A microenvironment with appropriate spatial and temporal signals will promote tissue regeneration. Porous β-TCP ceramic scaffolds have been developed to provide structural support in bone regeneration; however, these scaffolds lack bioactive components on the surface. ECM can serve as a source of growth factors, cytokines, chemokines and other biological signals, providing bioactive cues for cell proliferation and differentiation. As such, deposition of cell-derived ECM on a 3D porous β-TCP ceramic scaffold potentially creates a biomimetic microenvironment that promotes cellular development by combining biological cues and structural support.
In our previous study, we produced hMSC-derived ECM deposited on CaP scaffolds [27
]. In this study, we grew HUVEC on 3D porous β-TCP ceramic scaffolds and then decellularized the construct to generate HUVEC-derived ECM. This was followed by characterization using SEM, FTIR, XPS and immunochemistry. The results demonstrated the presence of ECM components on the surface of CaP scaffolds. It has been reported that endothelial derived ECM is enriched in proteoglycans laminin, collagen IV, and fibronectin. Fibronectin, collagen IV and laminin are thought to be important ECM protein components for cell adhesion, proliferation and differentiation [15
]. We further used these proteins as biomarkers for the presence of ECM. Our immunofluorescent staining experiments have shown the presence of collagen IV, fibronectin, and laminin on the β-TCP scaffolds.
After the characterization of ECM on the ceramic scaffold, we evaluated whether this HUVEC-derived ECM microenvironment of ECM/β-TCP composite scaffold can promote the osteogenic differentiation of hMSC in vitro. We seeded hMSC on the ECM/β-TCP and β-TCP only scaffolds. dsDNA content and ALP activity were measured to determine the extent of cell proliferation and differentiation while the expression of osteogenic genes was determined using real-time PCR. Our study did not demonstrate a significant difference between the ECM/β-TCP and β-TCP scaffold groups in dsDNA content, which provides an indirect measure of cell proliferation. Therefore, the presence of ECM on the scaffold did not significantly promote cell proliferation compared to the plain scaffold condition with incubation time. This may be because we used osteogenic medium, which promotes cells to differentiate but not to proliferate.
Our results do suggest that ECM plays a significant role in cell differentiation. ALP activity expression levels in ECM/β-TCP scaffolds were significantly higher than those in β-TCP only scaffolds. Bone-related genes were up-regulated in ECM/β-TCP groups compared to β-TCP only groups. The immunofluorescent staining for osteocalcin, a component of bone matrix, was shown to occur at a higher density within ECM/β-TCP scaffolds relative to β-TCP only scaffolds. These results imply that HUVEC-derived ECM promotes early osteogenic differentiation of hMSC in vitro.
This may be attributable to collagen, fibronectin and laminin proteins in HUVEC-derived ECM, which have previously been reported to stimulate osteogenic differentiation [12
]. However, Kaigler et al. reported that ECM derived from human dermal microvascular endothelial cells did not have any effect on hMSC’s osteogenic differentiation. In their experiments, they removed endothelial cells using urea from culture plates and immediately seeded hMSC on the remaining ECM [44
]. Villars et al. also reported that HUVEC-derived ECM had no effect on the ALP activity of hMSC [45
]. They used glycerol solution to remove cell materials and scraped ECM for dialysis, and then dialyzed ECM solution was homogenously coated for seeding hMSC. Our method is distinct in that we seeded HUVEC on scaffolds and decellularized the constructs using 0.5% Triton solution to remove cells and deposit ECM on the scaffolds. Our finding that HUVEC-derived ECM significantly promoted the osteogenic differentiation of hMSC is distinct from previous studies and this difference may be a result of the unique combination of ECM architecture and CaP scaffolds in a 3D spatial structure.
The early differentiation of hMSC may be mediated by the activation of the MAPK/ERK osteogenic signal pathway [12
]. HUVEC-derived ECM deposited on the surface of a scaffold provides new bioactive components, which may activate ERK1/2 expression through the MAPK/ERK signaling pathway mediated by integrins on the cell membrane of hMSC. To determine if the ECM activates the osteogenic differentiation of hMSC through the MAPK/ERK signaling pathway, we used the inhibitor PD98059 to block this MAPK/ERK signaling pathway. ALP activity and gene expression results showed that ALP activity was significantly inhibited and the osteogenic genes were significantly down-regulated in ECM/β-TCP group after PD98059 treatment. This inhibition did not occur in β-TCP group. Similarly, protein expression from Western blotting also showed that the phosphorylated ERK1/2 level in ECM/β-TCP group was significantly inhibited while protein expression in the β-TCP group was not. These results implicate the MAPK/ERK signaling pathway in activating hMSC osteogenic differentiation. ECM components activated this signaling pathway via intergrins on the hMSC membrane, thus down-streaming the osteogenic differentiation pathway of hMSC [15
]. This implies that ECM provides important biological cues for the differentiation of hMSC.
This study reinforces our understanding of cell-matrix interaction in ceramic scaffold-based tissue regeneration techniques. A biomimetic microenvironment provides bioactive cues, regulating the osteogenic behaviors of hMSC. Therefore, through the in vitro generation of a cell-derived ECM, the cellular function of a ceramic scaffold can be improved without any additional chemical modification or growth factor binding. This technique could also be used to enhance other tissue regeneration scaffolds. We believe that the clinical impact of this technique could be significant, as the treatment of significant bone defects remains and unsolved problem in modern medicine. Our ECM-modified scaffold can be tailored to defects of various shapes and sizes, is easily handled and stored in a clinical setting, and avoids the potential risk of infectious disease.
It is worth noting that in our previous experiments, we seeded hMSC onto 3D porous β-TCP scaffold and generated hMSC-derived ECM containing porous β-TCP scaffolds [27
]. In these studies, we did not observe enhancement of early osteogenic differentiation of hMSC by the hMSC-derived ECM. The difference is that we used an osteogenic medium for the first time in our current study and this combination of HUVEC-derived ECM and osteogenic medium may promote early osteogenic differentiation of hMSC. In the future, we will need to determine if the HUVEC-derived ECM alone will promote early osteogenic differentiation of hMSC in non-osteogenic medium and if the HUVEC-derived ECM will better promote early osteogenic differentiation of hMSC as compared to hMSC-derived ECM in osteogenic medium. Additional research is also indicated to investigate the in vivo
behaviors of this ECM/scaffold.