Functionalizing Collagen Membranes with MSC-Conditioned Media Promotes Guided Bone Regeneration in Rat Calvarial Defects

Abstract

Functionalizing biomaterials with conditioned media (CM) from mesenchymal stromal cells (MSC) is a promising strategy for enhancing the outcomes of guided bone regeneration (GBR). This study aimed to evaluate the bone regenerative potential of collagen membranes (MEM) functionalized with CM from human bone marrow MSC (MEM-CM) in critical size rat calvarial defects. MEM-CM prepared via soaking (CM-SOAK) or soaking followed by lyophilization (CM-LYO) were applied to critical size rat calvarial defects. Control treatments included native MEM, MEM with rat MSC (CEL) and no treatment. New bone formation was analyzed via micro-CT (2 and 4 weeks) and histology (4 weeks). Greater radiographic new bone formation occurred at 2 weeks in the CM-LYO group vs. all other groups. After 4 weeks, only the CM-LYO group was superior to the untreated control group, whereas the CM-SOAK, CEL and native MEM groups were similar. Histologically, the regenerated tissues showed a combination of regular new bone and hybrid new bone, which formed within the membrane compartment and was characterized by the incorporation of mineralized MEM fibers. Areas of new bone formation and MEM mineralization were greatest in the CM-LYO group. Proteomic analysis of lyophilized CM revealed the enrichment of several proteins and biological processes related to bone formation. In summary, lyophilized MEM-CM enhanced new bone formation in rat calvarial defects, thus representing a novel ‘off-the-shelf’ strategy for GBR.

The use of mesenchymal stromal cell secretome to enhance guided bone regeneration in comparison with leukocyte and platelet‐rich fibrin

“…Despite considerable biological variation within each treatment group, significantly greater 3D bone coverage was observed in MSC‐CM vs. PRF‐CM treated defects via micro‐CT after 2 and 4 weeks. The present data are consistent with that of our previous study where, using the same method of membrane functionalization, greater bone coverage was observed in MSC‐CM vs. native membrane treated defects (positive control) and untreated defects (negative control) (Shanbhag et al., 2023). After 4 weeks, untreated defects revealed <30% average bone formation, while application of a native membrane (GBR) increased bone formation to 58% and functionalization of the membrane with MSC‐CM further increased it to 79% (Shanbhag et al., 2023), which is similar to the average bone formation observed in the MSC‐CM group in the present study.…”

“…The present data are consistent with that of our previous study where, using the same method of membrane functionalization, greater bone coverage was observed in MSC‐CM vs. native membrane treated defects (positive control) and untreated defects (negative control) (Shanbhag et al., 2023). After 4 weeks, untreated defects revealed <30% average bone formation, while application of a native membrane (GBR) increased bone formation to 58% and functionalization of the membrane with MSC‐CM further increased it to 79% (Shanbhag et al., 2023), which is similar to the average bone formation observed in the MSC‐CM group in the present study. Since only a barrier membrane is applied without a bone substitute filling the defect, this model can be considered to assess the ‘true’ GBR potential of the defect and any enhancements of this inherent potential by added treatments, such as MSC‐CM.…”

“…This pattern of new bone formation based on the incorporation of membrane fibers has previously been reported when using collagen membranes (Feher et al., 2021). Although the exact mechanism and sequence of hybrid bone formation is yet unclear, our previous observations using electron microscopy have confirmed the presence of mineralized collagen membrane fibers within newly formed woven bone (Shanbhag et al., 2023). This suggests that hybrid bone formation may be an ‘intermediate’ stage, characterized by an early mineralization of membrane fibers, their incorporation into new woven bone, which is spared the task of producing those collagen fibers itself, and subsequent remodeling to mature parallel‐fibered bone.…”

“…Although the exact mechanism and sequence of hybrid bone formation is yet unclear, our previous observations using electron microscopy have confirmed the presence of mineralized collagen membrane fibers within newly formed woven bone (Shanbhag et al, 2023). This suggests that hybrid bone formation may be an Some limitations of the present study must be acknowledged.…”

“…Based on the 3D micro‐CT scans, defects from both experimental groups showing new bone formation in the central region were processed for undecalcified histology as previously described (Shanbhag et al., 2023). Briefly, specimens were dehydrated in ascending grades of alcohol and embedded in light‐curing resin (Technovit 7200 + 1% benzoyl peroxide, Kulzer & Co.).…”