Bone Regeneration Using Periodontal Ligament Cells Seeded on Silk Scaffold

Objectives: Bone replacement is one of the critical steps for management of oral bone defects. Many methods have been used to restore such defect. However, biocompatibility and increasing donor site morbidity are still among the main challenges. The current study aims to investigate the potential of using human periodontal ligament cells (hPDLCs) for bone formation, with Bombyx mori fibroin (BMF) silk as a scaffold material. Methods: Human periodontal ligament cells were isolated from human extracted molar teeth that were granted from Leeds Dental School tissue bank. Foam scaffolds were fabricated from BMF in cylindrical shapes. Sterilised scaffolds were subjected to surface modification with 20% Fetal bovine serum (FBS). Calcein fluorescence staining was used to label the cells before being seeded dynamically (4x103 cell/mm3) on BMF scaffolds. For in-vitro experiments, all samples were incubated in osteogenic medium for 21 days. Another group of seeded scaffolds were placed in diffusion chambers and implanted in peritoneal space of CD1 nude mice to evaluate the growth in-vivo. To study the cellular ingrowth, extracellular matrix formation and mineral deposition, all samples were prepared and stained for histological examination. Furthermore, the expression of osteogenic proteins e.g. collagen type I, osteopontin and osteocalcin was detected with immunohistochemical (IHC) stains. Results: After 24 hrs of in-vitro seeding, hPDLCs begin to spread over the scaffold surface as shown by fluorescence microscope images. Cell density increased over a period of 14 days. Histological examination demonstrated that both in-vitro and in-vivo samples showed ingrowth of cells and formation of abundant amounts of extracellular matrix and collagen bundles. Moreover, the presence of black-stained extracellular deposits was detected after staining with Von Kossa stain. Also, osteogenic proteins were detected in both groups. Conclusions: Human periodontal ligament cells that isolated from extracted teeth can proliferate and differentiate into osteoblast-like cells when grown in particular culturing conditions, also, they enhance bone-like tissue formation, both in-vitro and in-vivo

Periodontal regeneration capacity of human periodontal ligament-derived stromal cells

Restoring periodontal defect is still one of the clinical challenges; this is due to the complex structure and diversity of cell types in this joint. The advancement in tissue engineering and cell therapy, make it possible to recruit these approaches to overcome this challenge. This study aims to investigate the capacity of HPDLSCs to differentiation into the main periodontal cell types and enhance the regeneration process. The isolated HPDLSCs were cultured to be characterised for the presence progenitor cell using colony forming unit method. Flow cytometry was used to measure the expression of mesenchymal and hematopoietic cell markers. Furthermore, multilineage cells differentiation was induced. Cells then were seeded on silk scaffold and incubated both in vitro and in vivo in nude mice to examine cellular growth and differentiation. The isolated cells expressed a higher level of MSCs markers in comparison to Hematopoietic markers. Also, these cells showed the ability to proliferate and differentiate into osteogenic, fibrogenic, chondrogenic and adipogenic cues. In Vitro and In Vivo experiments demonstrated the ability of those cells to attach and spread on silk scaffold; In addition to the cellular activities that became evident through the formation of collagen fibres along with the deposition of extracellular minerals. In conclusion, HPDLSCs possess the essential progenitor cells that could differentiate into the primary periodontal cells; Thus, enhancing the periodontal regeneration process

Human periodontal ligament stromal cells: the isolation and characterisation

Restoring periodontal defect is still one of the most challenging clinical needs; this is due to the complex structure and diversity of cell types in this joint. The advancement in tissue engineering and cell therapy made it possible to utilise these approaches to overcome such a challenge. This study aimed to investigate the characteristics of isolated human periodontal ligament cells (HPDLCs), and the multi-linage differentiation capacity of these cells to enhance the regeneration process. Human periodontal ligament cells were isolated from healthy extracted third molar teeth. Colony forming unit-fibroblasts (CFU-f) was used to determine the presence of progenitor cells within the isolated cell population. Further characterisation was conducted by examining the expression levels of mesenchymal stem cells markers (CD29, CD73, STRO-1) as well as the levels of hematopoietic markers (CD34 and CD45), using flow cytometry. Multilineage cells differentiation was induced using adipogenic, chondrogenic, osteogenic and fibrogenic media. Then, the HPDLCs were seeded on 3D Bombyx mori silk fibroin scaffolds in vitro for five weeks and analysed using histology, immunohistochemistry and biochemical assays to confirm proliferation and differentiation processes. Afterwards, the samples were implanted into the peritoneal space of nude mice using diffusion chambers for further seven weeks. All samples were then retrieved from animal models and processed for histology. The data of CFU-f assay demonstrated attachment and proliferation of cultured cells subpopulation onto the vessel’s surface indicating the presence of progenitor stromal cells. Also, HPDLCs expressed higher levels of MSCs markers in comparison to the lower levels of hematopoietic markers. These cells showed the ability to proliferate and differentiate into adipogenic, chondrogenic, osteogenic and fibrogenic cues. Both, in vitro and in vivo experiments demonstrated the ability of those cells to attach and spread on silk scaffolds; as well as performing other cellular activities like formation of collagen fibres, along with the deposition of extracellular minerals. In conclusion, this study showed that HPDLCs contain the essential progenitor stromal cells, which have the capacity to differentiate into the main periodontal cells; this could indicate the role of these cells in enhancing the periodontal regeneration process