Effect Of Puerarin On Osteogenic Differentiation In Vitro And On New Bone Formation In Mid-palate Suture Of Rats

Malocclusion is one of the common clinical symptoms of orthodontics,its manifestation is often three-dimensional,including vertical(height),sagittal(length)and lateral(width)incongruities,the first two of which are easy to attract the attention of doctors and patients because of their intuitive impact on the aesthetic appearance of the face,while the problem formed by the transverse underdevelopment of the maxilla is mostly functional or non-intuitive and are often ignored by doctors and patients.Transverse underdevelopment of the maxilla is a common concomitant symptom of many malocclusions which manifested as narrow maxillary,dental arches,high arch of the palatal vault,crowding of dentition,crossbite of anterior or posterior teeth and oversized buccal gallery.Rapid maxillary expansion(RME)is often used in clinical work to correct this type of malocclusion.RME is a distraction osteogenesis(DO)surgical technique that generates new bone between separated bone segments via the application of continuous and stable force.During the RME,the mid-palatal suture is expanded by mechanical tension,osteogenesis is activated,osteoblasts(OB)proliferate and differentiate,new bone continues to deposit on the edge of the bone suture.Meanwhile,the width of dental arch increase,dentition crowding is relieved.In the process of continually remodeled of the skeleton,the equilibrium between bone formation by osteoblasts and bone resorption by osteoclasts is vital to the maintenance of bone integrity.In recent years,bone marrow mesenchymal stem cells(BMSCs)have been widely used in bone regeneration area due to their self-renewal,osteogenesis capabilities,and low immunogenicity.Numerous basic experiments and clinical trials have focused on the impact of BMSCs in bone tissue engineering and have explored it in depth.Traditional Chinese medicine is one of the brilliant treasures in our history.In recent years,the therapy of traditional Chinese medicine has been favored not only by domestic doctors and patients,but also by many international scholars.Puerarin(C21H20O10),isolated from several leguminous plants such as Puerarin tuberosa and Puerarin lobata,is a kind of isoflavone glycoside and easy to obtain and purify.Owing to its abundant pharmacological activities,puerarin has been adopted for the treatment of various disorders.Objectives:Puerarin is a phytoestrogen that possesses various pharmacological effect,and several research have revealed the relationship between puerarin and bone metabolism.This study was aimed to evaluate the potential influence of puerarin on the proliferation and osteogenic differentiation of rat BMSCs as well as on new bone formation following RME model in rats.Materials and methods:(1)Rat BMSCs were adopted and induced by osteogenesis and adipogenesis,the cell proliferation was detected by cell-counting kit-8(CCK-8)assay and clone experiment.Alkaline phosphatase(ALP)activity,alizarin red staining,the osteogenesis-related genes and protein expression levels were used to detect osteogenic differentiation of BMSCs.(2)Eighteen 6-week-old male Wistar rats were divided into 3 groups:group 1 without any treatment,group 2 received RME and saline solution(15 mg/kg/d),group 3 received RME and puerarin solution(15 mg/kg/d).After 2 weeks,Micro-computed tomography(Micro-CT),Hematoxylin and eosin(HE)staining,and Masson staining were used to detect the new bone formation and morphological changes.ALP and bone morphogenetic protein 2(BMP2)expression levels in mid-palatal suture were evaluated by immunohistochemical staining.Results:(1)Puerarin upregulates cell proliferation dose-dependently.ALP activity and mineralized matrix generation were clearly enhanced at certain specific puerarin concentrations(10-5 and 10-6 M);the expression levels of the osteoblast-related genes and proteins were increased.(2)The measurement of Micro-CT imaging and the histological examinations revealed that puerarin promoted bone regeneration in rat mid-palatal suture,up-regulated the expression of ALP and BMP2.Conclusions:Puerarin enhance BMSCs osteogenesis in vitro and exerts a beneficial impact on bone regeneration in rat mid-palatal suture in vivo.