Background: Articular cartilage has limited self-repair capacity, and current clinical treatment options for cartilage defects are inadequate. However, deer antler cartilage possesses unique regenerative properties, with the ability to rapidly repair itself. This rapid self-repair process is closely linked to the paracrine factors released by deer antler stem cells. These findings present potential for the development of cell-free therapies for cartilage defects in clinical settings. The aim of this study was to investigate a novel method for repairing cartilage. Methods: A rat model with articular cartilage defects was established through surgery. Hydrogels loaded with exosomes (Exos) derived from antler stem cells (ASC-Exos) were implanted into the rat cartilage defects. The extent of cartilage damage repair was assessed using histological methods. The effects of ASC-Exos on chondrocytes and rat bone marrow mesenchymal stem cells (BMSCs) were evaluated using cell viability assays, proliferation assays, and scratch assays. Additionally, the maintenance of the chondrocyte phenotype by ASC-Exos was assessed using real-time fluorescence quantitative PCR (qPCR) and western blot analysis. The protein components contained of the Exos were identified using data-independent acquisition (DIA) mass spectrometry. Results: ASC-Exos significantly promoted the repair of cartilage tissue damage. The level of cartilage repair in the experimental group (ASC-Exos) was higher than that in the positive control (human adipose-derived stem cells, hADSC-Exos) and negative control (dulbecco’s modified eagle medium) groups (p < 0.05). In vitro experiments demonstrated that ASC-Exos significantly enhanced the proliferation abilities of chondrocytes and the proliferation abilities and the migration abilities of BMSCs (p < 0.05). ASC-Exos up-regulated the expression levels of Aggrecan, Collagen II (COLII), and Sox9 mRNA and proteins in chondrocytes. Analysis of ASC-Exos protein components revealed the presence of active components such as Serotransferrin (TF), S100A4, and Insulin-like growth factor-binding protein 1 (IGF1). Conclusions: ASC-Exos have a significant effect on cartilage damage repair, which may be attributed to their promotion of chondrocyte and BMSCs proliferation and migration, as well as the maintenance of chondrocyte phenotype. This effect may be mediated by the presence of TF, S100A4, and IGF1.

Background: Deer antlers are the only known mammalian structure that undergoes full regeneration. In addition, it is peculiar because when growing, it contains vascularized cartilage. The differentiation of antler stem cells (ASCs) into chondrocytes while inducing endochondral extension of blood vessels is necessary to form antler vascularized cartilage. Therefore, antlers provide an unparalleled opportunity to investigate chondrogenesis, angiogenesis, and regenerative medicine. A study found that Galectin-1 (GAL-1), which can be used as a marker in some tumors, is highly expressed in ASCs. This intrigued us to investigate what role GAL-1 could play in antler regeneration. Methods: We measured the expression level of GAL-1 in antler tissues and cells by immunohistochemistry, WB and QPCR. We constructed antlerogenic periosteal cells (APCs, one cell type of ASCs) with the GAL-1 gene knocked out (APC^GAL-1−/−) using CRISPR-CAS9 gene editing system. The effect of GAL-1 on angiogenesis was determined by stimulating human umbilical vein endothelial cells (HUVECs) using APC^GAL-1−/− conditioned medium or adding exogenous deer GAL-1 protein. The effect of APC^GAL-1−/− on chondrogenic differentiation was evaluated compared with the APCs under micro-mass culture. The gene expression pattern of APC^GAL-1−/− was analyzed by transcriptome sequencing. Results: Immunohistochemistry revealed that GAL-1 was widely expressed in the antlerogenic periosteum (AP), pedicle periosteum (PP) and antler growth center. Western blot and qRT-PCR analysis using deer cell lines further supports this result. The proliferation, migration, and tube formation assays of human umbilical vein endothelial cells (HUVECs) showed that the proangiogenic activity of APC^GAL-1−/− medium was significantly decreased (P < 0.05) compared with the APCs medium. The proangiogenic activity of deer GAL-1 protein was further confirmed by adding exogenous deer GAL-1 protein (P < 0.05). The chondrogenic differentiation ability of APC^GAL-1−/− was impeded under micro-mass culture. The terms of GO and KEGG enrichment of the differentially expressed genes (DEGs) of APC^GAL-1−/− showed that down-regulated expression of pathways associated with deer antler angiogenesis, osteogenesis and stem cell pluripotency, such as the PI3K-AKT signaling pathway, signaling pathways regulating pluripotency of stem cells and TGF-β signaling pathway. Conclusions: Deer GAL-1, has strong angiogenic activity, is widely and highly expressed in deer antler. The APCs can induce angiogenesis by secreting GAL-1. The knockout of GAL-1 gene of APCs damaged its ability to induce angiogenesis and differentiate into chondrocytes. This ability is crucial to the formation of deer antler vascularized cartilage. Moreover, Deer antlers offer a unique model to explore explore how angiogenesis at high levels of GAL-1 expression can be elegantly regulated without becoming cancerous. Graphical

The hair follicle dermal papilla is critical for hair generation and de novo regeneration. When cultured in vitro, dermal papilla cells from different species demonstrate two distinguishable growth patterns under the conventional culture condition: a self-aggregative three dimensional spheroidal (3D) cell pattern and a two dimensional (2D) monolayer cell pattern, correlating with different hair inducing properties. Whether the loss of self-aggregative behavior relates to species-specific differences or the improper culture condition remains unclear. Can the fixed 2D patterned dermal papilla cells recover the self-aggregative behavior and 3D pattern also remains undetected. Here, we successfully constructed the two growth patterns using sika deer (Cervus nippon) dermal papilla cells and proved it was the culture condition that determined the dermal papilla growth pattern. The two growth patterns could transit mutually as the culture condition was exchanged. The fixed 2D patterned sika deer dermal papilla cells could recover the self-aggregative behavior and transit back to 3D pattern, accompanied by the restoration of hair inducing capability when the culture condition was changed. In addition, the global gene expressions during the transition from 2D pattern to 3D pattern were compared to detect the potential regulating genes and pathways involved in the recovery of 3D pattern and hair inducing capability.

Articular cartilage has a low capacity for self-regeneration. Therefore, stem cell therapy has been proposed and is gaining momentum. Antler can self-repair and its cartilaginous tissue can grow at an unprecedented rate (Up to 2 cm/day) and antler regeneration is based on antler stem cells. Therefore, we predicted that antler stem cells or their paracrine factors might be a good source for promoting cartilage repair. In this study, we prepared conditioned medium from antler stem cells (ASC-CM) and evaluated its effect. We implanted the medium into rat cartilage defects and assessed its capacity to promote cartilage repair. The protein composition of ASC-CM was analyzed using via DIA assay. ASC-CM can strongly promote chondrocyte proliferation in vitro; it significantly up-regulates the expression of chondrogenesis-related genes (Aggrecan, Col II, and Sox-9) and promotes glycosaminoglycan formation and type II collagen deposition in cartilaginous tissue; meanwhile up-regulate the apoptosis suppressor gene NAMPT and down-regulate apoptosis gene BAX. In vivo, cartilage defect in rats was significantly repaired using ASC-CM. The compositions of ASC-CM were got and some key proteins such as S100A4 were identified by bioinformatics analysis. The ASC-CM can promote the proliferation of rat-chondrocytes, maintain the chondrocyte phenotype, and inhibit apoptosis of rat-chondrocytes. Therefore, it can promote cartilage repair.

Periosteum is essential for bone regeneration and damage repair in mammals. Most species of deer family (Cervidae) develop two kinds of special periosteum, antler periosteum and pedicle periosteum, both supporting the complete regeneration of antler. Antler is the bone organ with the fastest growth rate in mammals. Along with the fast growth of antler, its external tissues such as blood vessels, nerves and the covering skin also grow rapidly. Currently, it is still unclear whether antler periosteum contributes to the fast growth of antler and how. It is also unclear why the regenerative capacity of antler periosteum is weaker than that of pedicle periosteum. In this study, the in vitro culture system for antler periosteal cells (AnPC) was constructed for the first time using the mid-beam antler periostea during antler fast-growth period. According to our results, the cultured AnPC expressed classical MSC markers, consistent with the pedicle periosteal stem cells (PPSC). However, the fluorescence intensities of the MSC markers on AnPC were significantly weaker than those on PPSC. In addition, AnPC showed much lower proliferation rates than PPSC. The proliferation rates of the AnPC also gradually decreased after successive passages, while the proliferation rates of the pedicle periosteal stem cells remained unchanged. These findings may partially explain the weaker regenerative capacity of antler periosteum. Further comparative global gene analysis revealed clearly the different gene expressed patterns between AnPC and PPSC. AnPC may mainly function on promoting angiogenesis, nerve growth and intramembrane bone formation during antler regeneration, whereas PPSC may primarily be involved in androgen signaling receptor pathway and PI3K-Akt signaling pathway and function on maintaining stem cell renewal.