An Animal Experimental Study On The Combination Of Bone Marrow Stromal Cells And Platelet-rich Plasma In Promoting Periodontal Tissue Regeneration

Objective: The purposes of this study were as follows: (1) In vitro to isolate and to culture bone marrow stromal cells (BMSCs), which would be used in the following experimental procedures. (2)In vitro to evaluate the influence of platelet-rich plasma (PRP), obtained by two-step centrifugation of whole blood, on BMSCs' proliferation, differentiation and extracellular matrix secreting in order to evaluate PRP's biological characters before in vivo transplantation. (3) To evaluate the regenerative capability of BMSCs, PRP alone or their combinations in promoting peridontal tissue regeneration by implanting them to periodontal buccal dehisences in order to assess the feasibility of using tissue engineering approaches in promoting periodontal tissue regeneration.Methods: (1) Bone marrow stromal cells, obtained by inspiriting bone marrow from beagle dog's iliac bone and isolated by gradul centrifugation, were culture-expanded in vitro. (2) Platelet-rich plasma was obtained by two-step centrifugation of whole vein blood, which was inspirited from the dog's small saphenous vein. The influence of PRP on BMSCs' proliferation, differentiation and extracellular matrix secreting were evaluated by detecting PRP- induced BMSCs' MTT incorporation rate, alkaline phosphate activity and total protein. For these purposes following methods such as MTT method, immunochemical staining, kinetic method and Coomassie brilliant blue stain-were utilized. (3) Using tricalcium phosphate (TCP) as scaffold, BMSCs, PRP alone or their combinations were implanted to the artifical buccal dehisecences. The experimental groups were as follows: TCP group, PRP/TCP group, BMSCs/TCP group and BMSCs/PRP/TCP group, and the sits without implantation (open flap) as control group. At 6 weeks and 12 weeks post-surgery, all animals were anesthetized and then sacrificed by perfusion with 4% paraformaldehyde in 0.1 M PBS through the carotid arteries. The studied teeth with surrounding soft and hard tissues were harvested, fixed in 10% buffered formalin for 10 days, decalcified in 10% formic acid for 60 days, trimmed, dehydrated, and embedded in paraffin, stained and observed as well as measured histogitically. Results: The results suggested that all experimental groups displayed periodontal tissue regeneration with a different degree while the control group showed significantly less periodontal tissue regeneration. The sites implanted with BMSCs, adding or without PRP, showed significantly tissue regeneration including functional periodontum, alveolar bone and cementum, especially acellular cementum at 12-week postoperation. However, the sites implanted with PRR only, showed alveolar bone regeneration without cementum formation, even at 12-week post-surgery, but it can accelerate bone-forming rate and bone maturation.Conclusion: (1)BMSCs, with or without PRP, in the local surgical sites, can be induced to form periodontal supporting tissue including functional periodontal liganment, alveolar bone, and especially acellular cementum, which can be strengthened by adding PRP to BMSCs. (2) PRP alone, without BMSCs added, can only promote alveolar bone regemeration without cementum formation, but it can accelerate bone-forming rate and bone maturation. (3)Utilizing tissue engineering approaches, the application of combinating BMSCs and PRP can premote periodontal tissue regeneration.