| BackgroundOsteoporosis is a major public health problem through its association with fragility fracture,especially as the aging population has been increased in the recent years.Osteoporosis can lead to osteogenic ability reduction,resulting in delayed bone healing or nonunion of the fractures.The evidence supporting the close relation of BMPs to bone metabolism and the identification of the double role of BMPs in the process of fracture healing and the pathogenesis of osteoporosis makes them attractive target molecules for the development of anabolic therapies for the prevention as well as for the treatment of osteoporotic fractures.Local delivery of rhBMP-2 is U.S.FDA approved to promote spinal fusion and fracture healing.Allow for BMP-2 expression is decreased and delayed in osteoporotic fractures,many experimental studies have begun to apply BMP-2 to promote and improve the healing of osteoporotic fractures,and satisfactory results have been achieved.While successful,the uses of rhBMP-2 have their drawbacks.The optimum release pattern of rhBMP-2 has not yet been established,and excess dosage with rhBMP-2 may be dangerous because rhBMP-2 have other activities such as organogenesis,cell differentiation,cell proliferation,and apoptosis.Moreover,the costly processes of recombinant protein production and purification make this therapy very expensive.Considering that rhBMP-2 side effects are dose-dependent,there is a great necessary to minimize large doses of rhBMP-2 currently used.One possible alternative to overcome this problem is synchronous drug combinations to enhance the potency of the rhBMP-2 used.The advantages of combining local delivery of rhBMP-2 with systemic or local anti-osteoporosis treatments have also been studied for enhancing bone formation.Meanwhile,osteoporosis is a systemic skeletal disease.Hence,appropriate simultaneous management of both the osteoporotic fracture and osteoporosis is logical and essential.The current clinical treatment regimens for osteoporosis are anti-resorptive drugs,which maintain bone mass by inhibiting osteoclast function,such as estrogen,estrogen receptor analogues,calcitonin and bisphosphates.However,recent reports on the negative effects of fracture healing and potential complications,including breast cancer,uterine bleeding and cardiovascular events have raised concern about their longterm use.Osteoporotic fractures are more common in middle-aged patients,and delayed fracture healing is more common in this group.Delayed healing may occur in up to 1/3 of femoral shaft and tibial fractures.Therefore,the goal of the new generation of drugs to improve osteoporosis fracture healing is not only to inhibit bone resorption,but also to stimulate the activity of osteoblasts and accelerate bone formation.In Chinese traditional medicine,the bone is governed by kidney.Therefore,"kidney deficiency" is regardedas the underlying cause of all skeletal pathologies.In traditional Chinese medicine's,the kidney is the general term for the endocrine function of the whole body,which is consistent with the endocrine function of the modern medical kidney in the regulation of calcium and phosphorus metabolism.Chinese herbal medicine has been widely used to prevent and treat diseases for thousands of years.Psoralen is a coumarin-like derivative extracted from the dried fruit of Psoralea corylifolia L,which is a well-known traditional Chinese medicine,described as "material for bone strengthening" based on the traditional Chinese medicine theory.It has been reported that Psoralen acts through the activation of BMPs signaling to promote osteoblast differentiation and could stimulate differentiation of bMSCs to osteoblasts.In vivo,Psoralen can promotes bone mass and has the effect to protect osteoporotic fracture in OVX mice.AimThe aim of the study was to evaluate the effect of combination therapy with BMP-2 and psoralen on fracture repair in ovariectomized mice.Serum measurements of bone alkaline and the undisturbed left femora were also performed to study whole bone metabolic.Materials and MethodsStudy one:One hundred and thirty 10-week-old female C57BL/6 mice were obtained from the Hubei Research Center of Laboratory Animal(Wuhan,China).When the mice reached twelve weeks of age,five mice were randomly designated as the sham-operated group in which mice received bilateral laparotomy but the ovaries were left in place.Except sham-operated group,all mice were bilateral ovariectomized(Ovx)via the dorsal approach.After ovariectomy,mice were left untreated for 6 weeks.Then five sham mice and five Ovx mice were sacrificed to measure the estrogen levels,and the femoral metaphysis was harvested for histological evaluation for confirmtion of successful establishment of the Ovx model.Study two:Once the successful establishment of the Ovx model was confirmed,all Ovx animals were randomly stratifed into one of the following groups: Model group,Psoralen group,rhBMP-2 group and Psoralen + rhBMP-2 group.A standardized fracture was created on the mid-diaphysis of each group respectively and stabilized with an intramedullary pin.The mice of the present study were sacrifced on day 10 and 21 after injury and their limbs were harvested for further analysis [x-ray,micro CT,histological,RT-qPCR,and mechanical analysis].Study three: At 3 weeks after fracture operation,the serum biochemical indexes of bone metabolism and estrogen levels were measured,and the histological changes of distal femur were observed to study the effects of psoralen combined with rhBMP-2 on osteoporosis in ovariectomized mice.ResultsStudy one:The bilateral OVX 6 weeks led to the body weight of mice was significantly increased(P<0.05),estrogen levels and wet weight of uterus significantly decreased than measured in sham mice(P<0.05),respectively.The effect of decreased estrogen levels could also been shown in a gonadal hypotrophy in OVX mice.Compared with sham mice,the bone trabeculae of mice in the OVX group were arranged sparsely,were disorderly and indicated significant resorption,were thinner or had disappeared altogether and the remaining connections were incomplete.These results confirmed the establishment of osteoporosis in OVX mice.Study two: Ten days after fracture,X-ray and histology analysis revealed more mineralized periosteal callus and proliferative osteoblasts cell could be observed in Psoralen+ rhBMP-2 group which formed better callus structure and even more radiopaque callus compared to all other groups.The Micro-CT surface analyses at day 21 after fracture approved the contact radiographic findings.The quantitative results revealed Psoralen+ rhBMP-2 group increased BV,BV/TV and TMD than other groups(P<0.05).In histology evaluations,Psoralen+ rhBMP-2 group displayed significantly more mineralized and lamellar callus compared with other groups which showed abundant amounts of chondroid callus fracture area and immature bone formation.More sparse osteoclasts were observed in Psoralen+rhBMP-2 group,but with similar shape in all groups.After healing for 21 days,immunohistochemical staining showed the AO value of BMP-2 positive in Psoralen+ rhBMP-2 and psoralen group was significantly higher than that in model group and rhBMP-2 group(P < 0.05).Compared with model group,rhBMP-2 group(P<0.05),Psoralen group(P<0.05)and Psoralen+ rhBMP-2 group(P<0.01)increased in MAR,respectively.Osterix and Osteocalcin mRNA expression levels were significantly higher in Psoralen+ rhBMP-2 group than in model group(P<0.05)at all time points examined.The highest values for maximum load,maximum stress and the torsional stiffness were seen Psoralen+ rhBMP-2 group and were significantly higher compared with model group(P<0.05).Study three:At 21 days,BALP showed a significantly higher concentration in Psoralen+ rhBMP-2 group compared with model group(P<0.05).BALP also increased in Psoralen and rhBMP-2 group,which,however,did not prove statistically significant compared with model group.CTX-1,a bone absorption marker,decreased significantly in Psoralen+ rhBMP-2 group and Psoralen group(P<0.05).Analysis of estrogen,no differences could be detected between among the groups(P>0.05).The day 21 time point after fracture,the bone trabeculae of the left distal femurs in rhBMP-2 group was similar with model group showing a notable sparseness in the trabecular area and trabecular numbe with trabecular in continuity.Psoralen+ rhBMP-2 group and Psoralen group,systemically treated with Psoralen displayed an significant improvement in the bone trabecula arrangemen compared with that of the model group;the number of trabeculae was increased and trabecular connections were enhanced.The osteoclast analysis showed that Psoralen+ rhBMP-2 group and Psoralen group had statistically smaller numbers of TRACP+ cells than the model group(P<0.05),whereas the rhBMP-2 group was not different.The result of this study showed that psoralen,as a natural phytoestrogen,may play a role in estrogen receptor selectivity,but does not significantly increase the level of estrogen in vivo.The combined use of psoralen can mobilize the local and systemic osteoblast activity of fracture,inhibit osteoclasts to a certain extent to reduce bone resorption and promotes the bone tissue microstructure in ovariectomized mice.Conclusion1.Using 12-week-old C57BL/6J female mice and ovariectomized for 6 weeks can better simulate the effect of estrogen deficiency on bone loss in postmenopausal women.It is a reliable method to make animal models of osteoporosis.2.The combination of psoralen and rhBMP-2 can achieve rapid targeted release of rhBMP-2 at the early stage of fracture,stimulate the proliferation and differentiation of osteoblasts.At the same time,continuous oral psoralen can obviously promote the expression level of Osteocalcin,Osterix genes and BMP-2 in the middle and late stage of fracture healing,collaborative increase the activity of osteoblasts and accelerate the repair of fracture.At the same time,psoralen can balance the function of osteoclasts in the fracture area,and does not cause excessive inhibition of osteoclasts,does not affect the process of osteoporosis,and has an early repair effect on the fracture of osteoporotic mice.This result indicates that the combination of psoralen and rhbmp-2 may reduce the dosage of rhbmp-2 and psoralen alone,and improve the clinical effect and cost-effectiveness.3.Psoralen,as a natural phytoestrogen,may play a role in the selectivity of estrogen receptor,not significantly increase the level of estrogen in vivo.The combined use of psoralen can mobilize the activity of osteoblasts both locally and systemically,and inhibit osteoclasts to a certain extent to reduce bone resorption and improve the microstructure of osteoporosis bone tissue. |
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