Innovative hydrogel delivery of bone marrow stromal cell-derived exosomes for enhanced bone healing

Abstract

Bone regeneration is a multifaceted process involving the well-coordinated interaction of cellular functions such as the regulation of inflammation, the formation of new blood vessels, and the development of bone tissue. Bone regeneration is a multifaceted process involving the well-coordinated interplay of multiple cellular activities, such as inflammation control, blood vessel and bone tissue. Zhang et al developed a multifunctional hydrogel system embedded with bone marrow stromal cell-derived exosomes to address the challenges of large bone defects. This innovative approach demonstrated the dual-role capability of bone marrow stromal cell-derived exosomes in directing cell fate by significantly enhancing both angiogenesis and osteogenic differentiation in vitro. The hydrogel system effectively promoted the polarization of macrophages towards the anti-inflammatory M2 phenotype, fostering an environment that supports bone repair. The effectiveness of this hydrogel was validated in a murine fracture model, which promoted significant bone regeneration and functional vascularization. Despite compelling evidence, this study highlights areas for further investigation, including detailed descriptions of experimental procedures, control group selection, long-term outcomes, and the evaluation of inflammation status in vivo. Addressing these limitations will enhance the robustness and impact of the findings.

Key Words: Bone regeneration; Hydrogel; Mesenchymal stem cell-derived exosomes; Inflammation; Angiogenesis

Core Tip: This study introduces a novel hydrogel system embedded with bone marrow stromal cell-derived exosomes that enhances bone regeneration by modulating inflammation and promoting angiogenesis. The dual-role capability of bone marrow stromal cell-derived exosomes in directing cell fate is a significant innovation, demonstrating enhanced angiogenesis and osteogenic differentiation. When validated in a murine fracture model, this approach showed promising potential for clinical application in the treatment of large bone defects. Further detailed investigations are needed to fully understand the therapeutic potential of this innovative strategy.

TO THE EDITOR

Bone regeneration is a multifaceted process that relies on the synchronized activity of several cellular mechanisms, such as inflammation control, angiogenesis, and bone formation. The novel strategy by Zhang et al[1], which involves incorporating bone marrow stromal cell-derived exosomes (BMSC-exos) into hydrogels, offers a promising solution to the pressing need for efficient bone healing methods, especially when dealing with extensive bone defects that pose significant challenges for orthopedic surgeons.

Key findings

The authors created a multifunctional hydrogel platform aimed at delivering BMSC-exos directly to the damaged area. This hydrogel showed a strong capability to shift macrophage polarization towards the anti-inflammatory M2 type, thus fostering a favorable environment for bone repair. Furthermore, embedding BMSC-exos in the hydrogel significantly boosted angiogenic and osteogenic activities in vitro, suggesting the dual role of BMSC-exos in regulating cell functions. This was evident from enhanced cell migration and increased expression of angiogenic and osteogenic markers in mouse osteoblast progenitor cells (mOPCs). The system’s performance was validated in a mouse fracture model, where it promoted notable bone regeneration and enhanced vascularization.

Advantages and constraints

Figures 4E, 4F, 5G, and 5H illustrate that the hydrogel combined with BMSC-exos significantly promoted osteogenesis and angiogenesis in mOPCs, indicating promising results that warrant deeper exploration into the mechanisms of cellular fate transitions. Exosomes have shown a versatile capacity to support multiple differentiation processes. For instance, exosomes derived from neural stem cells have been shown to promote the differentiation of recipient cells into both neurons and glial cells, demonstrating their versatility in neurogenesis[2,3]. Similarly, exosomes from tumor-associated macrophages can influence cancer progression by enhancing angiogenesis and modulating immune responses[4]. Exosomes from BMSCs have been shown to enhance osteogenic differentiation and angiogenesis both in vitro and in vivo, primarily via the delivery of miR-1260a[5]. These results underscore the multifunctional roles of exosomes in driving cellular differentiation and enhancing intercellular signaling, with their capacity to impact multiple pathways proving particularly advantageous.

Despite the positive outcomes of the BMSC-exo-loaded hydrogels in bone repair, there remain several key aspects that require further study: Firstly, the absence of comprehensive experimental details makes it difficult for others to reproduce the findings. While the authors mentioned, “For Transwell assays, after transfection and treatment with high glucose, mOPCs with different treatments were seeded into the upper chamber of 12-well transwell plates (2.5 × 10³ cells/well)”, it is not specified why the mOPCs underwent transfection or why they were exposed to high glucose conditions. Additionally, the specifics of the migration experiment, such as the concentration of exosomes used, the positioning (upper vs lower chamber), and the duration, require more clarity. In the angiogenesis assay, the authors maintained the mOPCs-loaded hydrogels for a 14-day culture period. However, the timing of BMSC-exos addition during the 14-day culture period was not clearly stated. I speculated that the authors meant to determine the role of BMSC-exo + hydrogel in inducing endothelial differentiation of mOPCs. While the results presented mainly focused on EdU immunofluorescence staining to indicate cell proliferation, the use of specific endothelial markers, such as CD31 or VE-cadherin, would have provided more robust evidence. The connection between the short-term tube formation assay and the long-term mOPCs differentiation in the hydrogel system was not well articulated, which may have contributed to misunderstandings regarding the experimental timeline and purpose. The rationale for using mOPCs cell-loaded hydrogels needs to be clarified, especially given the study’s focus on showcasing the osteogenic properties of hydrogel + BMSC-exos. The protocol for incorporating BMSC-exos into this experiment must be clearly outlined. In the sentence, “When cocultured with HUVECs, mOPCSs incubated on hydrogel + BMSC-exo exhibited enhanced proliferation and tube formation (Figure 5C and D)”, important details are missing[1]. The coculture procedure should be described, and it needs to be specified whether the tube structures were formed by human umbilical vein endothelial cells or mOPCs. Furthermore, the difference between Figure 4B and Figure 5E is not evident. In Figure 6F, the method used for protein expression analysis and the specific tissue type examined should be explicitly mentioned.

The multifunctional hydrogel used in this study consists of a biocompatible polymer matrix designed to facilitate the sustained delivery of BMSC-exos to the injury site. Key characteristics of the hydrogel, such as its biodegradability, porosity, and ability to modulate the immune response (particularly through the promotion of M2 macrophage polarization), are crucial for creating an optimal environment for bone regeneration. However, these critical details were not fully described in the study, limiting the understanding of how the hydrogel contributes to both structural support and biological functionality in the healing process.

On a positive note, the authors made an effort to evaluate the safety of the hydrogel. In Supplementary Figure 1, the authors state that the hydrogel did not negatively impact biological processes like osteogenesis, chondrogenesis, or adipogenesis. Although this assessment is important, the experimental design omits key specifics, including the hydrogel concentration and the inclusion of suitable control groups. Testing different hydrogel concentrations and assessing their effects on cell viability and proliferation would strengthen the validity of the findings. Additionally, while the authors mention that there were no adverse effects on the vital organs of mice, it is necessary to provide specifics regarding the method of hydrogel administration and the timeline for monitoring the mice to fully evaluate the safety profile.

The murine fracture model employed by Zhang et al[1], in their study, while widely accepted for preclinical bone regeneration research, presents certain limitations when applied to the evaluation of large bone defects. As the hydrogel system was specifically designed to address the challenges of large, load-bearing bone defects, the use of a murine model may not fully capture the complexities involved in human bone healing, particularly in terms of biomechanical stresses and structural integrity. That said, the use of mouse model does provide an important proof-of-concept for the biological efficacy of the hydrogel, particularly in demonstrating its ability to promote inflammation modulation, angiogenesis, and osteogenesis. The results from this model lay a solid foundation for future research. However, larger animal models such as rabbits or sheep would offer a more clinically relevant environment for evaluating the hydrogel’s performance in large bone defects. Such models would better simulate the load-bearing conditions and biomechanical challenges present in human applications, thereby allowing for a more comprehensive assessment of the clinical translatability of hydrogels.

The control groups was not optimal in demonstrating the added value of the hydrogel + BMSC-exos treatment compared to BMSC-exos alone. In Figure 4, the authors reported increased cell proliferation, migration, and osteogenesis in the hydrogel + BMSC-exo group relative to the hydrogel-only group. However, the lack of a BMSC-exo control group makes it difficult to assess whether the observed effects are truly due to the combined use of hydrogel and exosomes or just the exosomes themselves. The same issue applies to the analyses presented in Figures 5 and 6, where the inclusion of a BMSC-exo group would better illustrate any potential synergistic effects. The in vivo studies did not assess the inflammatory response, a key factor needed to fully comprehend the effects of the combined hydrogel and BMSC-exo treatment on bone regeneration.

CONCLUSION

This study demonstrates significant innovation in the field of bone regenerative medicine. However, the experiments lack detailed descriptions, and the selection of control groups does not does not effectively showcase the advantages of the hydrogel + BMSC-exos treatment over using BMSC-exos alone. Addressing these limitations, including evaluating the inflammation status in vivo, would enhance the robustness and impact of the findings.