Human periodontal ligament stromal cells and three-dimensional Bombyx mori fibroin silk scaffold for periodontal tissue regeneration

Restoring the structure and function of damaged periodontal tissues remains one of the clinical challenges in dentistry. The reason behind that could be attributed to the complex biology of the periodontium. Cellular therapy and tissue engineering were introduced as possible alternatives to overcome the limitations of current treatment modalities. Accordingly, this study aimed to investigate the potential of using primary human periodontal ligament stromal cells (hPDLSCs) with Bombyx mori fibroin (Bmf) silk scaffold for enhancing periodontal regeneration in vitro. The project also aimed to develop a novel mechanical stimulation bioreactor that can be a useful tool to study the effect of cyclic compressive/tensile stimulation on the behaviour of hPDLSCs in a 3D, in vitro environment.
Human periodontal ligament cells (hPDLCs) were isolated from extracted healthy third molar teeth of three donors. The hPDLCs (passage two) were characterised using Colony-forming unit-fibroblast (CFU-F) assay, multicolour flow cytometry to measure the expression of mesenchymal (CD29, CD73 and STRO-1) and haemopoietic (CD34 and CD45) stem cells markers, as well as trilineage (osteogenic, adipogenic and chondrogenic) differentiation.
The immunogenicity of Bmf silk scaffold was investigated by measuring the release of inflammatory cytokines (TNF-α and IL-1β) of THP-1 cell in response to their exposure to the scaffold. The scaffold cytocompatibility was then evaluated by culturing hPDLSCs with the scaffold for four weeks in basal/osteogenic media. The in vivo behaviour of hPDLSCs-Bmf silk construct was examined following seven weeks of intraperitoneal implantation (diffusion chamber model) in CD-1 nude mice.
A novel mechanical stimulation bioreactor was designed and manufactured in house. The bioreactor was used to apply a cyclic compressive loading on hPDLSCs-Bmf silk constructs for fourteen days at a 30 cycle/min for 15 min, twice daily. The proliferation and differentiation (osteogenic and cementogenic) of the control and experimental groups were compared.
Throughout the project, various techniques were used to evaluate cellular activities (viability, proliferation and differentiation), including biochemical assays, fluorescent cell labelling, histological and immunohistochemical examination.
The CFU-F assay revealed a significant difference among donors. Moreover, hPDLCs expressed high CD29 and CD73 (99.95% and 100%, respectively), while the level of STRO-1 was 1.05%. In contrast, the expression of CD34 was limited (0.73%). However, the expression of CD45 showed a remarkable variation among donors (28.73%). Cells of all donors had the capacity to differentiate into osteogenic, chondrogenic and adipogenic cues.
The Bmf silk scaffold induced low levels of inflammatory cytokines, which were comparable to those induced by 3D collagen-type 1 (COL-1) scaffold. Also, Bmf scaffold supported the proliferation and differentiation of hPDLSCs in vitro and in vivo.
With the novel bioreactor, it was possible to apply static/cyclic mechanical stimulation on eight samples simultaneously using different loading frequencies. Also, the results showed that compression stimulation enhanced the proliferation and differentiation of hPDLSCs in vitro.
In conclusion, the current findings confirm the presence of multipotent mesenchymal stromal cells MSCs within primary hPDLCs. However, it is crucial to evaluate the cells' characteristics before their implication for research/therapeutic purpose due to donor dependency. Additionally, Bmf silk material is a potential biocompatible scaffold that could support periodontal tissue regeneration in vitro and in vivo. Enrolling mechanical stimulation is crucial as it affects the behaviour of hPDLSCs. The manufactured bioreactor could be a feasible tool for studying cyclic compression on cellular activity in vitro. However, several parameters (e.g. type of stimulation, load, frequency, and mode of application) need to be further optimised to support the regeneration process positively.