Self‐Assembled Nanoparticles with Well‐Defined Oligosaccharide Promote Osteogenesis by Regulating Golgi Stress Response
Introduction Osteoporosis is a form of osteolytic disease caused by an imbalance in bone homeostasis, with diminished osteoblastic bone formation and augmented osteoclast activity resulting in reduced bone mass.  Globally, osteoporosis results in more than 8.9 million fractures annually, indicating that an osteoporotic fracture occurs approximately every 3 seconds. This requires prolonged medication and physical care, posing a burden on patients and healthcare systems.  While previous research has made significant contributions to osteoporosis treatment, recent research has shown that among the two main types of drugs used in pharmacotherapy, synthetic metabolic drugs have greater anti‐fracture efficacy and a larger increase in bone density compared to anti‐absorptive drugs. [ , , ] However the effects of anabolic drugs are short‐lived, and side effects such as atypical femoral fractures, cardiovascular events, colorectal cancer, and other cancers have been reported. [ , , , ] This emphasizes the need for multiple drug choices with satisfactory efficacy, high safety, and fewer side effects for osteoporosis treatment. The extracellular matrix (ECM) not only provides a robust scaffold for skeletal cells but also harbors promise for osteoblast performance. To address the aforementioned clinical imperative, here we present an approach capitalizing on the untapped potential of ECM constituents to recover bone health. Within the whole matrix molecule network, glycosaminoglycans (GAGs), distinguished by their linear polysaccharide nature and substantial molecular weight (MW), emerge as key orchestrators of cellular processes. Among the GAGs, chondroitin sulfate polysaccharides (CS) contribute an astonishing 90% of all GAGs in the skeleton matrix. It is composed of repeating disaccharide units consisting of D‐glucuronic acid (GlcA) and N ‐acetyl‐D‐galactosamine (GalNAc). CS deficiencies can lead to an imbalance in bone homeostasis in humans, as seen in Temtamy Preaxial Brachydactyly Syndrome accompanied with developmental delay and abnormal bone morphology.  Mice with impaired CS synthesis exhibit chondrodysplasia, progression of finger bifurcation, delayed endochondral osteogenesis, and decreased bone density.  However, the effect of CS on the regulation of bone homeostasis and its therapeutic potential in osteoporosis continues to linger within the realm of insufficient exploration. Moreover, the application of CS in vivo has been limited mainly because of the inherent defects in animal extraction materials, including broad MW distribution, batch differences, and protein contamination. As a high‐MW polysaccharide, natural CS adopts a random‐coil configuration in solution,  in which most of the functional polysaccharide can be buried inside losing the opportunity to interact with other molecules. To address these concerns, we have developed a novel approach utilizing chondroitin sulfate‐derived tetrasaccharide nanoparticles (CS4‐NP). These nanoparticles are designed to overcome the limitations of traditional CS by offering a precise molecular structure and consistent quality. The CS4‐NP are synthesized through a controlled process that ensures a narrow MW distribution and minimal batch‐to‐batch variability. This controlled synthesis allows for a more predictable and effective interaction with biological systems. Our study aims to investigate the significantly lower levels of CS in the serum of aged individuals diagnosed with osteoporosis and to demonstrate the therapeutic potential of CS4‐NP in restoring bone health. We hypothesize that CS4‐NP, with their defined structure and enhanced bioavailability, will effectively promote osteogenesis by restoring the ECM microenvironment and fostering osteoblast activity in osteoporotic mouse models. Furthermore, we have elucidated the underlying osteogenesis mechanism of this approach through the ATF4‐CSE signaling axis, which operates under the Golgi stress response. These findings provide a foundation for the future development of therapeutic strategies for osteoporosis with oligosaccharide‐based nanomaterials. 

Results 2.1 Abnormal Low Levels of CS in Patients with Osteoporosis and Ovariectomy‐Induced Osteoporosis Mouse Model Changes in the amount and structure of GAGs in the blood have been linked to the development of human diseases, with reduced levels often being indicative of disease onset.  To unravel the relationship between CS and osteoporosis, we analyzed human serum and mouse bone samples. The relevant parameters were evaluated in 100 eligible subjects. The participants with usable bone mineral density (BMD) values were divided into the following three groups according to the WHO criteria: healthy, osteopenia, and osteoporosis.  The osteoporosis group was further divided into the fracture osteoporosis and non‐fracture osteoporosis subgroups ( Figure ). The study participants had a median age of 69.45 (range: 61–81) (Table , Supporting Information) and a median serum CS level of 40.77 ng mL −1 (range: 22.04‐149.6). As depicted in Figure , serum CS levels were extremely lower in the osteopenia and osteoporosis groups compared to that in the normal control group (43.81 and 32.11 vs 111.4 ng mL −1 ). Figure presented scatter plots illustrating the correlation between serum CS levels and lumbar BMD, femoral neck BMD, and total hip BMD. The results showed a significant positive correlation between serum CS levels and all three BMD measurements (r  =  0.7084, P < 0.0001; r  =  0.6521, P < 0.0001; and r  =  0.5222, P < 0.0001, respectively). These findings demonstrated that higher levels of serum CS are associated with higher BMD at these sites. In addition, we assessed the diagnostic performance of CS in distinguishing between individuals with osteopenia or osteoporosis and normal controls. The area under the curve (AUC) was calculated to evaluate the accuracy of this discrimination. The AUC was 0.9650 (95% CI: 0.9267–1.000) for the osteopenia group and 0.8304 (95% CI: 0.7180–0.9428) for the osteoporosis group, indicating that serum CS levels can effectively differentiate patients with osteopenia or osteoporosis from healthy individuals (Figure ). These results highlighted the potential of serum CS as a valuable biomarker for assessing bone health and diagnosing osteoporosis. The strong correlation between CS levels and BMD measurements, as well as the high diagnostic accuracy demonstrated in this study, suggested the clinical utility of CS as a non‐invasive and easily accessible tool for osteoporosis screening and management. We examined CS synthase expression in the bones of mice with osteoporosis induced by ovariectomy (OVX) to compare it with normal controls (Figure ). In the osteoporotic mice, the expressions of C4st1 , C4st2 (4‐sulphated chondroitin), and Galnac4s6st (4,6‐sulphated chondroitin) were particularly down‐regulated. Analysis of the collected mouse bone samples revealed that the expression of CS in osteoporosis models decreased with the number of osteoblasts (OSTERIX‐positive region)  (Figure ). These results supported that the CS levels were reduced in both osteoporosis patients and mouse models. 2.2 Design and Preparation of CS4‐NP The correlation of osteoporosis with abnormally low levels of CS inspired us to explore the possibility of using CS as a potential treatment for osteoporosis. Given concerns about the reliability and safety of animal‐sourced natural CS, which limits its use as a clinical drug, there is a need to propose an alternative material that not only overcomes the drawbacks of CS but also exhibits similar properties ( Figure ). As a polysaccharide with high‐Mw, CS has been considered to adopt a random‐coil configuration in solution, in which most of the functional polysaccharide can be buried inside losing the opportunity to interact with other molecules. However, the polymeric properties of CS, including the high‐Mw, size, and hydrodynamic radius are considered to be helpful for the molecule to perform its function. Thus, in order to develop a new type of material that learns from CS, behaves like CS, and also overcomes the drawbacks of CS, we hypothesize that a new small molecule that has the functional repeating oligosaccharides of CS and the ability to self‐assemble into nanoparticles similar to CS, has the potential to be employed for osteoporosis treatment. Thus, an amphiphilic conjugate of CS4 and a short peptide has been designed and prepared. Here, fluorenyl‐9‐methoxycarbonyl‐L‐tyrosine (Fmoc‐Y) was chosen because its self‐assembling ability, forming π−π interactions between Fmoc groups and side‐chain phenyl rings.  Meanwhile, it is a simple bioinspired building block for the fabrication of functional materials and it has the potential to be applied to other functional scaffolds in the near future.  We employed a chemoenzymatic approach to prepare large amounts of chondroitin sulfate tetrasaccharide (CS4) (Figure ) and further modified it with Fmoc‐Y.  The synthesis route is simple, and cost‐effective and the obtained molecule can be widely used in vivo. Detailedly, large amounts of desulfated CS4 were generated through efficient enzymatic degradation of desulfated CS. Then azidation on the reducing end of desulfated CS4 was performed followed by click reaction with alkyne‐modified Fmoc‐Y, then sulfation on 6‐OH, resulting in CS4‐Y‐Fmoc conjugate. The successful formation of the CS4‐Y‐Fmoc conjugate was proven by using 1 H nuclear magnetic resonance spectroscopy ( 1 H NMR) (Figure , Supporting Information) and mass spectrometry (Figures , Supporting Information). Due to its amphiphilic nature, the CS4‐Y‐Fmoc conjugate self‐assembled into a nanoparticle (CS4‐NP) at the concentration of 1 mg mL −1 , wherein CS4 could be effectively expressed on the hydrophilic shell (Figure ). The CS4‐NP had a hydrated diameter of 86 ± 17 nm (Figure ) in solution, as determined through dynamic light scattering and zeta potential measurements (Figure ). Transmission electron microscopy (TEM) images indicated that the resulting CS4‐NP was spherical, with an approximately 44 nm diameter (Figure ), the discrepancy between TEM and DLS results can be understood by a polyelectrolyte effect due to the negatively charged CS4 corona.  Experimental results demonstrated that the CS4‐NP exhibited good stability in both water and simulated body fluid (Figure , Supporting Information). As experimental control groups, we also generated CSOs (chondroitin sulfate oligosaccharides), including chondroitin sulfate disaccharide (CS2), CS4, and chondroitin sulfate hexasaccharide (CS6) (see Supplemental Information II. for details). It is worth mentioning that, although CS4 can be made by chemical synthesis and other methods, it was challenging to prepare sufficient quantities of the CS4 with precise chemical structure including stereo‐ and regio‐selectivity for nanomedicine investigation. Subsequently, an in vitro study was conducted to investigate the regulatory effect of CS4‐NP on osteoblast differentiation. Enhanced matrix formation and improved mineralized matrix deposition were observed in cells treated with CS4‐NP compared to those treated with CS and CSO, as evidenced by alkaline phosphatase (ALP) staining, Alizarin red (AZR) staining, and real‐time PCR analysis at day 7 and day 14 (Figure ). These preliminary findings suggest the potential of CS4‐NP to promote osteoblast differentiation in vitro. 2.3 Enhanced Osteogenic Activity of Osteoblasts and Bone Mass by CS4‐NP To investigate the effects of CS4‐NP on ECM remodeling, we analyzed two major extracellular components of the bone ECM: collagen I (COL1) and non‐collagenous protein osteocalcin (OCN), which are pivotal in regulating bone mineralization.  The expression of Ocn and Col1 was found to be time‐dependent and concentration‐dependent in response to the nanoparticle treatment. CS4‐NP noticeably increased the expression of Ocn and Col1 compared to the control group at 3 h, 12 h, and 7 days of co‐culture ( Figure ). At the concentration of 100 µg mL −1 , protein expression levels showed a similar trend. The CS4‐NP group exhibited higher fluorescence intensity of OCN and COL1 than the control group, suggesting that CS4‐NP promotes ECM formation in vitro (Figure ). Building upon the understanding of the in vitro effects of nanoparticles, we now delve into their potential to enhance bone formation in vivo. Intravenous injections were administered at a dosage of 10 mg kg −1 body weight on a thrice‐weekly basis for a total of 8 weeks to the experimental cohorts. Micro‐CT scanning of the distal femur of mice showed increased trabecular bone volume / total volume (BV/TV), trabecular number (Tb. N.), trabecular thickness (Tb. Th.) and BMD, while decreased at trabecular separation (Tb. Sp.) in CS4‐NP‐treated mice, compared to those in NaCl‐treated, CS‐treated or CS4‐treated mice (Figure ). A similar bone mass increase phenotype of trabecular bone was detected in both male and female mice (Figure , Supporting Information). CS4‐NP highly increased OCN and COL1 expression in bone matrix of the primary and secondary ossification center compared with CS and CS4 (Figure ). Increased bone formation rates and expression of new collagen were also observed (Figure , Supporting Information). The drug, injected through the mice's tail vein, was rapidly distributed to organs throughout the body, including bone tissue (Figure , Supporting Information). Importantly, CS4‐NP did not exhibit any obvious toxicity in vivo, as confirmed through the analysis of serum biochemical markers such as ALT, AST, Glu‐G, and UREA (Figure , Supporting Information). Additionally, pathological alterations in vital organs, including the heart, lungs, liver, spleen, and kidneys, were not detected following treatment with CS4‐NP, as evidenced by Figure (Supporting Information). Our findings revealed that the presence of CS4‐NP facilitates the deposition of abundant collagen and matrix protein within the ECM, resulting in an increased3 bone mass. Moreover, CS4‐NP fostered the establishment of a stromal microenvironment characterized by superior biocompatibility and significantly enhanced osteoinductivity. These results underscored the remarkable potential of CS4‐NP in promoting osteoblast activity and facilitating bone formation in vivo. 2.4 Protective and Therapeutic Effects on OVX‐Induced Osteoporosis in Mice by CS4‐NP In light of the results on bone formation, we aimed to investigate the therapeutic potential of CS4‐NP in low‐bone‐mass diseases, using an ovariectomized mouse model that closely mimics the bone loss observed in postmenopausal women. To accomplish this, 8‐week‐old female mice were ovariectomized and left untreated for 8 weeks before treatment with CS4‐NP at a dose of 10 mg kg −1 body weight every 3 days for an additional 12 weeks ( Figure ). At the 2‐month mark following OVX surgery, female mice exhibited a noticeable decrease in bone mass compared to the SHAM group. However, Micro‐CT scanning revealed a considerable improvement in bone mass among mice treated with CS4‐NP compared to those treated with NaCl (Figure ). Quantitative analysis demonstrated an enhanced BV/TV and increased Tb. N., Tb. Th., and higher BMD in CS4‐NP‐treated mice, along with a reduction in SMI and Tb. Sp., when compared to those treated with NaCl, CS, and PTH (the classic drug for osteoporosis treatment) (Figure ; Figure , Supporting Information). Furthermore, histological examination using hematoxylin and eosin (H&E) staining illustrated improved bone microstructure in the femurs of CS4‐NP‐treated mice (Figure ). Comparable positive effects on bone trabeculae and bone mass were also observed in the vertebrae (Figure , Supporting Information). Additionally, CS4‐NP suggested a preventive effect when administered immediately after OVX, effectively inhibiting bone mass loss (Figures , Supporting Information). These findings confirmed the notion that CS4‐NP can effectively enhance bone mass in the context of oophorectomy‐induced osteoporosis. Evaluating cortical bone parameters is crucial for assessing bone quality and fracture risk. Mice treated with CS4‐NP showed increased BV/TV and cortical thickness compared to osteoporotic mice ( Figure ). Dynamic analysis revealed a higher mineral appositional rate (MAR) and bone formation rate per unit of bone surface area (BFR/BS) in CS4‐NP‐treated mice at the tissue level (Figure ). Elevated levels of newly synthesized collagen were observed in these mice through Goldner Masson staining. Additionally, treatment with CS4‐NP led to increased calcium salt content and improved mechanical strength as indicated by a significant increase in maximum load, stiffness, and maximum energy absorption (Figure ). Given that the maintenance of bone homeostasis hinges on the equilibrium between osteoblast‐mediated bone formation and osteoclast‐driven bone resorption, we also investigated the impact of CS4‐NP therapy on osteoclast numbers and activity. Notably, our findings indicate that CS4‐NP did not induce significant alterations in either the quantity or function of osteoclasts (Figure , Supporting Information). This implies that the observed increase in bone mass and enhancement in bone mechanical strength in CS4‐NP‐treated mice may primarily stem from augmented bone formation rather than inhibition of osteoclastic bone resorption. According to our research findings, CS4‐NP has demonstrated significant efficacy in reversing bone loss and structural deterioration in ovariectomized mice compared to full‐length CS and PTH. Specifically, it effectively promotes the process of bone formation, leading to increased bone density, as well as enhancing bone microstructure and overall biomechanical performance. 2.5 Promoting Effect of CS4‐NP Induced Golgi Stress Response on Bone Formation To elucidate the bone‐promoting mechanism of CS4‐NP, we compared bone tissue treated with CS4‐NP by RNA‐seq analysis (Figure , Supporting Information). Using the STRING protein interaction database,  compared with the control group, the increased genes formed a gene network centered on Col1a1, Col2a1, Matn1, and Sp7 ( Figure ). Meanwhile, Golgi cisternae were detected in the Gene Ontology enrichment analysis (Figure ). These findings imply an enhanced function of the Golgi apparatus, as the major manufactory for synthesizing collagen, glycosaminoglycans, and protein structures. TEM images revealed morphological changes in the Golgi apparatus of osteoblasts treated with CS4‐NP, indicating structural enlargement (Figure ). Recent studies have shown the vital role played by the Golgi apparatus in a range of cellular processes, including signal transduction, intracellular matrix secretion, and the maintenance of cell homeostasis.  The structural integrity of the Golgi apparatus is closely intertwined with its functional capabilities in these processes.  Moreover, changes in the morphology of the Golgi apparatus are often utilized as reliable markers of Golgi stress response and serve as a homeostatic mechanism initiated by the Golgi apparatus to enhance its function under stress or functional deficiency.  Therefore, our aim was to investigate whether CS4‐NP affected ECM remodeling and its potential to enhance bone formation by modulating the Golgi stress response. Undertaken to explore the influence of CS4‐NP on the Golgi apparatus and stress response, our in vitro experiments demonstrated the efficient internalization of CS4‐NP by cells (Figure , Supporting Information). The uptake was primarily facilitated through caveolin‐mediated endocytosis, as indicated by a significant reduction in internalization following pretreatment with genistein—a specific inhibitor of this process  (Figure ; Figure , Supporting Information). Subsequent organelle colocalization, enzyme activity, and lysosome function inhibition experiments further confirmed the Golgi‐biased localization of the nanoparticles (Figure ; Figures , Supporting Information). These results align with the understanding that caveolin‐mediated endocytosis directs substances toward the Golgi‐endoplasmic reticulum network, bypassing lysosomal degradation. [ , , ] Additionally, we found that CS4‐NP maintains their structural integrity within biological systems, as evidenced by Figure (Supporting Information), indicating their stability and potential for nanomedicine applications. The protein GM130, a recognized cis‐Golgi marker, was used to visually examine the perinuclear organization of the Golgi.  Incubation of MC3T3‐E1 cells with Golgi stressors, monensin (MON), and brefeldin A (BFA),  could intensively reduce the GM130 expression and induce Golgi rupture (Figure ). These stressors have been shown to cause Golgi stress and impair the structure and function of the Golgi apparatus, culminating in cell apoptosis, which is distinct from the Golgi stress response.  However, the addition of CS4‐NP did not disrupt the Golgi structure, which remained tightly surrounded by the nucleus and was slightly enlarged but compact (Figure ). Notably, CS4‐NP substantially increased the expression of Golgi stress response markers Acbd3, Atp2c, and Golga (Figure ), while no substantial change was noted in the genes associated with apoptosis (Figure , Supporting Information). Subsequently, we investigated whether inhibition of Golgi function would prevent the osteogenic effects of CS4‐NP. After disrupting Golgi function with BFA, the osteogenic effects of CS4‐NP appeared to diminish, and the secretion of ALP decreased (Figure ). Based on these observations, it was believed that CS4‐NP induced mild Golgi stress response without triggering cell apoptosis. In this context, CS4‐NP had the ability to regulate Golgi homeostasis and promote the process of osteogenesis. 2.6 Activation of the Downstream ATF4‐CSE Pathway on Stimulation of Osteoblast Function by CS4‐NP Next, we sought to explore the downstream signal transduction by which CS4‐NP stimulated Golgi stress response affects bone metabolism. Interestingly, it is reported that the Golgi stress response could promote Cystathionine‐γ‐lyase (CSE) expression through the activation of Activating transcription factor 4 (ATF4), a transcription factor in the regulation of osteoblast differentiation.  CSE is the primary enzyme responsible for synthesizing hydrogen sulfide (H2S) in osteoblasts, which could exert a significant role in bone.  Data from MC3T3‐E1 cells showed that CS4‐NP noticeably upregulated the expression of ATF4 and CSE ( Figure ). Moreover, in wild‐type (WT) mice, CS4‐NP promoted ALP activity (Figure ) and the expression of osteogenic genes (Alp, Col1a1, and Ocn) as well as bone proteoglycan genes (Acan, Bgn, and Dcn) (Figure ). Conversely, by using pre‐osteoblasts from CSE‐KO mice, the bone‐promoting effects of CS4‐NP did not work when CSE expression was suppressed (Figure ) (Figure , Supporting Information). Similar results were found in bone tissue in vivo (Figure ). The data indicates that CS4‐NP affects the Golgi stress response through ATF4/CSE signaling pathways, which are recognized for their role in regulating pre‐osteoblast differentiation and bone formation. A schematic representation of this operational model is depicted in Figure based on these findings. 

Abnormal Low Levels of CS in Patients with Osteoporosis and Ovariectomy‐Induced Osteoporosis Mouse Model Changes in the amount and structure of GAGs in the blood have been linked to the development of human diseases, with reduced levels often being indicative of disease onset.  To unravel the relationship between CS and osteoporosis, we analyzed human serum and mouse bone samples. The relevant parameters were evaluated in 100 eligible subjects. The participants with usable bone mineral density (BMD) values were divided into the following three groups according to the WHO criteria: healthy, osteopenia, and osteoporosis.  The osteoporosis group was further divided into the fracture osteoporosis and non‐fracture osteoporosis subgroups ( Figure ). The study participants had a median age of 69.45 (range: 61–81) (Table , Supporting Information) and a median serum CS level of 40.77 ng mL −1 (range: 22.04‐149.6). As depicted in Figure , serum CS levels were extremely lower in the osteopenia and osteoporosis groups compared to that in the normal control group (43.81 and 32.11 vs 111.4 ng mL −1 ). Figure presented scatter plots illustrating the correlation between serum CS levels and lumbar BMD, femoral neck BMD, and total hip BMD. The results showed a significant positive correlation between serum CS levels and all three BMD measurements (r  =  0.7084, P < 0.0001; r  =  0.6521, P < 0.0001; and r  =  0.5222, P < 0.0001, respectively). These findings demonstrated that higher levels of serum CS are associated with higher BMD at these sites. In addition, we assessed the diagnostic performance of CS in distinguishing between individuals with osteopenia or osteoporosis and normal controls. The area under the curve (AUC) was calculated to evaluate the accuracy of this discrimination. The AUC was 0.9650 (95% CI: 0.9267–1.000) for the osteopenia group and 0.8304 (95% CI: 0.7180–0.9428) for the osteoporosis group, indicating that serum CS levels can effectively differentiate patients with osteopenia or osteoporosis from healthy individuals (Figure ). These results highlighted the potential of serum CS as a valuable biomarker for assessing bone health and diagnosing osteoporosis. The strong correlation between CS levels and BMD measurements, as well as the high diagnostic accuracy demonstrated in this study, suggested the clinical utility of CS as a non‐invasive and easily accessible tool for osteoporosis screening and management. We examined CS synthase expression in the bones of mice with osteoporosis induced by ovariectomy (OVX) to compare it with normal controls (Figure ). In the osteoporotic mice, the expressions of C4st1 , C4st2 (4‐sulphated chondroitin), and Galnac4s6st (4,6‐sulphated chondroitin) were particularly down‐regulated. Analysis of the collected mouse bone samples revealed that the expression of CS in osteoporosis models decreased with the number of osteoblasts (OSTERIX‐positive region)  (Figure ). These results supported that the CS levels were reduced in both osteoporosis patients and mouse models. 

Design and Preparation of CS4‐NP The correlation of osteoporosis with abnormally low levels of CS inspired us to explore the possibility of using CS as a potential treatment for osteoporosis. Given concerns about the reliability and safety of animal‐sourced natural CS, which limits its use as a clinical drug, there is a need to propose an alternative material that not only overcomes the drawbacks of CS but also exhibits similar properties ( Figure ). As a polysaccharide with high‐Mw, CS has been considered to adopt a random‐coil configuration in solution, in which most of the functional polysaccharide can be buried inside losing the opportunity to interact with other molecules. However, the polymeric properties of CS, including the high‐Mw, size, and hydrodynamic radius are considered to be helpful for the molecule to perform its function. Thus, in order to develop a new type of material that learns from CS, behaves like CS, and also overcomes the drawbacks of CS, we hypothesize that a new small molecule that has the functional repeating oligosaccharides of CS and the ability to self‐assemble into nanoparticles similar to CS, has the potential to be employed for osteoporosis treatment. Thus, an amphiphilic conjugate of CS4 and a short peptide has been designed and prepared. Here, fluorenyl‐9‐methoxycarbonyl‐L‐tyrosine (Fmoc‐Y) was chosen because its self‐assembling ability, forming π−π interactions between Fmoc groups and side‐chain phenyl rings.  Meanwhile, it is a simple bioinspired building block for the fabrication of functional materials and it has the potential to be applied to other functional scaffolds in the near future.  We employed a chemoenzymatic approach to prepare large amounts of chondroitin sulfate tetrasaccharide (CS4) (Figure ) and further modified it with Fmoc‐Y.  The synthesis route is simple, and cost‐effective and the obtained molecule can be widely used in vivo. Detailedly, large amounts of desulfated CS4 were generated through efficient enzymatic degradation of desulfated CS. Then azidation on the reducing end of desulfated CS4 was performed followed by click reaction with alkyne‐modified Fmoc‐Y, then sulfation on 6‐OH, resulting in CS4‐Y‐Fmoc conjugate. The successful formation of the CS4‐Y‐Fmoc conjugate was proven by using 1 H nuclear magnetic resonance spectroscopy ( 1 H NMR) (Figure , Supporting Information) and mass spectrometry (Figures , Supporting Information). Due to its amphiphilic nature, the CS4‐Y‐Fmoc conjugate self‐assembled into a nanoparticle (CS4‐NP) at the concentration of 1 mg mL −1 , wherein CS4 could be effectively expressed on the hydrophilic shell (Figure ). The CS4‐NP had a hydrated diameter of 86 ± 17 nm (Figure ) in solution, as determined through dynamic light scattering and zeta potential measurements (Figure ). Transmission electron microscopy (TEM) images indicated that the resulting CS4‐NP was spherical, with an approximately 44 nm diameter (Figure ), the discrepancy between TEM and DLS results can be understood by a polyelectrolyte effect due to the negatively charged CS4 corona.  Experimental results demonstrated that the CS4‐NP exhibited good stability in both water and simulated body fluid (Figure , Supporting Information). As experimental control groups, we also generated CSOs (chondroitin sulfate oligosaccharides), including chondroitin sulfate disaccharide (CS2), CS4, and chondroitin sulfate hexasaccharide (CS6) (see Supplemental Information II. for details). It is worth mentioning that, although CS4 can be made by chemical synthesis and other methods, it was challenging to prepare sufficient quantities of the CS4 with precise chemical structure including stereo‐ and regio‐selectivity for nanomedicine investigation. Subsequently, an in vitro study was conducted to investigate the regulatory effect of CS4‐NP on osteoblast differentiation. Enhanced matrix formation and improved mineralized matrix deposition were observed in cells treated with CS4‐NP compared to those treated with CS and CSO, as evidenced by alkaline phosphatase (ALP) staining, Alizarin red (AZR) staining, and real‐time PCR analysis at day 7 and day 14 (Figure ). These preliminary findings suggest the potential of CS4‐NP to promote osteoblast differentiation in vitro. 

Enhanced Osteogenic Activity of Osteoblasts and Bone Mass by CS4‐NP To investigate the effects of CS4‐NP on ECM remodeling, we analyzed two major extracellular components of the bone ECM: collagen I (COL1) and non‐collagenous protein osteocalcin (OCN), which are pivotal in regulating bone mineralization.  The expression of Ocn and Col1 was found to be time‐dependent and concentration‐dependent in response to the nanoparticle treatment. CS4‐NP noticeably increased the expression of Ocn and Col1 compared to the control group at 3 h, 12 h, and 7 days of co‐culture ( Figure ). At the concentration of 100 µg mL −1 , protein expression levels showed a similar trend. The CS4‐NP group exhibited higher fluorescence intensity of OCN and COL1 than the control group, suggesting that CS4‐NP promotes ECM formation in vitro (Figure ). Building upon the understanding of the in vitro effects of nanoparticles, we now delve into their potential to enhance bone formation in vivo. Intravenous injections were administered at a dosage of 10 mg kg −1 body weight on a thrice‐weekly basis for a total of 8 weeks to the experimental cohorts. Micro‐CT scanning of the distal femur of mice showed increased trabecular bone volume / total volume (BV/TV), trabecular number (Tb. N.), trabecular thickness (Tb. Th.) and BMD, while decreased at trabecular separation (Tb. Sp.) in CS4‐NP‐treated mice, compared to those in NaCl‐treated, CS‐treated or CS4‐treated mice (Figure ). A similar bone mass increase phenotype of trabecular bone was detected in both male and female mice (Figure , Supporting Information). CS4‐NP highly increased OCN and COL1 expression in bone matrix of the primary and secondary ossification center compared with CS and CS4 (Figure ). Increased bone formation rates and expression of new collagen were also observed (Figure , Supporting Information). The drug, injected through the mice's tail vein, was rapidly distributed to organs throughout the body, including bone tissue (Figure , Supporting Information). Importantly, CS4‐NP did not exhibit any obvious toxicity in vivo, as confirmed through the analysis of serum biochemical markers such as ALT, AST, Glu‐G, and UREA (Figure , Supporting Information). Additionally, pathological alterations in vital organs, including the heart, lungs, liver, spleen, and kidneys, were not detected following treatment with CS4‐NP, as evidenced by Figure (Supporting Information). Our findings revealed that the presence of CS4‐NP facilitates the deposition of abundant collagen and matrix protein within the ECM, resulting in an increased3 bone mass. Moreover, CS4‐NP fostered the establishment of a stromal microenvironment characterized by superior biocompatibility and significantly enhanced osteoinductivity. These results underscored the remarkable potential of CS4‐NP in promoting osteoblast activity and facilitating bone formation in vivo. 

Protective and Therapeutic Effects on OVX‐Induced Osteoporosis in Mice by CS4‐NP In light of the results on bone formation, we aimed to investigate the therapeutic potential of CS4‐NP in low‐bone‐mass diseases, using an ovariectomized mouse model that closely mimics the bone loss observed in postmenopausal women. To accomplish this, 8‐week‐old female mice were ovariectomized and left untreated for 8 weeks before treatment with CS4‐NP at a dose of 10 mg kg −1 body weight every 3 days for an additional 12 weeks ( Figure ). At the 2‐month mark following OVX surgery, female mice exhibited a noticeable decrease in bone mass compared to the SHAM group. However, Micro‐CT scanning revealed a considerable improvement in bone mass among mice treated with CS4‐NP compared to those treated with NaCl (Figure ). Quantitative analysis demonstrated an enhanced BV/TV and increased Tb. N., Tb. Th., and higher BMD in CS4‐NP‐treated mice, along with a reduction in SMI and Tb. Sp., when compared to those treated with NaCl, CS, and PTH (the classic drug for osteoporosis treatment) (Figure ; Figure , Supporting Information). Furthermore, histological examination using hematoxylin and eosin (H&E) staining illustrated improved bone microstructure in the femurs of CS4‐NP‐treated mice (Figure ). Comparable positive effects on bone trabeculae and bone mass were also observed in the vertebrae (Figure , Supporting Information). Additionally, CS4‐NP suggested a preventive effect when administered immediately after OVX, effectively inhibiting bone mass loss (Figures , Supporting Information). These findings confirmed the notion that CS4‐NP can effectively enhance bone mass in the context of oophorectomy‐induced osteoporosis. Evaluating cortical bone parameters is crucial for assessing bone quality and fracture risk. Mice treated with CS4‐NP showed increased BV/TV and cortical thickness compared to osteoporotic mice ( Figure ). Dynamic analysis revealed a higher mineral appositional rate (MAR) and bone formation rate per unit of bone surface area (BFR/BS) in CS4‐NP‐treated mice at the tissue level (Figure ). Elevated levels of newly synthesized collagen were observed in these mice through Goldner Masson staining. Additionally, treatment with CS4‐NP led to increased calcium salt content and improved mechanical strength as indicated by a significant increase in maximum load, stiffness, and maximum energy absorption (Figure ). Given that the maintenance of bone homeostasis hinges on the equilibrium between osteoblast‐mediated bone formation and osteoclast‐driven bone resorption, we also investigated the impact of CS4‐NP therapy on osteoclast numbers and activity. Notably, our findings indicate that CS4‐NP did not induce significant alterations in either the quantity or function of osteoclasts (Figure , Supporting Information). This implies that the observed increase in bone mass and enhancement in bone mechanical strength in CS4‐NP‐treated mice may primarily stem from augmented bone formation rather than inhibition of osteoclastic bone resorption. According to our research findings, CS4‐NP has demonstrated significant efficacy in reversing bone loss and structural deterioration in ovariectomized mice compared to full‐length CS and PTH. Specifically, it effectively promotes the process of bone formation, leading to increased bone density, as well as enhancing bone microstructure and overall biomechanical performance. 

Promoting Effect of CS4‐NP Induced Golgi Stress Response on Bone Formation To elucidate the bone‐promoting mechanism of CS4‐NP, we compared bone tissue treated with CS4‐NP by RNA‐seq analysis (Figure , Supporting Information). Using the STRING protein interaction database,  compared with the control group, the increased genes formed a gene network centered on Col1a1, Col2a1, Matn1, and Sp7 ( Figure ). Meanwhile, Golgi cisternae were detected in the Gene Ontology enrichment analysis (Figure ). These findings imply an enhanced function of the Golgi apparatus, as the major manufactory for synthesizing collagen, glycosaminoglycans, and protein structures. TEM images revealed morphological changes in the Golgi apparatus of osteoblasts treated with CS4‐NP, indicating structural enlargement (Figure ). Recent studies have shown the vital role played by the Golgi apparatus in a range of cellular processes, including signal transduction, intracellular matrix secretion, and the maintenance of cell homeostasis.  The structural integrity of the Golgi apparatus is closely intertwined with its functional capabilities in these processes.  Moreover, changes in the morphology of the Golgi apparatus are often utilized as reliable markers of Golgi stress response and serve as a homeostatic mechanism initiated by the Golgi apparatus to enhance its function under stress or functional deficiency.  Therefore, our aim was to investigate whether CS4‐NP affected ECM remodeling and its potential to enhance bone formation by modulating the Golgi stress response. Undertaken to explore the influence of CS4‐NP on the Golgi apparatus and stress response, our in vitro experiments demonstrated the efficient internalization of CS4‐NP by cells (Figure , Supporting Information). The uptake was primarily facilitated through caveolin‐mediated endocytosis, as indicated by a significant reduction in internalization following pretreatment with genistein—a specific inhibitor of this process  (Figure ; Figure , Supporting Information). Subsequent organelle colocalization, enzyme activity, and lysosome function inhibition experiments further confirmed the Golgi‐biased localization of the nanoparticles (Figure ; Figures , Supporting Information). These results align with the understanding that caveolin‐mediated endocytosis directs substances toward the Golgi‐endoplasmic reticulum network, bypassing lysosomal degradation. [ , , ] Additionally, we found that CS4‐NP maintains their structural integrity within biological systems, as evidenced by Figure (Supporting Information), indicating their stability and potential for nanomedicine applications. The protein GM130, a recognized cis‐Golgi marker, was used to visually examine the perinuclear organization of the Golgi.  Incubation of MC3T3‐E1 cells with Golgi stressors, monensin (MON), and brefeldin A (BFA),  could intensively reduce the GM130 expression and induce Golgi rupture (Figure ). These stressors have been shown to cause Golgi stress and impair the structure and function of the Golgi apparatus, culminating in cell apoptosis, which is distinct from the Golgi stress response.  However, the addition of CS4‐NP did not disrupt the Golgi structure, which remained tightly surrounded by the nucleus and was slightly enlarged but compact (Figure ). Notably, CS4‐NP substantially increased the expression of Golgi stress response markers Acbd3, Atp2c, and Golga (Figure ), while no substantial change was noted in the genes associated with apoptosis (Figure , Supporting Information). Subsequently, we investigated whether inhibition of Golgi function would prevent the osteogenic effects of CS4‐NP. After disrupting Golgi function with BFA, the osteogenic effects of CS4‐NP appeared to diminish, and the secretion of ALP decreased (Figure ). Based on these observations, it was believed that CS4‐NP induced mild Golgi stress response without triggering cell apoptosis. In this context, CS4‐NP had the ability to regulate Golgi homeostasis and promote the process of osteogenesis. 

Activation of the Downstream ATF4‐CSE Pathway on Stimulation of Osteoblast Function by CS4‐NP Next, we sought to explore the downstream signal transduction by which CS4‐NP stimulated Golgi stress response affects bone metabolism. Interestingly, it is reported that the Golgi stress response could promote Cystathionine‐γ‐lyase (CSE) expression through the activation of Activating transcription factor 4 (ATF4), a transcription factor in the regulation of osteoblast differentiation.  CSE is the primary enzyme responsible for synthesizing hydrogen sulfide (H2S) in osteoblasts, which could exert a significant role in bone.  Data from MC3T3‐E1 cells showed that CS4‐NP noticeably upregulated the expression of ATF4 and CSE ( Figure ). Moreover, in wild‐type (WT) mice, CS4‐NP promoted ALP activity (Figure ) and the expression of osteogenic genes (Alp, Col1a1, and Ocn) as well as bone proteoglycan genes (Acan, Bgn, and Dcn) (Figure ). Conversely, by using pre‐osteoblasts from CSE‐KO mice, the bone‐promoting effects of CS4‐NP did not work when CSE expression was suppressed (Figure ) (Figure , Supporting Information). Similar results were found in bone tissue in vivo (Figure ). The data indicates that CS4‐NP affects the Golgi stress response through ATF4/CSE signaling pathways, which are recognized for their role in regulating pre‐osteoblast differentiation and bone formation. A schematic representation of this operational model is depicted in Figure based on these findings. 

Discussion Despite the increasing global demand for osteoporosis treatment, progress in this field has been limited over the past few decades. Identifying new targets in the skeletal system, such as key molecules in the bone ECM, is crucial for regulating the microenvironment of osteoblasts, and treating bone diseases. However, because of the intricate nature coupled with the challenges of assembling and regulating the ECM, only a limited number of attempts have been made to therapeutically induce bone repair using ECM components.  In order to explore the treatment strategy of bone loss based on extracellular matrix molecules and avoid, the synthesis difficulties and side effects caused by the complex molecular structure, we identified a novel saccharide biomaterial (CS4‐NP) that can induce bone formation in osteoporotic mice. This function is achieved by selectively stimulating the Golgi stress response and regulating osteoblast activity by the Golgi‐ATF4‐CSE axis. Our study demonstrated an effective and straightforward strategy for unmasking oligosaccharides to precisely regulate the activity of pre‐osteoblasts. Considering the inherent complexity and tedious procedure associated with oligosaccharide development, especially bottom‐up chemical synthesis, which significantly limited the amount of molecule resources for biomaterial design, we have adopted a chemical enzymatic solution to obtain chondroitin sulfate oligosaccharide chains. Additionally, we have utilized click chemistry to rapidly assemble gram‐scale quantities of nanomaterials that can be further applied in vivo, surpassing the limitations of previous studies. Through this approach, we have discovered specific osteogenic regulatory activities in the obtained oligosaccharide chain fragments, which have been masked under the polymerized chains.  The tetrasaccharide repeating unit of CS has been effectively exhibited by nanomaterial design showing higher efficacy than CS polysaccharide itself, which makes the undruggable CS polysaccharide druggable. The defined molecular structure of CS4‐NP offers better stability and bioavailability compared to polymeric CS. Their uniform size and targeted delivery to the Golgi apparatus—a crucial regulator of osteogenic signaling—contribute to their superior efficacy in promoting pre‐osteoblast differentiation and bone matrix mineralization. Our data further validated the biological mechanism of CS4‐NP action. RNA‐sequencing and subsequent experimental analyses demonstrated that treatment with CS4‐NP induced aggregation near the Golgi apparatus through calveolin‐mediated endocytosis. This process led to the up‐regulation of genes involved in ECM formation, collagen assembly, Golgi apparatus functionality, and other cellular structures associated with executive function. Subsequently, CS4‐NP induced morphological changes in the Golgi apparatus, and recent studies have shown that the structural integrity of the Golgi apparatus influences signaling pathway transmission, regulation of intracellular matrix secretion, and maintenance of cell homeostasis. Here, CS4‐NP mimics glycosaminoglycan structure to activate ECM remodeling of pre‐osteoblasts and stimulate mild Golgi stress responses to enhance the Golgi apparatus function, demonstrating a controllable therapeutic potential mechanism. Targeting cell stress improves cell homeostasis, matrix collagen content, and mineralization.  The specific stimulating organelle stress response of CS4‐NP may have new significance. ECM improvement targeted by cellular organelle stress responses has been attempted in treatments on bone diseases, for example, in a short‐limb dwarf mouse model treated with carbamazepine, a compound that stimulates autophagy and proteasome degradation, thereby reducing misfolded protein accumulation in the ER and universal periodic activation.  The Golgi stress response, a novel cellular stress response, has gained attention in recent years. Its pivotal role in apoptosis, anti‐apoptosis, and machinery for GAG synthesis has been progressively unveiled, making it a captivating subject for scientific research in the field of biotechnology. However, the specific mechanisms involved in bone formation by cellular organelle stress responses have not been well elucidated. Tomohiko Murakami et al. reported that bone morphogenetic protein 2 (BMP‐2) induces mild ER stress, revealing the mechanism by which ER stress‐induced signaling and Golgi processing co‐mediate bone formation.  In our study, we demonstrated that CS4‐NP can also induce mild Golgi stress response, showing bone increase, including increased type I collagen and osteocalcin in the bone matrix, and increased osteoblast activity. The morphology showed that the CS4‐NP treated Golgi apparatus was intact and slightly swollen, which was different from the Golgi apparatus rupture in apoptosis induced by MON and BFA. In addition, we also verified the downstream conduction molecules of the Golgi stress response. ATF4, a member of the ATF/cAMP response element (CRE) binding protein family, is typically involved in altered CSE transcription by binding to its CRE under various stress conditions (including oxidative stress, nutrient deficiency, Golgi stress, etc.).  The Golgi stress response stimulates the ATF4‐activated CSE system and cellular homeostasis. Using transgenic CSE mice, we demonstrated that CS4‐NP‐mediated bone formation is mediated via the ATF4‐CSE pathway. There is an ample scope for further investigation into CS4‐NP. While substantial impacts of CS4‐NP on bone remodeling have been observed in rodents, it is essential to validate these effects in primates. Additionally, more investigation is needed to assess the suitability of these oligosaccharides as alternative pharmaceutical formulations, such as orally administered or long‐acting injectable drugs. These formulations have the potential to provide a safer and more efficacious alternative to current treatments for osteoporosis. In conclusion, our study not only identified CS as a promising target molecule for the effective treatment of osteoporosis but also revealed the great potential of oligosaccharide‐based nanoparticles in treating chronic metabolic diseases. Notably, our approach innovatively utilizes low MW glycosaminoglycan mimics to actively stimulate bone regeneration by restoring the optimal ECM microenvironment required for bone formation. These findings strongly suggest that oligosaccharide drug design at the nanoscale can be used autonomously as an effective and versatile method in drug development and disease treatment. 

Conclusion Our findings have identified CS as a new target molecule for treating OP and have revealed, for the first time, the functions of nanoparticles in the treatment of chronic metabolic disease. This paper proposes that with a chemically well‐defined structure containing an active tetrasaccharide of chondroitin sulfate and a short hydrophobic peptide, the nanomedicine in the form of nanoparticles (CS4‐NP) assembled by such a small molecule can perform exceptional osteogenic functions and promote bone mass in mouse osteoporosis models. The results suggest that oligosaccharides can be used as independent functional molecules for drug development and disease treatment and may be particularly beneficial in restoring the ECM in senescent diseases. Generally, we posit that this direct and clear approach may offer an alternative avenue for the development of biomaterials related to glycosaminoglycans, demonstrating significant potential for application in medical contexts. 

Experimental Section Clinical Volunteer and Blood Collection A total of 100 volunteers were recruited from the communities in Shanghai (Ethics approval number: 2019‐LCYJ‐004). The inclusion criteria of subjects were as follows: i) age over 59, ii) BMD and T scores with a double energy x line absorber (ge prodigy, USA) were measured between 11 and 14, iii) no systemic diseases including diabetes, kidney diseases, thyroid gland, malignant tumors, gastrointestinal diseases, iv) no systematic anti‐osteoporosis therapy before. Age, height, weight, and other basic information were also collected and provided in Table (Supporting Information). Based on T scores and clinical symptoms, the participants were divided into three groups: osteoporosis (BMD T ‐score ≤ ‐2.5) or osteopenia (BMD T ‐score > ‐2.5 and ≤ ‐1) and healthy ( T ‐score > ‐1.0). Among them, the osteoporosis group was divided into the fracture (at least one place, one body part reached the checkpoint, clinical comprehensive diagnosis of fracture) and non‐fracture. All those participants were fully informed about the aims of the research with written informed consent according to the recommendations of the Ethics Committee of Tongji Hospital, School of Medicine, Tongji, University. The procedures for obtaining blood samples from both animals and humans were authorized by the Institutional Animal Care and Use Committee (IACUC). Five milliliters of whole blood were obtained from each participant. Each sample was split independently by erythrocyte solution (Beyotime, Shanghai, China), centrifuged at 450 g for 10 min, and then liquid supernatant was collected. On the last day of the animal trail, mice were anesthetized with isoflurane, and blood was collected from the orbital vein. Serum was collected after centrifugation and stored at −80 °C in the refrigerator until use. Enzyme‐Linked Immunosorbent Assay (ELISA) The concentration of chondroitin sulfate in the blood supernatant was quantified using the Human Chondroitin Sulfate ELISA kit (Xi Tang, F00457) according to the manufacturer's instructions. The optical density per well was measured using a 450 nm microplate reader (DENLEY DRAGON Wellscan MK 3, Thermo) with wavelength correction set at 630 nm. Subsequently, the concentration of CS in each sample was determined based on the standard curve. qRT‐PCR Analysis The bone tissue was aseptically extracted and pre‐cooled with PBS. 1000ul of Trizol per well was added to a pre‐cooled EP tube, crushed with a grinder, and placed on ice for 30 min. Total RNA extraction was then carried out using Trizol reagent (Invitrogen, MA, USA). RNA quantification was performed at 260 nm using the Syn‐energy Neo2 HTS multimode enzyme labeler (BioTek, VT, USA), followed by cDNA synthesis utilizing a first‐strand cDNA synthesis kit (Roche, Basel, Switzerland). PCR analysis involved a reaction mixture prepared with SYBR green master mix (Roche) and analyzed using Roche's Light Cycler 96 PCR system. Each sample underwent three replicate reactions. Omit the grinding process for the cell sample, while maintaining all other procedures unchanged. Primer details are available in Table (Supporting Information). Preparation of CS4‐NP CS4‐Y‐Fomc was dissolved in water at a concentration of 1 mg mL −1 after sonication for 10 mins and stayed at room temperature. (The preparation of CS4‐Y‐Fomc see Supplemental Information II). Characterization of CS4‐NP Optical images of CS4‐NP were obtained by transmission electron microscopy (TEM, HT7800, Hitachi). The particle size and zeta potentials of CS4‐NP were measured using Zetasizer Nano ZS90 (Malvern Instruments). All sample solutions were filtered through Millipore filters (220 nm) to remove the dust and kept at 25 °C. Cell Culture and Osteogenic Induction The mouse pre‐osteoblast cells and MC3T3‐E1 Subclone 14 cells (Cell Bank, Chinese Academy of Sciences, China) were cultured in α‐MEM medium supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin‐streptomycin (Hyclone) until reaching approximately full confluence. Cells from passages 3–7 were utilized for experiments. Osteogenic induction involved seeding cells at a density of 2×10 4 cells per well in 24‐well plates followed by replacing the regular culture medium with osteogenic induction medium after an initial incubation period of 24 h. The new medium contained dexamethasone (10 nM), β‐glycerophosphate (5 mM), and ascorbic acid (50 µg mL −1 ) in α‐MEM with 1% penicillin‐streptomycin; it was refreshed every three days, and cell cultures lasted either seven or twenty‐one days before subsequent observation. Immunofluorescence Cells on the slide were fixed using 4% paraformaldehyde (PFA) for 30 min, underwent antigen retrieval in citrate buffer at room temperature for 1 h, and then blocked with a 10% goat serum solution at 37 °C for 1 h. Next, the cells were incubated overnight at 4 °C with primary antibodies diluted in PBS: anti‐Collagen Type I/COL1A1 antibody (Boster, PB0981,1:100), and anti‐GM130 (Abcam, ab133612,1:500). Subsequently, the slides were incubated with corresponding fluorophore‐conjugated antibodies for 1 h. DAPI was used to stain the nuclei. Microscopic images were acquired using a confocal microscope (Leica Q500MC), and ImageJ software (Version: 2.3.0/1.53q) was utilized for quantifying the fluorescence intensity of the samples. In the case of in vivo tissue samples, 4 µm thick sections were prepared and then subjected to dewaxing, rehydration with deionized water, and antigen retrieval. The staining procedure was performed following the same protocol as immunocytochemistry. The primary antibodies used were as follows: anti‐chondroitin sulfate antibody (CS‐56) (Abcam, ab11570, 1:100), anti‐SP7/Osterix antibody (Abcam, ab22552, 1:100), anti‐type I collagen/COLIAI antibody (Boster, PB0981, 1:100), and Anti‐osteocalcin antibody (Abcam, ab133612, 1:200). ALP and ARS Staining After osteogenic induction, cells were subjected to BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, C3206) and ARS solution (Sigma, A5533) staining according to the manufacturer's instructions. Briefly, the cells were fixed in 4% PFA for 30 min, washed, and then incubated in the ALP staining buffer for 10 min. For ARS analysis, the fixed cells were stained with a 0.1% ARS solution for 30 min. Plate images were captured using an Olympus microscope and quantitative data was analyzed using ImageJ. in vivo Bone Forming Male/female 8‐week‐old Hauschka Ha/ICR mice (SIPEIFU, China) were used in animal experiments following the guidelines of the Institutional Animal Care and Use Committees of Tongji University (Approval No. [2021]‐DW‐034). Twenty‐four mice were randomly divided into 4 groups:1) NaCl‐group (treated with NaCl); 2) CS group (treated with chondroitin sulfate polysaccharides, 10 mg kg −1 ); 3) CS4 group (treated with tetrasaccharide, 10 mg kg −1 ); 4) CS4‐NP group (treated with CS4‐NP nanoparticle, 10 mg kg −1 ). Each group was injected intravenously 3 times a week for 8w. Microcomputed Tomography Analysis The femur and fifth lumbar vertebra (L5) were extracted from mice, fixed in 4% paraformaldehyde for 48 h, and then examined using a Skyscan 1176 (Skyscan, Aartselaar, Belgium). The scanner operated at a voltage of 50 kV, current of 400 µA, and resolution of 8.88 µm per pixel. Trabecular bone parameters including Tb.BV/TV, BMD, Tb.Th, Tb.N, Tb.Sp, and SMI were evaluated within a specific region representing trabecular bone.  Toxicity Assays Mice were administered CS4‐NP at a dosage of 10 mg kg −1 , and the short‐ and long‐term toxicity on vital organs was assessed at multiple time points (1h, 5d, 14d, and 28d). Systematic collection of heart, liver, spleen, lung, kidney, and serum samples for analysis followed the administration. H&E staining was performed for histopathology, 28 days after administration. The expression of serum biochemical factors was detected by the Mindray automatic biochemical analyzer (Mindray, BS‐240VET, China). Osteoporosis Model Induction and Treatment Before the osteoporosis surgery, the abdominal surface hair of the mice was removed by shaving knife. Mice were first anesthetized with isoflurane and then continuously anesthetized with isoflurane. Under sterile conditions, surgical scissors were used to cut 0.5‐1 cm along the midline of the abdomen, then the skin and muscle were bluntly separated. Ligation was performed on the fallopian tubes above and below the uterine horn. Shorten the uterine horn and remove the ovaries. The adipose tissue was pushed back into the abdominal cavity, the laminae and muscle layers were sutured together, and the skin was sutured later. 8 weeks after the operation, the mice were randomly divided into 4 groups: 1) SHAM‐group (opened the abdomen but did not remove the ovaries. Administer a placebo); 2) OVX group (OVX and NaCl group); 3) CS group (OVX and CS group); 4) CS4‐NP group (OVX and CS4‐NP group). Each group was injected intravenously 3 times a week for 12 weeks and bone tissue and main organs were harvested for follow‐up analysis. Histological Analysis Mouse tissues were promptly fixed in 4% PFA for 24 h upon collection. Subsequently, the bone tissue samples experienced a 28‐day immersion in 10% EDTA solution with regular refreshment every 3 days. After undergoing dehydration, clarification, and waxing processes, the bone tissue was subsequently embedded in paraffin wax and sectioned to a thickness of 4 µm (Leica, rm2235, Dublin, Ireland). Following this, H&E (Wellbio, WH2145) and Goldner Masson (Solarbio, G1340‐7) staining procedures were performed to evaluate tissue structure and collagen deposition. After Goldner Masson staining, mature bone was stained blue and new bone was stained red, and the expression intensity of the red dye was quantified. Double Labeling Analysis A double labeling analysis was performed to evaluate the impact of CS4‐NP on the rate of cortical bone formation. Xylenol orange and calcein were administered at doses of 10 and 20 mg kg −1 , respectively, with a one‐week interval. After a 12 h period from the injection of calcein green, all mice were euthanized. The femurs were collected, fixed in a 4% PFA solution for 24 h, embedded in light‐cured resin (EXAKT 7200 VLC) for 13.5 h, and then sectioned to a controlled thickness between 15 and 20 µm using an EXAKT microsectioning and grinding system. Finally, the slides were stained with DAPI (Sigma) and imaged under a confocal microscope. Three‐Point Bending Test Improved three‐point bending devices were recently used in this study.  Before the formal experiment, the femur was wrapped with saline‐soaked gauze to prevent dehydration and stored in a small sealed freezer bag at ‐20 °C. Specifically, the femur, which exhibits a physiological curvature directed superiorly, was securely mounted onto a support bracket with a span of 6 mm. A bending load was then applied perpendicularly at the midpoint between the supports at a constant displacement rate of 1.0 mm s −1 until failure was observed. The ultimate force, stiffness, and energy absorption capacity were quantified based on the resulting load‐deformation curve. High‐Throughput RNA‐Seq and Data Analysis 8‐week‐old female mice underwent ovariectomy and were injected with CS4‐NP/NaCl. RNA from bone tissue was extracted and sequenced. mRNA isolation, cDNA synthesis, and library sequencing were done. Differential expression analysis was performed with a threshold of p < 0.05 and |log2 (fold change) | > 1. Volcano maps were generated using the “ggpubr” and “ggthemes” packages in R software to visualize the differential expression profiles. Venn diagrams were used to screen for differentially expressed genes. GO and KEGG analyses were conducted to gain insight into the biological effects of these genes, with annotation, visualization, and comprehensive databases. The STRING database was used to infer protein‐protein interactions in the DEGs enrichment pathway. Transmission Electron Microscope (TEM) Analysis Three representative right femurs from each experimental group were fixed in a 2.5% glutaraldehyde solution, followed by ethanol dehydration, embedding in Agar 100 epoxy resin (Agar Scientific, AGR1045), and preparation into 70 nm thin sections. Subsequently, these sections underwent staining with 2% uranyl acetate and lead citrate before examination and recording using a TEM (JOL‐JEM 1400 Plus, Japan Electron Optic, Tokyo, Japan) to visualize organelles including Golgi apparatus and endoplasmic reticulum. Cell Uptake Assay Cells were inoculated on 24‐well plates overnight at a density of 2 × 10 4 cells per dish. The cells were then treated with 100ug mL −1 fluorescent CS4‐NP for 4 h. In the study of the endocytosis pathway, endocytosis pathway inhibitors (genistein (MCE, HY‐14596); chlorpromazine (Sigma, C0982); rottlerin (MCE, HY‐18980)) were pre‐incubated for 2 h and then fluorescent CS4‐NP was added. Subsequently, the cells were fixed and the cytoskeleton was stained by phalloidine (Yeasen, 40734ES75,1:100), and finally observed and recorded by laser confocal microscopy. Golgi Stress Response Test Cells were inoculated on 24‐well plates overnight at a density of 2 × 10 4 cells per dish. The following day, monensin (MCE, HY‐N4302, 0.05 µM, 1 h); brefeldin A (MCE, HY‐16592100 10 µg mL −1 , 1 h), CS4‐NP or DMSO were added to cells for 1h. Then, cells were rinsed with PBS, harvested, and analyzed. In the Golgi inhibition test, the same concentration of brefeldin A was pretreated for 1 h, and then the cells were cultured in the osteogenic induction medium. Statistical Analysis The mean ± SD was utilized for data reporting. Statistical significance between the two groups was assessed using a two‐tailed Student's t ‐test, while differences among multiple groups were determined through one‐way ANOVA with Dunnett's multiple comparisons test. Proportional confidence intervals were calculated using the Wilson/Brown method. Statistical significance was considered at a P value of < 0.05. GraphPad Prism (Version 9.4.0, GraphPad Software Inc, CA, USA) and Origin (OriginLab, MA, USA) conducted the statistical analyses; the figure legend provides sample size ( n ) for each analysis. 

A total of 100 volunteers were recruited from the communities in Shanghai (Ethics approval number: 2019‐LCYJ‐004). The inclusion criteria of subjects were as follows: i) age over 59, ii) BMD and T scores with a double energy x line absorber (ge prodigy, USA) were measured between 11 and 14, iii) no systemic diseases including diabetes, kidney diseases, thyroid gland, malignant tumors, gastrointestinal diseases, iv) no systematic anti‐osteoporosis therapy before. Age, height, weight, and other basic information were also collected and provided in Table (Supporting Information). Based on T scores and clinical symptoms, the participants were divided into three groups: osteoporosis (BMD T ‐score ≤ ‐2.5) or osteopenia (BMD T ‐score > ‐2.5 and ≤ ‐1) and healthy ( T ‐score > ‐1.0). Among them, the osteoporosis group was divided into the fracture (at least one place, one body part reached the checkpoint, clinical comprehensive diagnosis of fracture) and non‐fracture. All those participants were fully informed about the aims of the research with written informed consent according to the recommendations of the Ethics Committee of Tongji Hospital, School of Medicine, Tongji, University. The procedures for obtaining blood samples from both animals and humans were authorized by the Institutional Animal Care and Use Committee (IACUC). Five milliliters of whole blood were obtained from each participant. Each sample was split independently by erythrocyte solution (Beyotime, Shanghai, China), centrifuged at 450 g for 10 min, and then liquid supernatant was collected. On the last day of the animal trail, mice were anesthetized with isoflurane, and blood was collected from the orbital vein. Serum was collected after centrifugation and stored at −80 °C in the refrigerator until use. 

The concentration of chondroitin sulfate in the blood supernatant was quantified using the Human Chondroitin Sulfate ELISA kit (Xi Tang, F00457) according to the manufacturer's instructions. The optical density per well was measured using a 450 nm microplate reader (DENLEY DRAGON Wellscan MK 3, Thermo) with wavelength correction set at 630 nm. Subsequently, the concentration of CS in each sample was determined based on the standard curve. 

The bone tissue was aseptically extracted and pre‐cooled with PBS. 1000ul of Trizol per well was added to a pre‐cooled EP tube, crushed with a grinder, and placed on ice for 30 min. Total RNA extraction was then carried out using Trizol reagent (Invitrogen, MA, USA). RNA quantification was performed at 260 nm using the Syn‐energy Neo2 HTS multimode enzyme labeler (BioTek, VT, USA), followed by cDNA synthesis utilizing a first‐strand cDNA synthesis kit (Roche, Basel, Switzerland). PCR analysis involved a reaction mixture prepared with SYBR green master mix (Roche) and analyzed using Roche's Light Cycler 96 PCR system. Each sample underwent three replicate reactions. Omit the grinding process for the cell sample, while maintaining all other procedures unchanged. Primer details are available in Table (Supporting Information). 

CS4‐Y‐Fomc was dissolved in water at a concentration of 1 mg mL −1 after sonication for 10 mins and stayed at room temperature. (The preparation of CS4‐Y‐Fomc see Supplemental Information II). 

Optical images of CS4‐NP were obtained by transmission electron microscopy (TEM, HT7800, Hitachi). The particle size and zeta potentials of CS4‐NP were measured using Zetasizer Nano ZS90 (Malvern Instruments). All sample solutions were filtered through Millipore filters (220 nm) to remove the dust and kept at 25 °C. 

The mouse pre‐osteoblast cells and MC3T3‐E1 Subclone 14 cells (Cell Bank, Chinese Academy of Sciences, China) were cultured in α‐MEM medium supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin‐streptomycin (Hyclone) until reaching approximately full confluence. Cells from passages 3–7 were utilized for experiments. Osteogenic induction involved seeding cells at a density of 2×10 4 cells per well in 24‐well plates followed by replacing the regular culture medium with osteogenic induction medium after an initial incubation period of 24 h. The new medium contained dexamethasone (10 nM), β‐glycerophosphate (5 mM), and ascorbic acid (50 µg mL −1 ) in α‐MEM with 1% penicillin‐streptomycin; it was refreshed every three days, and cell cultures lasted either seven or twenty‐one days before subsequent observation. 

Cells on the slide were fixed using 4% paraformaldehyde (PFA) for 30 min, underwent antigen retrieval in citrate buffer at room temperature for 1 h, and then blocked with a 10% goat serum solution at 37 °C for 1 h. Next, the cells were incubated overnight at 4 °C with primary antibodies diluted in PBS: anti‐Collagen Type I/COL1A1 antibody (Boster, PB0981,1:100), and anti‐GM130 (Abcam, ab133612,1:500). Subsequently, the slides were incubated with corresponding fluorophore‐conjugated antibodies for 1 h. DAPI was used to stain the nuclei. Microscopic images were acquired using a confocal microscope (Leica Q500MC), and ImageJ software (Version: 2.3.0/1.53q) was utilized for quantifying the fluorescence intensity of the samples. In the case of in vivo tissue samples, 4 µm thick sections were prepared and then subjected to dewaxing, rehydration with deionized water, and antigen retrieval. The staining procedure was performed following the same protocol as immunocytochemistry. The primary antibodies used were as follows: anti‐chondroitin sulfate antibody (CS‐56) (Abcam, ab11570, 1:100), anti‐SP7/Osterix antibody (Abcam, ab22552, 1:100), anti‐type I collagen/COLIAI antibody (Boster, PB0981, 1:100), and Anti‐osteocalcin antibody (Abcam, ab133612, 1:200). 

After osteogenic induction, cells were subjected to BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, C3206) and ARS solution (Sigma, A5533) staining according to the manufacturer's instructions. Briefly, the cells were fixed in 4% PFA for 30 min, washed, and then incubated in the ALP staining buffer for 10 min. For ARS analysis, the fixed cells were stained with a 0.1% ARS solution for 30 min. Plate images were captured using an Olympus microscope and quantitative data was analyzed using ImageJ. 

Male/female 8‐week‐old Hauschka Ha/ICR mice (SIPEIFU, China) were used in animal experiments following the guidelines of the Institutional Animal Care and Use Committees of Tongji University (Approval No. [2021]‐DW‐034). Twenty‐four mice were randomly divided into 4 groups:1) NaCl‐group (treated with NaCl); 2) CS group (treated with chondroitin sulfate polysaccharides, 10 mg kg −1 ); 3) CS4 group (treated with tetrasaccharide, 10 mg kg −1 ); 4) CS4‐NP group (treated with CS4‐NP nanoparticle, 10 mg kg −1 ). Each group was injected intravenously 3 times a week for 8w. 

The femur and fifth lumbar vertebra (L5) were extracted from mice, fixed in 4% paraformaldehyde for 48 h, and then examined using a Skyscan 1176 (Skyscan, Aartselaar, Belgium). The scanner operated at a voltage of 50 kV, current of 400 µA, and resolution of 8.88 µm per pixel. Trabecular bone parameters including Tb.BV/TV, BMD, Tb.Th, Tb.N, Tb.Sp, and SMI were evaluated within a specific region representing trabecular bone.  

Mice were administered CS4‐NP at a dosage of 10 mg kg −1 , and the short‐ and long‐term toxicity on vital organs was assessed at multiple time points (1h, 5d, 14d, and 28d). Systematic collection of heart, liver, spleen, lung, kidney, and serum samples for analysis followed the administration. H&E staining was performed for histopathology, 28 days after administration. The expression of serum biochemical factors was detected by the Mindray automatic biochemical analyzer (Mindray, BS‐240VET, China). 

Before the osteoporosis surgery, the abdominal surface hair of the mice was removed by shaving knife. Mice were first anesthetized with isoflurane and then continuously anesthetized with isoflurane. Under sterile conditions, surgical scissors were used to cut 0.5‐1 cm along the midline of the abdomen, then the skin and muscle were bluntly separated. Ligation was performed on the fallopian tubes above and below the uterine horn. Shorten the uterine horn and remove the ovaries. The adipose tissue was pushed back into the abdominal cavity, the laminae and muscle layers were sutured together, and the skin was sutured later. 8 weeks after the operation, the mice were randomly divided into 4 groups: 1) SHAM‐group (opened the abdomen but did not remove the ovaries. Administer a placebo); 2) OVX group (OVX and NaCl group); 3) CS group (OVX and CS group); 4) CS4‐NP group (OVX and CS4‐NP group). Each group was injected intravenously 3 times a week for 12 weeks and bone tissue and main organs were harvested for follow‐up analysis. 

Mouse tissues were promptly fixed in 4% PFA for 24 h upon collection. Subsequently, the bone tissue samples experienced a 28‐day immersion in 10% EDTA solution with regular refreshment every 3 days. After undergoing dehydration, clarification, and waxing processes, the bone tissue was subsequently embedded in paraffin wax and sectioned to a thickness of 4 µm (Leica, rm2235, Dublin, Ireland). Following this, H&E (Wellbio, WH2145) and Goldner Masson (Solarbio, G1340‐7) staining procedures were performed to evaluate tissue structure and collagen deposition. After Goldner Masson staining, mature bone was stained blue and new bone was stained red, and the expression intensity of the red dye was quantified. 

A double labeling analysis was performed to evaluate the impact of CS4‐NP on the rate of cortical bone formation. Xylenol orange and calcein were administered at doses of 10 and 20 mg kg −1 , respectively, with a one‐week interval. After a 12 h period from the injection of calcein green, all mice were euthanized. The femurs were collected, fixed in a 4% PFA solution for 24 h, embedded in light‐cured resin (EXAKT 7200 VLC) for 13.5 h, and then sectioned to a controlled thickness between 15 and 20 µm using an EXAKT microsectioning and grinding system. Finally, the slides were stained with DAPI (Sigma) and imaged under a confocal microscope. 

Improved three‐point bending devices were recently used in this study.  Before the formal experiment, the femur was wrapped with saline‐soaked gauze to prevent dehydration and stored in a small sealed freezer bag at ‐20 °C. Specifically, the femur, which exhibits a physiological curvature directed superiorly, was securely mounted onto a support bracket with a span of 6 mm. A bending load was then applied perpendicularly at the midpoint between the supports at a constant displacement rate of 1.0 mm s −1 until failure was observed. The ultimate force, stiffness, and energy absorption capacity were quantified based on the resulting load‐deformation curve. 

8‐week‐old female mice underwent ovariectomy and were injected with CS4‐NP/NaCl. RNA from bone tissue was extracted and sequenced. mRNA isolation, cDNA synthesis, and library sequencing were done. Differential expression analysis was performed with a threshold of p < 0.05 and |log2 (fold change) | > 1. Volcano maps were generated using the “ggpubr” and “ggthemes” packages in R software to visualize the differential expression profiles. Venn diagrams were used to screen for differentially expressed genes. GO and KEGG analyses were conducted to gain insight into the biological effects of these genes, with annotation, visualization, and comprehensive databases. The STRING database was used to infer protein‐protein interactions in the DEGs enrichment pathway. 

Three representative right femurs from each experimental group were fixed in a 2.5% glutaraldehyde solution, followed by ethanol dehydration, embedding in Agar 100 epoxy resin (Agar Scientific, AGR1045), and preparation into 70 nm thin sections. Subsequently, these sections underwent staining with 2% uranyl acetate and lead citrate before examination and recording using a TEM (JOL‐JEM 1400 Plus, Japan Electron Optic, Tokyo, Japan) to visualize organelles including Golgi apparatus and endoplasmic reticulum. 

Cells were inoculated on 24‐well plates overnight at a density of 2 × 10 4 cells per dish. The cells were then treated with 100ug mL −1 fluorescent CS4‐NP for 4 h. In the study of the endocytosis pathway, endocytosis pathway inhibitors (genistein (MCE, HY‐14596); chlorpromazine (Sigma, C0982); rottlerin (MCE, HY‐18980)) were pre‐incubated for 2 h and then fluorescent CS4‐NP was added. Subsequently, the cells were fixed and the cytoskeleton was stained by phalloidine (Yeasen, 40734ES75,1:100), and finally observed and recorded by laser confocal microscopy. 

Cells were inoculated on 24‐well plates overnight at a density of 2 × 10 4 cells per dish. The following day, monensin (MCE, HY‐N4302, 0.05 µM, 1 h); brefeldin A (MCE, HY‐16592100 10 µg mL −1 , 1 h), CS4‐NP or DMSO were added to cells for 1h. Then, cells were rinsed with PBS, harvested, and analyzed. In the Golgi inhibition test, the same concentration of brefeldin A was pretreated for 1 h, and then the cells were cultured in the osteogenic induction medium. 

The mean ± SD was utilized for data reporting. Statistical significance between the two groups was assessed using a two‐tailed Student's t ‐test, while differences among multiple groups were determined through one‐way ANOVA with Dunnett's multiple comparisons test. Proportional confidence intervals were calculated using the Wilson/Brown method. Statistical significance was considered at a P value of < 0.05. GraphPad Prism (Version 9.4.0, GraphPad Software Inc, CA, USA) and Origin (OriginLab, MA, USA) conducted the statistical analyses; the figure legend provides sample size ( n ) for each analysis. 

The authors declare no conflict of interest. 

P.N. and L.Z. contributed equally to this work. P.N. performed in data curation, investigation, conceptualization, writing–original draft, writing–review and editing. L.Z. performed in data curation, supervision, visualization, writing–review and editing. J.Y., Y.D., X.X., and S.L. performed in data curation, supervision, visualization, writing–review and editing. L.S. performed in project administration, resources, supervision, writing–review and editing. G.C. and Y.S. performed in funding acquisition, project administration, resources, supervision, writing–review and editing. All authors have given approval to the final version of the manuscript. 

Supporting Information 

