Icariin attenuates hypoxia-induced oxidative stress and apoptosis in osteoblasts and preserves their osteogenic differentiation potential in vitro.

OBJECTIVES: Icariin, a prenylated flavonol glycoside isolated from traditional Chinese medicinal herb of the genus Epimedium, has been demonstrated to be a potential alternative therapy for osteoporosis, and its action mechanism so far has been mainly attributed to its phytoestrogenic property. As blood supply to bone is considerably reduced with ageing and by the menopause, we hypothesized that icariin treatment would reduce bone loss by preventing ischaemia-induced hypoxic damages to bone. MATERIALS AND METHODS: To investigate effects of icariin treatment on cultured rat calvarial osteoblasts exposed to hypoxic conditions (2% oxygen). RESULTS: Compared to normoxic control, cell viability decreased with time to 50% by 48 h in the hypoxic group, and icariin attenuated the reduction, dose dependently, with 10(-6) and 10(-5)  m concentrations showing significant protective effects. Icariin also inhibited increase of lactate dehydrogenase activity in culture media. Measurements on oxidative stress, cell cycling and cell survival indicated that icariin protected osteoblasts by reducing production of reactive oxygen species and malondialdehyde, increasing superoxide dismutase activity, arresting the cell cycle and inhibiting apoptosis. Icariin also preserved osteogenic differentiation potential of the hypoxic cells in a dose-dependent manner, compared to the hypoxia alone group, as revealed by increased levels of RUNX-2, OSX and BMP-2 gene expression, alkaline phosphatase activity, and formation of mineralized nodules. CONCLUSIONS: Our results demonstrated that icariin attenuated oxidative stress and apoptosis and preserved viability and osteogenic potential of osteoblasts exposed to hypoxia in vitro, and suggested that its anti-osteoporotic effect may be attributed to its anti-hypoxic activity and phytoestrogenic properties.

Comparison between 8-prenylnarigenin and narigenin concerning their activities on promotion of rat bone marrow stromal cells' osteogenic differentiation in vitro.

A number of recent studies have suggested that flavonols (a class of phytochemical with many biological activities), might exert protective effects against post-menopausal bone loss. In the present study, we compared naringenin (NG) and 8-prenylnaringenin (PNG), two major naturally occurring flavonols, on in vitro differentiation of osteoblasts and bone resorbing activity, of rat bone marrow stromal cells (BMSCs). Our results indicated that both compounds, at 10(-6)  m, enhanced BMSCs' differentiation. Then effects of the two compounds at 10(-6)  m on ALP activity, osteocalcin secretion and calcium deposition, were compared over a time course. Numbers and areas of colonies stained for ALP (CFU-F(ALP) ) expression, and mineralized bone nodules, were histochemically analysed after 12 days and 16 days osteogenic induction, respectively. Expression of BMP-2, OPG, OSX, RUNX-2 genes and p38MAPK protein were examined using real-time PCR and western blotting, respectively. The data presented indicate that PNG, significantly enhanced the rat BMSCs' differentiation and mineralization through the BMP-2/p38MAPK/Runx2/Osterix signal pathway, greater than did NG. In conclusion, PNG has a more pronounced ability to enhance osteoblast differentiation and mineralization, than NG.

Icariin inhibits the osteoclast formation induced by RANKL and macrophage-colony stimulating factor in mouse bone marrow culture.

Icariin is a prenylated flavonol glycoside contained in the herb Epimedium, which has long been used to improve bone fracture healing or prevent osteoporosis because of the belief that the herb has bone-strengthening action. We have previously demonstrated that icariin enhances the osteogenic differentiation of rat bone marrow stromal cells, and partially explained the bone-strengthening mechanism of the herb. In the present study, the effect of icariin on osteoclastogenesis and bone resorption activity was investigated in mouse bone marrow culture. It was found that icariin dose-dependently inhibited the growth and differentiation of hemopoietic cells from which osteoclasts were formed. Far less TRAP+ multinuclear cells appeared in the 10 microM icariin group than in the control. The bone resorption pits formed in the 10 microM icariin group was also significantly less than that of the control. RT-PCR analysis showed that the gene expression of TRAP, RANK and CTR was obviously lower than that of the control. It can be concluded that icariin has the ability to inhibit the formation and bone resorption activity of osteoclasts, which suggests that icariin should be the effective component for the bone-strengthening action of herb Epimedium.

Icariin enhances the osteogenic differentiation of bone marrow stromal cells but has no effects on the differentiation of newborn calvarial osteoblasts of rats.

Since the total flavonoid extract (TFE) of Epimedium herb was found to prevent osteoporosis induced by ovariectomy in rats, we have been attempting to identify the exact compound responsible for the bone-strengthening activity. In this experiment, four flavonoid extracts were obtained from Epimedium sagittatum (Siebold & Zucc.) Maxim, which contained 25.3%, 51.2%, 82.3% and 99.2% icariin respectively. They were separately supplemented into the culture media of newborn rat calvarial osteoblasts (ROB) or primary rat bone marrow stroma cells (rMSCs) at 0.1, 1, 10 and 100 microg/ml respectively, in order to observe their effects on the cells. Not any appreciable effect was found on the differentiation of ROB, but an enhancing effect on the osteogenic differentiation of rMSCs was found, and the enhancing degree was icariin-dependent, that is, a higher concentration of icariin in the extract caused more mineralized bone nodules and higher calcium deposition levels. The gene expressions involved in osteogenesis were also improved which was revealed by RT-PCR, including alkaline phosphatase, bone matrix protein (osteocalcin, osteopontin, bone sialoprotein) and cytokines (TGF-beta1 and IGF-I). The effect of icariin on cell proliferation was assayed by the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Icariin inhibited the proliferation of rMSCs and ROB when its concentration was higher than 10(-5) microM (6.7 microg/ml), no stimulative effect was found. The above results indicated that icariin may exert bone-strengthening activity by enhancing the osteogenic differentiation of MSCs, which partially explains the anti-osteoporosis action of Epimedium herb.

Icariin, a flavonoid from the herb Epimedium enhances the osteogenic differentiation of rat primary bone marrow stromal cells.

The herb Epimedium has long been used in Traditional Chinese Medicine to treat bone fracture and prevent osteoporosis. Researchers believe that the flavonoids contained in the herb are the effective component for this activity. However, no single flavonoid has been studied for its effect on bone-related cells. In the present study, icariin, one of the major flavonoids of the herb, supplemented the primary culture medium of rat bone marrow stromal cells (rMSCs) at 0.1 microM , 1 microM and 10 microM respectively. It was found that icariin stimulated the proliferation of rMSCs and increased the number of CFU-F stained positive for alkaline phosphatase in a dose-dependent manner. Icariin also dose-dependently increased the alkaline phosphatase activity, osteoalcin secretion and calcium deposition level of rMSCs during osteogenic induction. The addition of 10 microM icariin caused four times more mineralized bone nodules to be formed by rMSCs than in the control. The results demonstrated that icariin should be an effective component for bone-strengthening activity, and one of the mechanisms is to stimulate the proliferation and enhance the osteogenic differentiation of MSCs.

The serum of rats administered flavonoid extract from Epimedium sagittatum but not the extract itself enhances the development of rat calvarial osteoblast-like cells in vitro.

The flavonoid extract from Epimedium sagittatum (FES) has been found by us to be effective in preventing osteoporosis induced by ovariectomy in rats. In the present study, the effect of FES on the development of rat calvarial osteoblast-like (ROB) cells was investigated. No appreciable effect was observed when ROB cells were exposed to FES in vitro. However, serum isolated from rats administered FES orally was able to significantly stimulate the proliferation as well as the osteoblastic differentiation of ROB cells compared to serum from control rats. The results indicate that the serum of rats administered FES contains active metabolites ofFES that enhance the development of osteoblasts, while the original form of FES itself is inactive.

[Repair of soft tissue defect with combined skin flap].

OBJECTIVE: To investigate the clinical result in repair of soft tissue defect with combined skin flap vascularized by pedicle on the one end and vascular anastomosis on the other end. METHODS: From October 1990 to August 1995, 5 cases with soft tissue defect at the extremities and 1 cases with sacral bed sore were repaired by the combined skin flaps transfer, ranged from 15 cm x 30 cm to 16 cm x 70 cm in defect, among them, 5 cases with myocutaneous flap and 1 case with skin flap, and the size of the combined skin flaps was 15 cm x 40 cm to 12 cm x 80 cm. RESULTS: All the flaps were survived with satisfactory effect. Followed up 3 to 6 years, there was no obvious complication. CONCLUSION: Transfer of combined skin flaps vascularized by pedicle and vascular anastomosis is suitable to repair the soft tissue defect, especially in large area defect.

An implant of a composite of bovine bone morphogenetic protein and plaster of paris for treatment of femoral shaft nonunions.

Sixteen patients with nonunion of femoral shaft fractures were treated by implantation of a composite of bovine bone morphogenetic protein and plaster of Paris (bBMP/PLP). There was an average of 1.5 surgical procedures per patient attempting union prior to bBMP/PLP implantation The implant was prepared in an aggregate of 50 mg bBMP in 1 gram of plaster of Paris. It was implanted directly to fill the fracture gaps and packed around the fracture lines. Seventeen operations were done on the 16 patients. Union was obtained in 16 of the 17 operations. The average time of union for the 16 successful operations was 5.7 months. There were no significant clinically manifested postoperative complications.

The second dorsal metacarpal flap with vascular pedicle composed of the second dorsal metacarpal artery and the dorsal carpal branch of radial artery.

A pedicle second dorsal metacarpal flap, comprising the second dorsal metacarpal artery, the partial carpal arterial arch of the dorsal hand, and the dorsal carpal branch of radial artery, was designed. The flap may be rotated through two axes of rotation, one at the entry of carpal branch of radial artery into the first dorsal interosseous muscle and one at the entry of the recurrent cutaneous branch arising from the second dorsal metacarpal artery into the skin. The method introduced can increase as much as possible the length of the vascular pedicle of the second dorsal metacarpal flap. Thus, it can cover a small defect at a more distant area. If the width of this flap is not more than 3 cm, the donor site can be closed directly. The flap had been used in five cases with no necrosis of the flap or complication of the donor site. In further investigation of the postoperative patients, no stiffness and tightness have been observed through a short period of rehabilitation of the hand. The results are satisfactory. The anatomy, the operative technique, and three selective cases are described here.

The sartorius myocutaneous island flap.

The vascular supply to the distal part of sartorius m. was studied in 37 limbs by dissection under magnification and after black ink and latex injections. The muscle or myocutaneous island flap pedicled on the saphenous artery (SA) is supplied by a retrograde circulation through anastomoses of the SA with the perforating branches of the posterior tibial artery and the medial inferior genicular artery. Three different vascular patterns were defined. Retrograde injection also showed good perfusion of the SA. The flap is useful for covering around the knee, the proximal and middle thirds of the lower leg, and the end of the amputation stump. Raising of the flap has not resulted in any functional or cosmetic defect. Eighteen operations have so far been performed, six using the muscle island flap and twelve using the myocutaneous island flap, and all survived completely.

Free medial gastrocnemius myocutaneous flap transfer with neurovascular anastomosis to treat Volkmann's contracture of the forearm.

Some articles have reported the use of medial gastrocnemius transfer to repair soft-tissue defects caused by trauma and inflammation (Morris, 1978; Feldman et al., 1978; Arnold and Mixter, 1983). However, we have not found any report describing the use of free medial gastrocnemius myocutaneous flap transfer with neurovascular anastomosis to treat Volkmann's contracture of the forearm. Since 1982, 20 cases of Volkmann's contracture of the forearm have been treated by transfer of medial gastrocnemius flap with neurovascular anastomosis. These 20 patients ranged in age from 6-18 years, with a mean of 10 years. There were 18 males and 2 females. All cases resulted from ischaemia of the extrinsic and intrinsic muscles of the forearm and hand. The most common causes were severe supracondylar fractures or, less frequently, fracture-dislocations of the elbow. Six to twenty months after operation, the transplanted muscles exhibited normal electromyographic potentials, good volume and contractile power with satisfactory functional recovery of the forearms. The deformities of the hand and wrist were also corrected. In this paper we describe the anatomy of the donor site and the operative procedure.

[Significance of CT scanning in diagnosis of herniated lumbar disc].

During March 1986 to September 1988, 52 patients complaining lumbago and/or sciatica diagnosed by CT scanning as herniated lumbar disc were operated upon. The accuracy of CT diagnosis for lumbar disc herniation proved by surgery was 47 cases (90.4%). CT findings and surgical findings did not accord in 5 cases. Of these, some were because of technical problems of the CT scanning and some were faults of surgical technique. The merits and faults of CT scanning for diagnosis of protrusion of lumbar disc and its CT manifestations were discussed.