mL.Cultivation GFP ± rat pups BMSCs in vitro were trained to P3 generation.The experiment was carried out in 3 groups.The sensory and motor nerve homogenates of 500 μL at the above 6 concentration gradients were added during cultivation respectively in the sensory nerve group (n =18) and the motor nerve group(n =18) while 500 μL of osteoblast inducing conditional media was added in the control group(n =3).Cell proliferation quantity detection and alkaline phosphatase (ALP) staining were used to assess the proliferation and differentiation of BMSCs after 14 days.Results According to the results of CCK-8,the cellular absorbance values at concentrations of 1.0 and 0.1 mg/mL homogenate in the sensory nerve group (1.957 ±0.065 and 1.751±0.073) were significantly greater than in the control group (1.145±0.087) while the cellular absorbance value at concentration of 10.0 mg/mL homogenate in the motor nerve group (0.304 ± 0.619) was significandy smaller than in the control group (1.145 ± 0.087) (P ＜ 0.05).According to the ALP staining,the amounts of cellular calcium nodules in the sensory and motor nerve groups (2.667 ± 0.816 and 3.000 ± 0.632,respectively) were significantly smaller than in the control group (11.833 ± 1.471) (P ＜ 0.05).Conclusion Sensory nerve homogenate is different from motor nerve homogenate in that it may promote proliferation of BMSCs and inhibit osteogenesis differentiation of BMSCs in a certain rage of concentrations.