Advancing skeletal health and disease research with single-cell RNA sequencing.

Orthopedic conditions have emerged as global health concerns, impacting approximately 1.7 billion individuals worldwide. However, the limited understanding of the underlying pathological processes at the cellular and molecular level has hindered the development of comprehensive treatment options for these disorders. The advent of single-cell RNA sequencing (scRNA-seq) technology has revolutionized biomedical research by enabling detailed examination of cellular and molecular diversity. Nevertheless, investigating mechanisms at the single-cell level in highly mineralized skeletal tissue poses technical challenges. In this comprehensive review, we present a streamlined approach to obtaining high-quality single cells from skeletal tissue and provide an overview of existing scRNA-seq technologies employed in skeletal studies along with practical bioinformatic analysis pipelines. By utilizing these methodologies, crucial insights into the developmental dynamics, maintenance of homeostasis, and pathological processes involved in spine, joint, bone, muscle, and tendon disorders have been uncovered. Specifically focusing on the joint diseases of degenerative disc disease, osteoarthritis, and rheumatoid arthritis using scRNA-seq has provided novel insights and a more nuanced comprehension. These findings have paved the way for discovering novel therapeutic targets that offer potential benefits to patients suffering from diverse skeletal disorders.

Formononetin Inhibits Mast Cell Degranulation to Ameliorate Compound 48/80-Induced Pseudoallergic Reactions.

Formononetin (FNT) is a plant-derived isoflavone natural product with anti-inflammatory, antioxidant, and anti-allergic properties. We showed previously that FNT inhibits immunoglobulin E (IgE)-dependent mast cell (MC) activation, but the effect of FNT on IgE-independent MC activation is yet unknown. Our aim was to investigate the effects and possible mechanisms of action of FNT on IgE-independent MC activation and pseudoallergic inflammation. We studied the effects of FNT on MC degranulation in vitro with a cell culture model using compound C48/80 to stimulate either mouse bone marrow-derived mast cells (BMMCs) or RBL-2H3 cells. We subsequently measured ß-hexosaminase and histamine release, the expression of inflammatory factors, cell morphological changes, and changes in NF-κB signaling. We also studied the effects of FNT in several in vivo murine models of allergic reaction: C48/80-mediated passive cutaneous anaphylaxis (PCA), active systemic anaphylaxis (ASA), and 2,4-dinitrobenzene (DNCB)-induced atopic dermatitis (AD). The results showed that FNT inhibited IgE-independent degranulation of MCs, evaluated by a decrease in the release of ß-hexosaminase and histamine and a decreased expression of inflammatory factors. Additionally, FNT reduced cytomorphological elongation and F-actin reorganization and attenuated NF-κB p65 phosphorylation and NF-κB-dependent promoter activity. Moreover, the administration of FNT alleviated pseudoallergic responses in vivo in mouse models of C48/80-stimulated PCA and ASA, and DNCB-induced AD. In conclusion, we suggest that FNT may be a novel anti-allergic drug with great potential to alleviate pseudoallergic responses via the inhibition of IgE-independent MC degranulation and NF-κB signaling.

Biomimetic natural biomaterials for tissue engineering and regenerative medicine: new biosynthesis methods, recent advances, and emerging applications.

Biomimetic materials have emerged as attractive and competitive alternatives for tissue engineering (TE) and regenerative medicine. In contrast to conventional biomaterials or synthetic materials, biomimetic scaffolds based on natural biomaterial can offer cells a broad spectrum of biochemical and biophysical cues that mimic the in vivo extracellular matrix (ECM). Additionally, such materials have mechanical adaptability, microstructure interconnectivity, and inherent bioactivity, making them ideal for the design of living implants for specific applications in TE and regenerative medicine. This paper provides an overview for recent progress of biomimetic natural biomaterials (BNBMs), including advances in their preparation, functionality, potential applications and future challenges. We highlight recent advances in the fabrication of BNBMs and outline general strategies for functionalizing and tailoring the BNBMs with various biological and physicochemical characteristics of native ECM. Moreover, we offer an overview of recent key advances in the functionalization and applications of versatile BNBMs for TE applications. Finally, we conclude by offering our perspective on open challenges and future developments in this rapidly-evolving field.

Stepwise Differentiation of Mesenchymal Stem Cells Augments Tendon-Like Tissue Formation and Defect Repair In Vivo.

UNLABELLED: : Tendon injuries are common and present a clinical challenge, as they often respond poorly to treatment and result in long-term functional impairment. Inferior tendon healing responses are mainly attributed to insufficient or failed tenogenesis. The main objective of this study was to establish an efficient approach to induce tenogenesis of bone marrow-derived mesenchymal stem cells (BMSCs), which are the most common seed cells in tendon tissue engineering. First, representative reported tenogenic growth factors were used as media supplementation to induce BMSC differentiation, and the expression of teno-lineage transcription factors and matrix proteins was compared. We found that transforming growth factor (TGF)-ß1 significantly induced teno-lineage-specific gene scleraxis expression and collagen production. TGF-ß1 combined with connective tissue growth factor (CTGF) elevated tenomodulin and Egr1 expression at day 7. Hence, a stepwise tenogenic differentiation approach was established by first using TGF-ß1 stimulation, followed by combination with CTGF for another 7 days. Gene expression analysis showed that this stepwise protocol initiated and maintained highly efficient tenogenesis of BMSCs. Finally, regarding in situ rat patellar tendon repair, tendons treated with induced tenogenic BMSCs had better structural and mechanical properties than those of the control group, as evidenced by histological scoring, collagen I and tenomodulin immunohistochemical staining, and tendon mechanical testing. Collectively, these findings demonstrate a reliable and practical strategy of inducing tenogenesis of BMSCs for tendon regeneration and may enhance the effectiveness of cell therapy in treating tendon disorders. SIGNIFICANCE: The present study investigated the efficiency of representative tenogenic factors on mesenchymal stem cells' tenogenic differentiation and established an optimized stepwise tenogenic differentiation approach to commit tendon lineage differentiation for functional tissue regeneration. The reliable tenogenic differentiation approach for stem cells not only serves as a platform for further studies of underlying molecular mechanisms but also can be used to enhance cell therapy outcome in treating tendon disorders and develop novel therapeutics for tendon injury.

The effects of secretion factors from umbilical cord derived mesenchymal stem cells on osteogenic differentiation of mesenchymal stem cells.

Factors synthesized by mesenchymal stem cells (MSCs) contain various growth factors, cytokines, exosomes and microRNAs, which may affect the differentiation abilities of MSCs. In the present study, we investigated the effects of secretion factors of human umbilical cord derived mesenchymal stem cells (hUCMSCs) on osteogenesis of human bone marrow derived MSCs (hBMSCs). The results showed that 20 µg/ml hUCMSCs secretion factors could initiate osteogenic differentiation of hBMSCs without osteogenic induction medium (OIM), and the amount of calcium deposit (stained by Alizarin Red) was significantly increased after the hUCMSCs secretion factors treatment. Real time quantitative reverse transcription-polymerase chain reaction (real time qRT-PCR) demonstrated that the expression of osteogenesis-related genes including ALP, BMP2, OCN, Osterix, Col1α and Runx2 were significantly up-regulated following hUCMSCs secretion factors treatment. In addition, we found that 10 µg hUCMSCs secretion factors together with 2×10(5) hBMSCs in the HA/TCP scaffolds promoted ectopic bone formation in nude mice. Local application of 10 µg hUCMSCs secretion factors with 50 µl 2% hyaluronic acid hydrogel and 1×10(5) rat bone marrow derived MSCs (rBMSCs) also significantly enhanced the bone repair of rat calvarial bone critical defect model at both 4 weeks and 8 weeks. Moreover, the group that received the hUCMSCs secretion factors treatment had more cartilage and bone regeneration in the defect areas than those in the control group. Taken together, these findings suggested that hUCMSCs secretion factors can initiate osteogenesis of bone marrow MSCs and promote bone repair. Our study indicates that hUCMSCs secretion factors may be potential sources for promoting bone regeneration.

Partial loss of Smad7 function impairs bone remodeling, osteogenesis and enhances osteoclastogenesis in mice.

Smad7 is well demonstrated as a negative regulator of TGF-ß signaling. Its alteration in expression often results in diseases such as cancer and fibrosis. However, the exact role of Smad7 in regulating bone remodeling during mammalian development has not been properly delineated. In this study we performed experiments to clarify the involvement of Smad7 in regulating osteogenesis and osteoclastogenesis both invivo and invitro. Genetically engineered Smad7(ΔE1) (KO) mice were used, whereby partial functional of Smad7 is lost by deleting exon I of the Smad7 gene and the truncated proteins cause a hypomorphic allele. Analysis with µCT imagery and bone histomorphometry showed that the KO mice had lower TbN, TbTh, higher TbSp in the metaphysic region of the femurs at 6, 12, 24weeks from birth, as well as decreased MAR and increased osteoclast surface compared with the WT mice. In vitro BM-MSC multi-lineage differentiation evaluation showed that the KO group had reduced osteogenic potential, fewer mineralized nodules, lower ALP activity, and reduced gene expression of Col1A1, Runx2 and OCN. The adipogenic potential was elevated in the KO group with more formation of lipid droplets, and increased gene expression of Adipsin and C/EBPα. The osteoclastogenic potential of KO mice BMMs was elevate, with emergence of more osteoclasts, larger resorptive areas, and increased gene expression of TRAP and CTR. Our results indicate that partial loss of Smad7 function in mice leads to compromised bone formation and enhanced bone resorption. Thus, Smad7 is acknowledged as a novel key regulator between osteogenesis and osteoclastogenesis.

Prevention of osteopenia and dyslipidemia in rats after ovariectomy with combined aspirin and low-dose diethylstilbestrol.

OBJECTIVE: To study whether effect of aspirin plus low-dose diethylstilbestrol is more effective and safer than high diethylstilbestrol dose alone on prevention of ovariectomy-induced osteopenia and dyslipidemia. METHODS: Thirty-eight 4-month-old female SD rats were divided into baseline (BAS) group (n=6), sham operation group (n=8) and ovariectomy (OVX) group (n=24). The OVX group was further divided into vehicle treatment group (n=8), diethylstilbestrol (30 µg/kg•d) treatment group (OVX+D30 group, n=8), and aspirin (9 mg/kg•d) plus diethylstilbestrol (10 µg/kg•d) treatment group (OVX+A-D10 group, n=8). Their left tibiae were collected for the bone histomorphometric analysis in undecalcified sections. Left femurs were collected for the bone mineral density measurement. RESULTS: The body weight and serum cholesterol were increased, while uterine weight and cancellous bone mass were decreased in OVX rats compared with the SHAM group. Cancellous bone mass was significantly increased, while body weight and bone resorption parameters were decreased in both A-D10 and D30 treatment group compared with OVX group. The rats treated with A-D10 showed significantly increased in bone formation parameters and decreased in serum triglyceride compared with the D30-treated rats. CONCLUSION: Aspirin plus low-dose diethylstilbestrol can effectively prevent osteopenia by reducing bone resorption, and is thus a better treatment modality for preventing dyslipidemia than high-dose diethylstilbestrol alone.

Selectivity of N170 in the left hemisphere as an electrophysiological marker for expertise in reading Chinese.

OBJECTIVE: The left-lateralized N170, an event-related potential component consistently shown in response to alphabetic words, is a robust electrophysiological marker for reading expertise in an alphabetic language. In contrast, such a marker is lacking for expertise in reading Chinese, because the existing results about the lateralization of N170 for Chinese characters are mixed, reflecting complicated factors such as top-down modulation that contribute to the relative magnitudes of N170 in the left and right hemispheres. The present study aimed to explore a potential electrophysiological marker for reading expertise in Chinese with minimal top-down influence. METHODS: We recorded N170 responses to Chinese characters and three kinds of control stimuli in a content-irrelevant task, minimizing potential top-down effects. RESULTS: Direct comparison of the N170 amplitude in response to Chinese characters between the hemispheres showed a marginally significant left-lateralization effect. However, detailed analyses of N170 in each hemisphere revealed a more robust pattern of left-lateralization - the N170 in the left but not the right hemisphere differentiated Chinese characters from control stimuli. CONCLUSION: These results suggest that the selectivity of N170 (a greater N170 in response to Chinese characters than to control stimuli) within the left hemisphere rather than the hemispheric difference of N170 with regard to Chinese characters is an electrophysiological marker for expertise in reading Chinese.

Left-lateralized early neurophysiological response for Chinese characters in young primary school children.

Adult readers consistently show an enhanced early event-related potential (ERP) response, N170, for visual words compared with other stimuli at left posterior electrodes. Developmental studies with words in alphabetic languages showed that this neurophysiological specialization for print develops rapidly from 6 to 10-years of age and becomes established around 10-11 years of age. Here we report for the first time the development of the word-related N170 in Chinese children learning to read Chinese, a logographic writing system radically different from alphabetic scripts in visual and linguistic features. We recorded ERP responses elicited by Chinese characters and line drawings of common objects in three groups of primary school children at 7, 9, and 11 years of age as well as college students. Results showed that the amplitude of N170 evoked by Chinese characters in the 7-year-old group was significantly larger than that in the 11-year-old group and the adult readers. Remarkably, all four age groups - even the youngest group - showed an increased and left-lateralized N170 response for Chinese characters, as compared with line drawings, suggesting that a relatively specialized mechanism for processing Chinese characters is already emergent by as early as 7 years of age. Our results, combined with studies of non-Chinese child readers suggest that the developmental pattern of word-related N170 is highly similar across different scripts, possibly reflecting increased visual processing expertise that children acquire through everyday reading.

[Phylogenetic analysis of enterovirus 71 isolated from patients with hand, foot and mouth disease in Guangdong and Fujian provinces, 2000-2001].

OBJECTIVE: To identify enterovirus 71 (EV71) strains isolated from patients with hand, foot and mouth disease (HFMD) in Guangdong and Fujian provinces from 2000 to 2001 by using phylogenetic analysis. METHODS: All 25 samples were first tested for enteric viruses by RT-PCR using enterovirus specific primers EV-1 and EV-2, and then were identified for EV71 by RT-PCR using EV71 specific primers 159S and 162A. The amplicons of 485bp segment (part of the VP1 gene) were cloned into pGEM-T and sequenced. A phylogenetic tree was constructed by comparison of the sequences with other 12 EV71 strains isolated from China, Japan, Hungary, and the United States including the prototype BrCr. RESULTS: The positive rate of EV71 was about 20%. The sequence analysis showed that the new isolate (GZH2000) shared 94%-96% nucleotide identity with three strains isolated in 1998 and 2000, and 91% with a strain isolated in 1987 from Chinese mainland, but shared only 82%-84% homology with EV71 isolates studied abroad. CONCLUSIONS: EV71 is one of the important pathogens of HFMD in south China. The strains isolated from mainland were closely related with most isolates from Taiwan, but different from most EV71 strains reported abroad. The symptoms of EV71 infection in mainland were not as intensive as those described in Taiwan's outbreak.

Diversity of rotavirus strains among children with acute diarrhea in China: 1998-2000 surveillance study.

As part of a national rotavirus surveillance activity, we collected fecal specimens from 3,177 children with acute diarrhea in 10 regions of China between April 1998 and April 2000 and screened them for rotavirus. Rotavirus was detected in 41% (n = 1,305) of specimens, and in these, G1 was the predominant serotype (72.6%), followed by G3 (14.2%), G2 (12.1%), G4 (2.5%), G9 (0.9%), and G untypeable (0.7%). Among 327 G-typed strains tested for P genotype, 14 different P-G combinations were identified, with the globally common strains P[8]G1, P[4]G2, P[8]G3, and P[8]G4 representing 75.6% of all typed rotavirus strains. Among the uncommon strains, 11 were P[6]G9, and others included P[6]G1, P[6]G3, and five novel P-G combinations (P[9]G1, P[4]G1, P[4]G3, P[4]G4, and P[8]G2). Our results indicate that while the common rotavirus strains remain predominant, the diversity of strains is much greater than was previously recognized.