Jihua Chai1, Runze Jin1, Guohua Yuan2, Valerie Kanter3, Richard J Miron4, Yufeng Zhang5. 1. The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China. 2. The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China; Department of Pediatric Dentistry, School and Hospital of Stomatology, Wuhan University, Wuhan, China. 3. Department of Endodontics, University of California Los Angeles, Los Angeles, California. 4. The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China; Department of Periodontology, School of Dental Medicine, University of Bern, Bern, Switzerland. Electronic address: richard.miron@zmk.unibe.ch. 5. The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China; Department of Dental Implantology, School and Hospital of Stomatology, Wuhan University, Wuhan, China.
Abstract
INTRODUCTION: Platelet-rich plasma (PRP) has been widely used in regenerative dentistry for over 2 decades. Nevertheless, previous studies have shown that its growth factor content is released over a short time period, and the application of anticoagulants limits its regenerative potential. Therefore, a second-generation platelet concentrate (liquid platelet-rich fibrin [PRF]) was developed without the use of anticoagulants and with shorter centrifugation times. The purpose of the present study was to compare the cellular regenerative activity of human dental pulp cells (hDPCs) when cultured with either liquid PRF or traditional PRP. METHODS: The regenerative potential of hDPCs isolated from healthy human third molars (18-22 years, n = 5) was investigated in both normal and inflammatorylike conditions (lipopolysaccharide [LPS]) and assessed for their potential for dentin repair. The effects of liquid PRF and PRP were assessed for cellular migration, proliferation, and odontoblastic differentiation using a transwell assay, scratch assay, proliferation assay, alkaline phosphatase assay, alizarin red staining, and real-time polymerase chain reaction for genes encoding collagen type 1 alpha 1, dentin sialophosphoprotein, and dentin matrix protein 1, respectively. The effects of both platelet concentrates were also assessed for their ability to influence nuclear translocation of nuclear factor kappa B (p65) by immunofluorescence, and reverse-transcription polymerase chain reaction for genes encoding interleukin-1β, tumor necrosis factor alpha, and nuclear factor kappa B (p65) during an inflammatory condition. RESULTS: Both PRP and liquid PRF increased the migration and proliferation of hDPCs when compared with the control group, whereas liquid PRF showed a notable significant increase in migration when compared with PRP. Furthermore, liquid PRF induced significantly greater alkaline phosphatase activity, alizarin red staining, and a messenger RNA expression of genes encoding collagen type 1 alpha 1, dentin sialophosphoprotein, and dentin matrix protein 1 when compared with PRP. When hDPCs were cultured with LPS to stimulate an inflammatory environment, a marked decrease in dentin-related repair was observed. When liquid PRF was cultured within this inflammatory environment, the reduced regenerative potential in this LPS-produced environment was significantly and markedly improved, facilitating hDPC regeneration. The messenger RNA expression of inflammatory markers including tumor necrosis factor alpha, interleukin-1β, and p65 were all significantly decreased in the presence of liquid PRF, and, furthermore, liquid PRF also inhibited the transport of p65 to the nucleus in hDPCs (suggesting a reduced inflammatory condition). CONCLUSIONS: The findings from the present study suggest that liquid PRF promoted greater regeneration potential of hDPCs when compared with traditional PRP. Furthermore, liquid PRF also attenuated the inflammatory condition created by LPS and maintained a supportive regenerative ability for the stimulation of odontoblastic differentiation and reparative dentin in hDPCs.
INTRODUCTION: Platelet-rich plasma (PRP) has been widely used in regenerative dentistry for over 2 decades. Nevertheless, previous studies have shown that its growth factor content is released over a short time period, and the application of anticoagulants limits its regenerative potential. Therefore, a second-generation platelet concentrate (liquid platelet-rich fibrin [PRF]) was developed without the use of anticoagulants and with shorter centrifugation times. The purpose of the present study was to compare the cellular regenerative activity of human dental pulp cells (hDPCs) when cultured with either liquid PRF or traditional PRP. METHODS: The regenerative potential of hDPCs isolated from healthy human third molars (18-22 years, n = 5) was investigated in both normal and inflammatorylike conditions (lipopolysaccharide [LPS]) and assessed for their potential for dentin repair. The effects of liquid PRF and PRP were assessed for cellular migration, proliferation, and odontoblastic differentiation using a transwell assay, scratch assay, proliferation assay, alkaline phosphatase assay, alizarin red staining, and real-time polymerase chain reaction for genes encoding collagen type 1 alpha 1, dentin sialophosphoprotein, and dentin matrix protein 1, respectively. The effects of both platelet concentrates were also assessed for their ability to influence nuclear translocation of nuclear factor kappa B (p65) by immunofluorescence, and reverse-transcription polymerase chain reaction for genes encoding interleukin-1β, tumor necrosis factor alpha, and nuclear factor kappa B (p65) during an inflammatory condition. RESULTS: Both PRP and liquid PRF increased the migration and proliferation of hDPCs when compared with the control group, whereas liquid PRF showed a notable significant increase in migration when compared with PRP. Furthermore, liquid PRF induced significantly greater alkaline phosphatase activity, alizarin red staining, and a messenger RNA expression of genes encoding collagen type 1 alpha 1, dentin sialophosphoprotein, and dentin matrix protein 1 when compared with PRP. When hDPCs were cultured with LPS to stimulate an inflammatory environment, a marked decrease in dentin-related repair was observed. When liquid PRF was cultured within this inflammatory environment, the reduced regenerative potential in this LPS-produced environment was significantly and markedly improved, facilitating hDPC regeneration. The messenger RNA expression of inflammatory markers including tumor necrosis factor alpha, interleukin-1β, and p65 were all significantly decreased in the presence of liquid PRF, and, furthermore, liquid PRF also inhibited the transport of p65 to the nucleus in hDPCs (suggesting a reduced inflammatory condition). CONCLUSIONS: The findings from the present study suggest that liquid PRF promoted greater regeneration potential of hDPCs when compared with traditional PRP. Furthermore, liquid PRF also attenuated the inflammatory condition created by LPS and maintained a supportive regenerative ability for the stimulation of odontoblastic differentiation and reparative dentin in hDPCs.