| Alveolar bone deficiency attributed to severe periodontitis, trauma and tumor impedesdentition restoration, mastication efficiency and teeth aesthetics and is a common difficultyfaced by dentists. Patients can only improve their quality of life if alveolar bone gainedregeneration. Most of the materials commonly used now merely replace the innate alveolarbone instead of biophysically regenerating new alveolar bone--something that could only beachieved through tissue engineering techniques proposed by current studies.One kind of adult multipotent stem cell PDLPC (periodontal ligament progenitor cells)harvested from periodontal tissues exhibits the periodontal lineage-specificity that isresponsible for maintaining the periodontal tissue regenerative capacity and highly expressesmesenchymal stem cell surface markers such as CD44, CD146and STRO-1. A favorablescaffold is needed to mimic the complicated environment and produce physiologicallyequivalent engineered tissue with harbored stem cells that are required by regenerative oralmedicine. One such ideal scaffold suited for the repopulation of progenitor cells is autologousbioactive platelet concentrate platelet-rich fibrin (PRF).To explore the suitability of PRF as a scaffold for PDLPCs in the regeneration of alveolarbone, the biological activities of PDLPCs treated with PRF and their underlying mechanismswere investigated.Enzymatic digestion tissue block method and cloning rings were employed to isolate theprogenitors capable of clone formation from periodontal explants. STRO-1, CD146and CD44were highly expressed on the surface of these progenitors and there was an absence ofexpression of CD45, CD11and CD34. PDLPCs are able to proliferate actively and increaseboth Alkaline Phosphatase (ALP) and matrix mineralization under osteogenic inductiveconditions which demonstrated their potential to be seed cells.Histological sectioning, staining and scanning electronic microscopy were utilized todemonstrate the superstructure of PRF. In addition, ELISA was employed to quantify thegrowth factor’s release during21days. HE stained paraffin sections revealed3portions ofPRF: fibrin clot without cells, buffy coat as platelet and leukocytes and red blood cells. The PRF showed a fiber-like structure within which the mean diameter of the space was5.6μmwith a variety of cells, such as red blood cells, leukocytes and platelets, meshed togetherinside. Growth factors were released for at least14days. Transforming growth factor-β1(TGF-β1) and platelet derived growth factor-AB (PDGF-AB) reached their release peakwithin the first7days and after that the release was reduced day by day until21day whenrelease ended. The release of insulin-like growth factor-1(IGF-1) reached its peak at day1anddecreased significantly after reaching its peak. The combination of its fibrous structure andrelease of growth factors showed PRF to be a powerful bioscaffold with an integratedreservoir of growth factors ideal for tissue regeneration.To determine the effect of PRF on the biological behaviors of dental progenitors,PDLPCs, dental follicle progenitor cells (DFPCs) and alveolar bone progenitor cells (ABPCs)were treated with PRF+osteogenic medium and PRF+DMEM comparing osteogenicmedium or DMEM alone. The radioactive-like attachment of PDLPCs to the PRF at themargin was revealed under microscope in comparison with the absence of any suchconnection between the cells and the PRF at the margin in the other2types of dentalprogenitor cells. PDLPCs became highly stretched on surface of PRF and some tinycytoplasm processes inserted into space among fibers. PRF stimulated a dramatic increase(P<0.01) ranging from9-24fold and30-90%separately in cell migration and proliferation ina dose-dependent pattern. PDLPCs and DFPCs showed higher susceptibility to PRF in cellmigration and proliferation studies in comparison with ABPCs. Both PRF-conditionedmedium and osteogenic medium resulted in osteogenic differentiation in all three dentalprogenitors. PRF+osteogenic medium showed the strongest inductive effect on celldifferentiation followed by osteogenic medium and PRF alone. In addition, the application ofPRF alone induced the osteogenic differentiation only in ABPCs and PDLPCs whichillustrates the cell-specific effect of PRF.PRF was absorbed quickly and replaced by collagen in vivo quickly without cells seededon. After seeding cells, the subcutaneous implant of PRF with PDLPCs was maintained after21days in vivo. H&E stained paraffin sections revealed that there was no PRF left but instead,only dense connective tissue without calcification within which fibroblast-like cells werearranged regularly. While, after placing PDLPCs/PRF complex into critical-size calvariadefects, the defects disappeared, and after8weeks of healing, newly formed tissues were ofsimilar density as bone under μ-CT. Von Kossa stained ground sections revealed that thedefects were closed by calcified bone-like tissue. These experiments in vivo demonstrated that the PDLPCs/PRF complex could be remodeled into either hard or soft tissue depending on theenvironmental signaling in vivo.Real-time PCR and Western Blot were conducted to gain detailed insight into themechanisms of the osteogenic inductivity of PRF on PDLPCs. The experiments showed PRFenhanced osteogenic differentiation of PDLPCs through upregulating ERK1/2. After theadministration of ERK1/2inhibitor U0126, ERK1/2level decreased significantly (P<0.01)while the expression of osteogenic markers such as OPN and OCN were suppressed19.13%and22.0%in comparison with control group (P<0.01). The expression of RUNX2, COL1andALP were decreased to68.37%,45.22%and43.02%(P<0.05). This experiment demonstratedthat ERK1/2was involved in osteogenic differentiation of PDLPCs treated by PRF.Together, this research illustrates for the first time that PRF enhances PDLPCSosteogenic differentiation through activating the ERK1/2pathway and the PDLPCs/PRFcomplex can be remodeled to calcified tissue in the appropriate environment in vivo. Thisresearch clearly demonstrates the importance of choosing the appropriate seed cells andbioscaffold in the field of regenerative oral medicine and should allow for the futuredevelopment of highly important clinical treatments. |
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