ABSTRACT
The emergence of SARS-CoV-2, the virus responsible for COVID-19, has profoundly reshaped global health priorities, with far-reaching consequences beyond respiratory illness. While much of the scientific focus has centered on pulmonary complications, increasing evidence suggests that the virus exerts significant effects on skeletal health, particularly through its interaction with osteoblasts, the bone-forming cells responsible for maintaining structural integrity. Given the delicate balance of bone remodeling—a process governed by osteoblast-mediated deposition and osteoclast-driven resorption—understanding how SARS-CoV-2 disrupts this equilibrium has become an urgent area of investigation. The significance of this topic lies in its implications for long-term skeletal health, especially among COVID-19 survivors, who may face an increased risk of bone loss, fragility, and related complications. This study delves into the direct and indirect effects of SARS-CoV-2 on osteoblast differentiation and function, uncovering mechanisms that may contribute to the virus’s impact on bone health.
To explore these effects, mesenchymal stem cells (MSCs) derived from human umbilical cords were used as precursor cells to osteoblasts, providing a controlled system for studying differentiation under SARS-CoV-2 exposure. The study utilized well-established osteoblast differentiation protocols, employing markers such as alkaline phosphatase (ALP), collagen deposition, and calcium mineralization to assess the extent of cellular development. In parallel, adipocyte differentiation assays were conducted to determine whether SARS-CoV-2 influences MSC lineage commitment. Viral infection experiments were carried out using both the ancestral strain (Wh) and the Omicron variant (BA.5), with infections performed at multiplicities of infection (MOI) of 0.1 and 1.0. The research also incorporated flow cytometry, RT-qPCR, and cytokine profiling to evaluate ACE2 receptor expression, viral replication kinetics, inflammatory responses, and redox balance. Additionally, to distinguish between the effects of active viral replication and structural viral components, experiments included UV-inactivated SARS-CoV-2 and neutralization assays using antibodies targeting the viral Spike (S) protein.
The findings reveal a striking dichotomy in how SARS-CoV-2 interacts with osteoblast precursor cells compared to fully differentiated osteoblasts. MSCs, despite being susceptible to infection, failed to produce infectious viral particles, indicating an abortive infection that does not support viral replication. However, once MSCs differentiated into osteoblasts, they became permissive to productive SARS-CoV-2 infection, with viral RNA detected in culture supernatants, alongside evidence of active viral transcription and de novo viral particle release. This suggests that osteoblasts, unlike their progenitors, provide a suitable environment for viral replication, a phenomenon that may have far-reaching implications for bone health in infected individuals.
A key observation was the significant suppression of osteoblast differentiation following SARS-CoV-2 exposure. ALP activity, collagen deposition, and calcium mineralization were all markedly reduced at 14 and 21 days post-infection, irrespective of viral strain or MOI. This inhibitory effect was not due to cytotoxicity, as cell viability remained comparable between infected and uninfected cells, ruling out a direct cell-killing mechanism. Instead, the data suggest that SARS-CoV-2 actively interferes with the molecular pathways governing osteoblastogenesis. Notably, the virus altered the expression of two critical transcription factors that regulate MSC lineage commitment: RUNX2, which drives osteoblast differentiation, was suppressed, while PPARγ, which promotes adipogenesis at the expense of osteogenesis, was upregulated. This shift in the RUNX2/PPARγ balance hints at a potential mechanism whereby SARS-CoV-2 not only inhibits bone formation but may also predispose MSCs toward adipogenic differentiation.
Further analysis of the inflammatory microenvironment revealed that SARS-CoV-2 infection triggered a surge in IL-6 secretion during osteoblast differentiation, with levels peaking at 7 days post-infection before declining to baseline by day 21. Given IL-6’s well-documented role in suppressing osteoblast function and promoting osteoclast-mediated bone resorption, its sustained elevation likely contributes to the observed impairment of differentiation. Concurrently, the virus upregulated RANKL expression, a key mediator of osteoclastogenesis, suggesting a dual mechanism by which SARS-CoV-2 simultaneously suppresses bone formation and enhances bone resorption, exacerbating the risk of skeletal deterioration. Interestingly, despite the activation of these pro-resorptive pathways, the virus failed to induce IFNβ1 transcription in infected cells, a notable finding given the role of type I interferons in modulating osteoblast activity.
Oxidative stress emerged as another contributing factor to the inhibition of osteoblast differentiation. Mitochondrial reactive oxygen species (mROS) levels were significantly elevated in MSCs exposed to SARS-CoV-2, with ROS accumulation peaking at 7 and 14 days post-infection. Given that ROS at physiological levels promote osteoblast differentiation, while excessive oxidative stress disrupts the process by degrading key transcription factors such as RUNX2, this observation aligns with the broader inhibitory effects of the virus on bone-forming cells. The interplay between viral infection, oxidative stress, and inflammatory cytokines likely constitutes a multi-layered mechanism through which SARS-CoV-2 disrupts bone homeostasis.
To determine whether these effects were contingent upon active viral replication, additional experiments were conducted using UV-inactivated SARS-CoV-2, which retains structural integrity but lacks replicative capacity. Remarkably, inactivated virus was equally effective in suppressing osteoblast differentiation, suggesting that a structural viral component—rather than intracellular viral replication—is responsible for impairing bone formation. Neutralization assays provided further insight into this mechanism: when SARS-CoV-2 or UV-inactivated virus was pre-incubated with an anti-Spike (S) protein antibody, the inhibitory effects on osteoblastogenesis were significantly reduced. This implicates the viral Spike protein as a key mediator of osteoblast dysfunction, potentially through its interaction with host cell receptors or downstream signaling pathways.
Contrary to expectations, the virus did not influence adipocyte differentiation. Despite upregulating PPARγ, SARS-CoV-2 exposure did not enhance lipid droplet accumulation in MSC-derived adipocytes, suggesting that while the virus disrupts osteogenesis, it does not actively promote adipogenesis. This finding reinforces the notion that SARS-CoV-2 skews MSC lineage commitment away from osteoblast differentiation without necessarily enhancing alternative fates.
The implications of these findings extend beyond the immediate context of COVID-19 infection. Given the central role of osteoblasts in maintaining bone density and strength, disruptions to their function could have long-term consequences for survivors, particularly older individuals already at risk for osteoporosis. The interplay between viral infection, cytokine dysregulation, oxidative stress, and transcriptional reprogramming suggests a complex, multifaceted mechanism through which SARS-CoV-2 undermines skeletal integrity. The observation that inactivated virus can exert these effects further raises concerns about the potential for viral proteins, even in the absence of active infection, to contribute to post-viral musculoskeletal complications. Understanding these mechanisms not only sheds light on an underexplored consequence of COVID-19 but also opens avenues for targeted therapeutic interventions aimed at mitigating virus-induced bone loss.
In sum, this study provides compelling evidence that SARS-CoV-2 impairs osteoblast differentiation and function through a combination of viral-host interactions, inflammatory signaling, oxidative stress, and transcriptional dysregulation. The permissiveness of osteoblasts to viral replication, coupled with the virus’s ability to suppress bone-forming activity, highlights an overlooked aspect of COVID-19 pathophysiology with significant clinical implications. The findings underscore the need for continued research into the long-term skeletal effects of SARS-CoV-2 and the development of potential countermeasures to protect bone health in affected individuals.
The emergence of Coronavirus Disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has reshaped global health priorities, often progressing to severe pneumonia in many patients. In regions with limited healthcare resources and a high prevalence of chronic illnesses, the rising case numbers remain a pressing challenge. While many countries have transitioned into the post-epidemic phase, the need for close clinical monitoring of recovering patients has never been more critical. Among the myriad of complications associated with COVID-19, the impact on bone health has garnered increasing attention. This article delves into the intricate mechanisms through which SARS-CoV-2 affects bone health, focusing on osteoblast differentiation, bone matrix deposition, and the long-term skeletal consequences in COVID-19 survivors.
Table 1: Comprehensive Summary of the Effects of SARS-CoV-2 on Bone Health and Osteoblast Differentiation
Section | Sub-Section | Sub-Sub-Section | Details |
---|---|---|---|
Introduction | COVID-19 and Bone Health | Impact on Vulnerable Populations | COVID-19 has reshaped global health priorities, leading to severe pneumonia in some cases. Post-recovery clinical monitoring is critical, particularly for individuals with chronic conditions and frail populations, as these groups face higher risks of systemic complications, including bone loss. |
Skeletal Remodeling | Role of Osteoblasts and Osteoclasts | The skeleton undergoes continuous remodeling, with osteoblasts facilitating bone matrix deposition and osteoclasts contributing to resorption. This balance is crucial for maintaining bone health. SARS-CoV-2 may disrupt this process. | |
Materials and Methods | Isolation of MSCs | Source and Processing | MSCs were isolated from human umbilical cords using α-MEM, 10% platelet lysate, and incubated in a 5% CO₂ atmosphere. Cells were expanded until passage 2-3, characterized by positive markers (CD105, CD73, CD90) and negative markers (CD45, CD34, CD14, CD19, HLA-DR). |
Osteoblast and Adipocyte Differentiation | Protocol for Differentiation | MSCs were cultured in osteoblast differentiation medium (β-glycerophosphate, dexamethasone, ascorbic acid) or adipocyte differentiation medium (IBMX, dexamethasone, insulin). Osteoblast differentiation occurred by day 14–21, while adipocyte differentiation completed within 7–10 days. | |
SARS-CoV-2 Infection | Viral Strains Used | SARS-CoV-2 ancestral strain (Wh) and Omicron (BA.5) were used at MOIs of 0.1 and 1.0. Infection involved a 4-hour incubation followed by multiple PBS washes. | |
ACE2 Surface Expression | Flow Cytometry Analysis | ACE2 expression was analyzed using flow cytometry. Osteoblasts showed higher but non-significant levels of ACE2 compared to precursor MSCs, suggesting alternative viral entry mechanisms. | |
Results | SARS-CoV-2 Replication in Osteoblasts | Viral Load Kinetics | Differentiated osteoblasts were permissive to SARS-CoV-2 infection, as viral RNA levels increased over time, confirmed via RT-qPCR targeting ORF1ab and nucleocapsid genes. The presence of subgenomic RNA indicated active viral replication. |
Abortive Infection in MSCs | Failure to Produce Infectious Particles | MSCs exposed to SARS-CoV-2 synthesized viral subgenomic RNA but failed to produce infectious virions, classifying the infection as abortive. This suggests an incomplete viral replication cycle. | |
Effects on Osteoblast Differentiation | Suppression of ALP, Collagen, and Calcium Deposition | SARS-CoV-2 exposure significantly reduced osteoblast differentiation, as measured by ALP activity, collagen deposition (Sirius Red staining), and calcium deposition (Alizarin Red S staining). These effects persisted at 14 and 21 days post-infection. | |
Redox Imbalance in MSCs | Mitochondrial ROS Production | MSCs exposed to SARS-CoV-2 exhibited increased mitochondrial ROS (mROS) levels at 7 and 14 dpi, suggesting oxidative stress contributes to impaired osteoblast differentiation. | |
Modulation of Transcription Factors | RUNX2 and PPARγ Expression | RUNX2, a key osteoblast transcription factor, was downregulated at 1 dpi but normalized by 21 dpi. In contrast, PPARγ, an adipogenic regulator, was upregulated by BA.5 at 1 and 21 dpi, suggesting a shift favoring adipogenesis over osteogenesis. | |
Inflammatory Cytokine Expression | IL-6 and RANKL Upregulation | IL-6, a cytokine known to suppress osteoblast function, was elevated at 1 dpi and remained high at 7 dpi before returning to baseline at 21 dpi. RANKL, which promotes osteoclastogenesis, was upregulated at both 1 and 21 dpi, indicating increased bone resorption. | |
IFNβ1 Expression | Lack of Type I Interferon Response | Unlike other viral infections, SARS-CoV-2 did not significantly alter IFNβ1 transcription levels in MSCs or osteoblasts, suggesting that IFNβ1 is not involved in the observed suppression of osteoblast differentiation. | |
UV-Inactivated SARS-CoV-2 Effects | Structural Viral Components Affect Differentiation | UV-inactivated SARS-CoV-2 inhibited osteoblast differentiation as effectively as live virus, indicating that a viral structural component (rather than active replication) plays a role in disrupting differentiation. | |
Role of the Spike Protein | Neutralization Assay | Pre-incubation of SARS-CoV-2 with anti-Spike (S) protein antibodies reduced the virus’s ability to inhibit osteoblast differentiation. This confirms that the S protein contributes to the suppression of osteoblastogenesis. | |
Effects on Adipocyte Differentiation | No Significant Impact on Fat Accumulation | Despite inhibiting osteoblast differentiation, SARS-CoV-2 did not enhance adipocyte differentiation. PPARγ expression increased, but lipid droplet accumulation remained unchanged. | |
Discussion | Potential Mechanisms of Bone Loss | Direct vs. Indirect Effects | The observed inhibition of osteoblast differentiation and increased osteoclastogenesis suggest that SARS-CoV-2 contributes to bone loss via direct (infection-mediated) and indirect (inflammatory and metabolic) mechanisms. |
Oxidative Stress and Bone Health | ROS as a Mediator of Inhibition | Elevated ROS levels observed in MSCs suggest that oxidative stress plays a key role in inhibiting osteoblast differentiation post-SARS-CoV-2 infection. | |
Cytokine-Mediated Bone Resorption | IL-6 and RANKL Pathways | IL-6 upregulation promotes RANKL expression, enhancing osteoclast differentiation and bone resorption. The persistence of RANKL upregulation suggests a long-term impact on bone health. | |
Implications for Long-Term Bone Health | Osteoporosis Risk in COVID-19 Survivors | Given that osteoblast suppression and increased osteoclast activity contribute to bone loss, COVID-19 survivors—especially elderly or frail individuals—may be at increased risk of developing osteoporosis. | |
Conclusion | Summary of Findings | Long-Term Skeletal Impact | SARS-CoV-2 exposure impairs osteoblast differentiation, promotes osteoclastogenesis via IL-6 and RANKL, and increases ROS production. These effects suggest a heightened risk of bone density loss and osteoporosis in COVID-19 survivors. |
Future Research Directions | Unanswered Questions | Further studies are needed to identify alternative SARS-CoV-2 entry receptors in osteoblasts, investigate the role of immune cell interactions in bone loss, and explore potential therapeutic interventions to mitigate post-COVID skeletal complications. |
The Dynamic Nature of Bone Remodeling
The skeleton is a dynamic organ system that undergoes continuous remodeling through the coordinated actions of osteoblasts and osteoclasts. Osteoclasts facilitate bone resorption through acidification and the secretion of lysosomal enzymes. In contrast, osteoblasts are responsible for the deposition of bone matrix and are thought to facilitate the calcification and mineralization of the bone matrix. This intricate process involves the deposition and mineralization of the bone matrix, both of which are essential for strengthening and maintaining the skeleton. A key player in facilitating mineralization is alkaline phosphatase (ALP). Type I collagen, the primary organic component of bone, constitutes 90% of the organic matrix. The proper deposition of the organic matrix is crucial for aligning the mineral matrix precisely, thereby ensuring the structural integrity and functionality of bone.
SARS-CoV-2 and Bone Health: Emerging Concerns
Evidence suggests that both the disease and its treatments exert lasting negative effects on the bone health of survivors, particularly among the elderly and frail. This raises concerns about heightened risks of bone loss in these vulnerable populations. Intriguingly, research shows that the extent of bone loss—whether local or systemic—aligns closely with the severity of the inflammatory response. Alarmingly, this inflammation-induced bone loss can persist even after successful treatment and resolution of the primary infection. Recent findings support this connection, showing that reduced vertebral bone mineral density (BMD) is associated with worse outcomes in COVID-19 patients, underscoring its potential as a key marker of disease severity. Furthermore, preclinical studies have shed additional light on the harmful impact of SARS-CoV-2 on bone and muscle health.
Mechanisms of SARS-CoV-2-Induced Bone Damage
Recent studies have begun to elucidate how SARS-CoV-2 infection causes bone damage. Findings demonstrate that ancestral and Omicron variants of SARS-CoV-2 induce an increase in osteoclast numbers, leading to excessive bone resorption. Currently, it remains unexplored whether SARS-CoV-2 infection affects osteoblast differentiation and function. This study explores the direct effects of SARS-CoV-2 on osteoblast differentiation and function, using mesenchymal stem cells (MSCs) derived from human umbilical cords as precursor cells. The findings demonstrate that SARS-CoV-2 exposure inhibits osteoblast differentiation. These results reveal a novel mechanism through which SARS-CoV-2 may directly impair bone health, potentially contributing to long-term skeletal consequences in COVID-19 survivors.
Experimental Design and Methodology
The study employed a comprehensive experimental design to investigate the effects of SARS-CoV-2 on osteoblast differentiation and function. Mesenchymal stem cells (MSCs) were isolated from human umbilical cords and expanded in culture. These MSCs were then differentiated into osteoblasts and adipocytes using specific differentiation media. The cells were exposed to both ancestral and Omicron variants of SARS-CoV-2 at varying multiplicities of infection (MOI). Viral load, cell viability, and various markers of osteoblast differentiation were assessed using a range of techniques, including flow cytometry, RT-qPCR, and spectrophotometric assays.
Key Findings
- Osteoblasts Are Permissive for Productive SARS-CoV-2 Replication: The study found that both ancestral and Omicron variants of SARS-CoV-2 could productively infect and replicate in differentiated osteoblasts, leading to the release of new viral particles. This was despite low levels of ACE2 expression in these cells, suggesting alternative mechanisms of viral entry.
- Abortive Infection in MSCs: In contrast to differentiated osteoblasts, MSCs exhibited an abortive infection, where viral replication was initiated but did not result in the production of new infectious particles. This suggests that the differentiation state of the cells plays a crucial role in determining the outcome of SARS-CoV-2 infection.
- Inhibition of Osteoblast Differentiation: Exposure to SARS-CoV-2 significantly inhibited osteoblast differentiation, as evidenced by reduced ALP activity, calcium deposition, and collagen deposition. This inhibition was not due to a loss of cell viability, indicating a direct effect of the virus on the differentiation process.
- Role of Reactive Oxygen Species (ROS): The study found that SARS-CoV-2 infection led to an increase in mitochondrial ROS production, which may contribute to the inhibition of osteoblast differentiation. High levels of ROS are known to inhibit osteoblast differentiation by oxidizing key transcription factors such as RUNX2.
- Modulation of Transcription Factors: SARS-CoV-2 infection modulated the expression of key transcription factors involved in osteoblast and adipocyte differentiation. Specifically, the virus inhibited RUNX2 expression while upregulating PPARγ, leading to a shift in the differentiation potential of MSCs towards adipogenesis at the expense of osteogenesis.
- Cytokine Production: The study found that SARS-CoV-2 infection induced the production of IL-6, a cytokine known to inhibit osteoblast differentiation. Additionally, the virus upregulated the expression of RANKL, a key mediator of osteoclastogenesis, further contributing to bone loss.
- Role of the Spike Protein: The study demonstrated that the Spike (S) glycoprotein of SARS-CoV-2 plays a crucial role in inhibiting osteoblast differentiation. Neutralization of the S protein using specific antibodies reversed the inhibitory effects of the virus on osteoblast differentiation.
- UV-Inactivated Virus: The study found that UV-inactivated SARS-CoV-2 could also inhibit osteoblast differentiation, suggesting that structural components of the virus, rather than its replicative capacity, are responsible for the observed effects.
- Adipocyte Differentiation: In contrast to its effects on osteoblast differentiation, SARS-CoV-2 did not significantly alter adipocyte differentiation, indicating a specific impact on the osteogenic lineage.
Discussion and Implications
The findings of this study have significant implications for our understanding of the long-term consequences of COVID-19 on bone health. The inhibition of osteoblast differentiation by SARS-CoV-2, coupled with the induction of osteoclastogenesis, suggests a potential mechanism for the increased bone loss observed in COVID-19 survivors. This is particularly concerning for elderly and frail individuals, who are already at increased risk for osteoporosis and fractures.
The study also highlights the role of ROS and cytokine production in mediating the effects of SARS-CoV-2 on bone health. The increase in ROS production following viral infection may contribute to the inhibition of osteoblast differentiation, while the upregulation of IL-6 and RANKL may promote osteoclastogenesis and bone resorption. These findings suggest that targeting these pathways could be a potential therapeutic strategy for mitigating the long-term skeletal consequences of COVID-19.
Furthermore, the study underscores the importance of the Spike protein in mediating the effects of SARS-CoV-2 on osteoblast differentiation. The ability of neutralizing antibodies against the S protein to reverse the inhibitory effects of the virus on osteoblast differentiation suggests that targeting the S protein could be a viable therapeutic approach.
Limitations and Future Directions
While this study provides valuable insights into the mechanisms through which SARS-CoV-2 affects bone health, it is not without limitations. The use of MSCs derived from umbilical cords may not fully recapitulate the behavior of MSCs from other sources, such as bone marrow. Additionally, the absence of a microenvironment context, such as cell-to-cell interactions or the extracellular matrix, limits the ability to study the modulation of the observed phenomena. Future studies should aim to address these limitations by using MSCs from different sources and incorporating more complex in vitro models that better mimic the in vivo environment.
Moreover, the study did not investigate the potential role of immune cells in modulating the effects of SARS-CoV-2 on bone health. Given the importance of the immune response in COVID-19, future research should explore the interactions between immune cells and bone cells in the context of SARS-CoV-2 infection.
Conclusion
In conclusion, this study provides compelling evidence that SARS-CoV-2 infection has a direct and detrimental impact on bone health by inhibiting osteoblast differentiation and promoting osteoclastogenesis. The findings highlight the importance of monitoring bone health in COVID-19 survivors, particularly in vulnerable populations, and suggest potential therapeutic targets for mitigating the long-term skeletal consequences of the disease. As the world continues to grapple with the ongoing effects of the COVID-19 pandemic, understanding and addressing the full spectrum of its impact on human health remains a critical priority.
resource: https://www.mdpi.com/1999-4915/17/2/143