The effect of aqueous extract of Prunus dulcis on tibial bone healing in the rabbit.

BACKGROUND: Bone fractures are medical emergencies that require prompt intervention to help return bone to its normal function. Various methods and treatments have been utilized to increase the speed and efficiency of bone repair. This study aimed to investigate the treatment effects of Prunus dulcis aqueous extract on tibial bone healing in rabbits. METHODS: All animals were distributed in five groups with six rats in each group, including the sham group, the control group in which tibial lesion was made and received distilled water, treatment groups with 150 mg kg-1, 300 mg kg-1 doses of Prunus dulcis extract, and osteocare treated group. Biochemical blood factors including calcium, phosphorus, and alkaline phosphatase (on days 0, 10, 30, and 50), biomarkers of oxidative stress such as GPx, CAT, and MDA (on days 10 and 30), radiological evaluation, histopathological parameters, and osteocalcin immunohistochemical expression were assessed. RESULTS: The data showed calcium levels in the treatment groups increased significantly from day 10 to day 50, respectively, and blood phosphorus levels decreased from day 10 to day 50 in the treatment groups. Alkaline phosphatase initially increased and then decreased in treatment groups. In the treatment groups, GPx and CAT levels significantly increased, and the serum amount of MDA reduced. The best antioxidant results were related to the extract-treated group with a higher dose. Radiographic score was significantly higher in the treatment groups than the control group on day 30. Based on the pathological findings, the healing occurred faster in the extract-treated group with a higher dose. Osteocalcin expression was significantly higher in the control group than that in the treatment groups. CONCLUSIONS: Treatment with Prunus dulcis extract with a dosage of 300 mg/kg accelerated tibial bone healing in rabbits.

Guhong Injection promotes fracture healing by activating Wnt/beta-catenin signaling pathway in vivo and in vitro.

Guhong Injection (GHI), composed of aceglutamide and safflower aqueous extract, has been applied to the clinical treatment of orthopedic diseases, but the relevant mechanism by which GHI exerts effects on bone remodeling has not been reported. In the present study, we investigated the effects of various concentrations of GHI (2.5, 5 and 10 ml/kg) in accelerating rat tibia healing progress by observing haematoxylin and eosin (HE) stained sections, detecting the activity of bone metabolism biochemical markers such as bone morphogenetic protein-2 (BMP-2), transforming growth factor-beta (TGF-beta), osteocalcin (OC) and C-terminal crosslinking telopeptide of type Ⅰ collagen (CTX-1) in rat serum, as well as measuring the expressions of collagen I (COL-1) and collagen II (COL-2) in rat tibia. Also, we investigated the effects of different concentrations of GHI (30, 60 and 90 µl/ml) on the proliferation and differentiation of osteoblasts (OBs) through proliferating cell nuclear antigen (PCNA), alkaline phosphatase (ALP) and type I collagen (COL-1). At the same time, the expression of important factors of Wnt/beta-catenin signaling pathway including Wnt-3a, beta-catenin, disheveled-1 (Dvl-1), glycogen synthase kinases-3beta (GSK-3beta), lymphoid enhancing factor-1 (LEF-1) and axis inhibition protein-2 (Axin-2) after GHI intervention was detected by quantitative real-time PCR (q-PCR), immunohistochemistry and Western blotting. In vivo, rats of tibia fracture model treated with intraperitoneal injection (ip) of GHI had more mature fibroblasts, as well as shorter period formation of new bone. The levels of BMP-2, TGF-beta and OC in rat serum were significantly up-regulated, while the level of CTX was down-regulated. After 4 weeks of drug treatment, the level of COL-1 in the rat tibia increased, but there was no significant change in the level of COL-2. In vitro, after drug intervention, the number of OBs increased significantly, the activities of PCNA, ALP and COL-1 were enhanced. Treatment with GHI increased the mRNA and protein expression of Wnt-3a, beta-catenin, Dvl-1 and LEF-1, and decreased the expression of mRNA of Axin-2 and GSK-3beta. All results demonstrate that GHI accelerates the proliferation of OBs and shortens the recovery time of bone structure, and the Wnt/beta-catenin signaling pathway is involved in the regulation process.

Effects of hyperbaric oxygen therapy on open tibial fractures in rabbits after transient seawater immersion.

OBJECTIVE: To explore the effect and mechanism of hyperbaric oxygen (HBO2) therapy of open tibial fractures in rabbits after transient seawater immersion. METHODS: Forty-eight (48) New Zealand rabbits were randomly and averagely divided into an HBO2 therapy group (Group A) and a control group (Group B). All rabbits were subjected to unilateral open tibial fractures, while immersed in artificial seawater (20-22 °C) for three hours prior to debridement and external fixation. Group A was treated with HBO2 at 2 atmospheres absolute (ATA) for 50 minutes once daily for two weeks; Group B received postoperative routine treatments only. The fracture zone in each group was compared by radiological, histological and immunohistochemical examinations. RESULTS: In Group A, bony callus and mature osteocytes without infiltration of inflammatory cells were observed in the fracture zone. Vascular endothelial growth factor (VEGF) was expressed mainly in the cytoplasm of osteoblasts, chondrocytes and osteocytes, and exhibited significant changes at different time points. The gray value of bony callus in Group A was 190.58 ± 7.52; that of Group B was 144 ± 8.11. Difference between the groups was statistically significant (P ⟨ 0.01). The content of malondialdehyde (MDA) in Group A was significantly lower than Group B (P ⟨ 0.01), and the activity of superoxide dismutase (SOD) in Group A was higher than Group B (P ⟨ 0.01) at four weeks. There were no significant differences in MDA content and SOD activity between groups at eight and 12 weeks. CONCLUSIONS: HBO2 treatment of open tibial fractures in seawater can reduce the inflammatory reaction and reperfusion injury, and promote osteocytic proliferation and fracture healing.

Effects of Potentilla fulgens as a Prophylactic Agent in Tibial Defects in Rats.

OBJECTIVE: To investigate the effects of Potentilla fulgens as a prophylactic agent on tibial defects in the rat. STUDY DESIGN: Twenty-eight male Wistar albino rats weighing 200-215 g each were divided into 3 experimental groups. The tibial bone defect group served as the control group. The experimental groups were Potentilla fulgens with tibial defect (14 days) and Potentilla fulgens with tibial defect (28 days). Extract of Potentilla fulgens was mixed with water (400 mg/kg/day) and given to groups 14 and 28 as drinking water. The histopathological and immunohistochemical characteristics of each tibial bone cavity within each group were observed. The trabecular new bone formation was evaluated by expression rate of osteonectin and osteopontin. RESULTS: In the Potentilla fulgens + tibial defect group (14 days), trabecular bone had started combining extensive new bone formation, osteocyte cells were evident, and lamellar bone was formed. Osteoblasts showed a positive reaction with osteonectin. Osteopontin expression was positively observed between fibrous structures and in the osteoblast and osteocyte cells. This can be considered indicative of new bone formation. In the Potentilla fulgens + tibial defect group (28 days), an increase in expansion in trabecular bone and myeloid tissue was observed. Osteoblastic activity and osteocyte cells began to be observed in new bone fragments. CONCLUSION: In our study we show that Potentilla fulgens extract provided a protective effect on new bone formation and aided in the development of osteocytes and secretion of matrix in osteoblasts. Additionally, we show the inductive effect of the extract on new bone formation. In particular, the expression of osteopontin and osteonectin was also supported with the Western blot technique on the development of osteoblasts and osteocytes, showing a similar trend with our results.

In vivo heat-stimulus-triggered osteogenesis.

Several studies have reported that heat stress stimulates the activity of osteoblastic cells in vitro. However, few have addressed the effects of heat stress on osteogenesis in vivo, nor have the optimal temperatures for bone formation been determined. The aim of the present study was to investigate the effects of hyperthermia treatment on osteogenesis in a rat tibial defect model. Forty-four Sprague Dawley rats were divided into two groups with or without hyperthermia treatment. A 3-mm circular defect in the proximal tibia filled with magnetite cationic liposomes embedded in alginate beads was subjected to hyperthermia treatment (43-46 °C). Radiological assessment at 2 weeks after the treatment showed that significantly stimulated osteogenesis was observed in the hyperthermia group as compared to the control group (p = 0.003). Histomorphometrical analysis at 2 weeks revealed a significant increase of newly formed bone in the hyperthermia group, compared with the control group (p < 0.001). Area of newly formed bone in each hyperthermia group was significantly increased as compared with the control group (43 °C; p = 0.005, 44 °C; p = 0.019, 45 °C; p = 0.003, and 46 °C; p = 0.003, respectively). Alkaline phosphatase was overexpressed at the surfaces of newly formed bone adjacent to magnetite cationic liposome implantation. Our results demonstrate for the first time that heat stimulus accelerates osteogenesis in vivo, and may thus be of interest as a novel and promising tool to induce osteogenesis clinically as well.

Femoral and tibial stress fractures associated with vitamin D insufficiency.

We describe a case highlighting the need to consider hypovitaminosis-D when investigating background causation and treatment of femoral and tibial stress fractures. The case also suggests that prescribing calcium and vitamin D supplementation may help with fracture healing in soldiers presenting with stress fractures who may have unrecognised hypovitaminosis-D which if left untreated may delay fracture healing.

Effects of biosilicate(®) scaffolds and low-level laser therapy on the process of bone healing.

OBJECTIVE: This study aimed to investigate the in vivo tissue performance of the association of Biosilicate(®) scaffolds and low-level laser therapy (LLLT) in a tibial bone defects model in rats. BACKGROUND DATA: Many studies have been demonstrating the osteogenic potential of Biosilicate and LLLT. However, there is a need to investigate the effects of both treatments for bone consolidation. METHODS: The animals were divided into control group (CG), Biosilicate scaffold group (BG), and Biosilicate scaffolds plus LLLT group (BLG). Animals were euthanized after 15, 30, and 45 days post-injury. RESULTS: The histological analysis revealed that all the experimental groups showed inflammatory infiltrate and granulation tissue, at the area of the defect at day 15. After 30 days, CG still showed granulation tissue and bone ingrowth. Both Biosilicate groups presented newly formed bone and interconected trabeculae. At 45 days, CG showed immature newly formed bone. A more mature newly formed bone was observed in BG and BLG. On day 15, BG demonstrated a statistically higher expression of cyclooxygenase (COX)-2 compared with CG and BLG. No statistically significant difference was observed in COX-2 immunoexpression among the groups at 30 and 45 days. Similar expression of bone morphogenetic protein (BMP)-9 was demonstrated for all experimental groups at 15 and 30 days. At 45 days, the BMP-9 immunoexpression was statistically upregulated in the BLG compared with the CG and BG. No statistically significant difference was observed in the receptor activator of nuclear factor kappa-B ligand (RANKL) immunoexpression among the groups in all periods evaluated. Biosilicate groups presented a decrease in biomechanical properties compared with CG at 30 and 45 days post-surgery. CONCLUSIONS: Our findings suggest that Biosilicate presented osteogenic activity, accelerating bone repair. However, laser therapy was not able to enhance the bioactive properties of the Biosilicate.

Adjunctive hyperbaric oxygen therapy in the treatment of atrophic tibial nonunion with Ilizarov external fixator: a radiographic and scintigraphic study in rabbits.

OBJECTIVE: The aim of this experimental study was to determine the effects of adjunctive hyperbaric oxygen therapy (HBO) on atrophic tibial nonunion treatment using Ilizarov external fixator. METHODS: Twenty New Zealand white rabbits were randomly divided into two equal groups. A circular external fixator was applied to the right tibia of all the rabbits. A 5-mm bone block was resected and a tibial pseudarthrosis was obtained after a 6-month waiting period. The experimental group rabbits (n=10) underwent daily 2.5 ATA HBO therapy for 2 hours for 20 days and the control group rabbits (n=10) did not receive any corresponding treatment. Osteoblastic activity was evaluated with bone scintigraphy on days 30 and 90. Fracture healing was evaluated by plain radiographs on days 30 and 90. RESULTS: On Day 30, radiological scores were statistically similar in both groups (p=0.167). However, on Day 90, the experimental group displayed significantly higher radiological scores (p<0.001). Osteoblastic activity was also higher in the experimental group on both scintigraphic assessments (p=0.005 and p=0.001). CONCLUSION: The results of this study suggest that HBO can be used as a supplementary therapy in the management of atrophic tibial nonunion.

Raman spectroscopy validation of DIAGNOdent-assisted fluorescence readings on tibial fractures treated with laser phototherapy, BMPs, guided bone regeneration, and miniplates.

OBJECTIVES: We aimed to assess through Raman spectroscopy and fluorescence the levels of calcium hydroxyapatite (CHA) and lipids and proteins in complete fractures treated with internal rigid fixation (IRF) treated or not with laser phototherapy (LPT) and associated or not with bone morphogenetic proteins (BMPs) and guided bone regeneration (GBR). BACKGROUND: Fractures have different etiologies and treatments and may be associated with bone losses. LPT has been shown to improve bone healing. METHODS: Tibial fractures were created on 15 animals and divided into five groups. LPT started immediately after surgery, repeated at 48-h intervals. Animal death occurred after 30 days. RESULTS: Raman spectroscopy and fluorescence were performed at the surface. Fluorescence data of group IRF + LPT + Biomaterial showed similar readings to those of the group IRF-no bone loss. Significant differences were seen between groups IRF + LPT + Biomaterial and IRF + LPT; IRF + LPT + Biomaterial; and IRF + Biomaterial; and between IRF + LPT + Biomaterial and IRF. CH groups of lipids and proteins readings showed decreased levels of organic components in subjects treated with the association of LPT, biomaterial, and GBR. Pearson correlation showed that fluorescence readings of both CHA and CH groups of lipids and proteins correlated negatively with the Raman data. CONCLUSIONS: The use of both methods indicates that the use of the biomaterials associated with infrared LPT resulted in a more-advanced and higher quality of bone repair in fractures treated with miniplates and that the DIAGNOdent may be used to perform optical biopsy on bone.

In vitro & in vivo assessment of a herbal formula used topically for bone fracture treatment.

AIM OF THE STUDY: A novel topical paste used for fracture healing (FH), consisting of the extracts of six herbs, Radix Dipsaci, Ramulus Sambucus Williamsii, Rhizoma Notoginseng, Flos Carthami, Rhizoma Rhei and Fructus Gardeniae, was developed according to the classical theory of traditional Chinese medicine. This study aimed to determine the effectiveness of this formula, and some of its important chemical components in the promotion of fracture healing. The transdermal transport of FH was also examined. MATERIALS AND METHODS: The osteogenic, angiogenic and nitric oxide suppressing effects of FH and its important chemical marker components were assessed by using osteoblastosacroma UMR-106 cells, human umbilical vein endothelial cells (HUVEC) and murine macrophage RAW264.7 cells, respectively. The bone healing effects of the FH paste and its transdermal absorption were determined using a rabbit fracture model. The callus sizes, bone specific alkaline phosphatase levels and biomechanical properties of the healed bone were assessed. RESULTS: FH significantly increased the cell proliferation in UMR-106 and HUVEC cells and inhibited the nitric oxide production in murine macrophage in dose-dependent manner. Its important chemical components asperosaponin VI, ginsenoside Rg1 and emodin were shown to be acting positively in the respective in vitro studies. FH paste significantly improved the bone healing in the rabbit fracture model, as was indicated by the increases in callus size at weeks 2-5, and the elevations in bone specific alkaline phosphatase activities at weeks 5-6. The analysis using LC/MS/MS also showed the presence of important chemical marker components of the FH formula in the plasma after 8 weeks of topical treatment. CONCLUSION: This study presents the first scientific evidence of the efficacy of a herbal paste in the promotion of fracture healing. There were evidences of transdermal transport of the chemical components, control the inflammation through nitric oxide inhibition, promotion of angiogenesis, and bone healing in the in vitro tests, as well as in the experimental animal.

Effects of receptor activator of NFkappaB (RANK) signaling blockade on fracture healing.

As dominant regulators of osteoclastogenesis and bone resorption, receptor activator of NFkappaB (RANK), receptor activator of NFkappaB ligand, and OPG have been identified as ideal drug targets for the treatment of metabolic bone disease. One concern regarding the therapeutic use of RANK signaling inhibitors is their effect on fracture healing. Therefore we tested if uncoupling and osteoclast depletion via RANK blockade affects callus formation, maturation and matrix remodeling, as well as union rates in a mouse tibia fracture model. Low dose (1 mg/kg i.p.) RANK:Fc therapy had no effect on callus formation, matrix maturation and remodeling, and resulted in 100% bony union by day 28. High dose RANK:Fc treatment (10 mg/kg i.p.) effectively eliminated osteoclasts at the fracture site on day 14, with no significant effects on fracture healing. When therapy was discontinued, normal numbers of osteoclasts were observed at the fracture site by day 28. However, continuous therapy resulted in a large osteopetrotic callus consisting of both mineralized and unmineralized matrix that was void of osteoclasts, but bony union was unaffected at day 28. We also evaluated this process in the complete absence of RANK signaling using RANK -/- mice. These animals exhibited significant radiographic and histologic evidence of callus formation, indicating that RANK signaling is not required for fracture callus formation. However, only 33% of RANK -/- animals formed bony unions compared to 100% of the osteopetrotic control mice. This defect was most likely a result of decreased blood flow, as evidenced by fewer blood vessels in the RANK -/- animals. Together, these data imply that osteoclast depletion via inhibition of RANK signaling is a viable option for the treatment of pathological bone loss since no adverse effects on fracture healing are observed when therapy is discontinued.

Ethanol and its effects on fracture healing and bone mass in male rats.

Operatively induced, standardized tibia fractures in 42 10-week-old male rats were fixed with intramedullary nails. 21 of the rats were fed liquid containing 15% ethanol. 5 weeks after inducing the fracture, the rats were killed and the total body bone mineral density (BMD) was analyzed with the DEXA technique, and the mechanical properties of the fractured and the unfractured tibiae as well as the ipsi- and contralateral femoral shaft and femoral neck were tested. The rats given a liquid containing 15% ethanol were found to have significantly lower total BMD and total calcium than the controls. We also found a significantly lower bending moment and bending stiffness both in the fractured and unfractured tibiae among rats fed on ethanol. The energy absorption until refracture was less in rats fed on ethanol. Posttraumatic osteopenia was present, as judged by the mechanical tests of the ipsilateral femoral shaft and the femoral neck in all animals. There was no difference in this respect between the animals fed on ethanol and the controls. We found that ethanol disturbs bone metabolism which reduces the mechanical properties of the tibiae and femora of rats, but the healing process of an induced tibial shaft fracture was not affected.

[Experimental study on the effect of Chinese traditional medicine "bone growth fluid" in the change of trace elements in bone lengthening area].

In order to study the effect of Chinese traditional medicine, "Bone Growth Fluid", on bone formation in bone lengthening, the limb lengthening model was made on goat to observe bone formation in the distracted area, and the content of the trace elements was determined. The bone-lengthening operation was carried out on the upper metaphysis of left tibia. The animals were divided into two groups following operation. From 2nd day afteroperation, "Bone Growth Fluid", 10 ml per kilogram body weight, was given daily to goats in the experimental group, and same amount of normal saline was given to goats in another group as control. The results showed-that new bone formation and bone remodeling in the experimental group appeared earlier than that in the control group, and the content of the trace elements was also improved. So Chinese Traditional medicine, "Bone Growth Fluid", could accelerate the accumulation of the trace elements in callus on the distracted sites and it might play some role in the promotion of osteogenesis and bone remodeling in bone lengthening.

[Effect of radix Salviae miltiorrhizae on calcium, zinc, copper content in serum, callus and bony tissue in early stage of healing process in rat closed tibial fracture].

Changes of calcium, zinc, copper contents in serum, callus and bony tissue in the early stage of the healing process of rat closed tibial fracture, also the changes of them with radix Salviae miltiorrhizae (RSM) treatment were studied. It was found that calcium, zinc contents and Zn/Cu ratio increased significantly and the rise of serum copper content was inhibited by the administration of RSM after fracture. Zn/Cu ratio in fracture callus was correlated to the calcium content in fracture callus. These findings suggested that the effect of the promotion of RSM on fracture healing was related to the increased zinc content in serum, also related to the acceleration of mobilization of zinc in fractured bone, and to the acceleration of fracture callus formation and mineralization process by the increased zinc and Zn/Cu ratio in the callus of the fracture.

[The calcium and phosphorus content of the bone tissue in ovariectomized rats with a fracture of the tibia: the effect of vitamin K and vitamin D metabolites]. / Soderzhanie kal'tsiia i fosfora v kostnoi tkani u ovariéktomirovannykh krys s perelomom bol'shoi bertsovoi kosti: vliianie vitamina K i metabolitov vitamina D.

Calcium and phosphorus content in the bone tissue of ovariectomized rats with a shinbone fracture has been estimated. It was found out that unlike pseudo-operated animals whose fracture was accompanied by a decrease of calcium and phosphorus content and density in the right proximal epiphysis of the femur, in case of ovariectomized rats those parameters increased. Under those conditions a decrease in the epiphysis density and calcium and phosphorus content both in the shinbone and in the femur of the left extremity was similar both in pseudo-operated and ovariectomized rats. Administration of 1 alpha (OH)D3, 24R, 25(OH)2D3 and vitamin K to ovariectomized animals with a shinbone fracture produced no positive effect on the bone tissue, which anyhow, did not exclude a possibility of a positive effect in other doses and for a longer period of time.

Energy metabolism in fracture healing. Measurement of adenosine triphosphate in callus to monitor progress.

We measured the adenosine triphosphate (ATP) content of callus at various intervals during healing in 78 fractured tibiae in 10- to 12-week-old rabbits. The results, compared with the level in normal tissues, showed a high rate of energy metabolism in the early phase of fracture healing, which persisted until the callus was corticalised and remodelling had started. The ATP content could provide a more sensitive index to monitor fracture healing in animal studies. Our findings lend support to the need for nutritional supplements for patients with multiple fractures.

[Concentration of 99mTc-tin-phosphate complexes in soft tissues]. / Zur Frage der Weichteilkonzentration von 99mTc-Zinn-Phosphat-Komplexen

The concentration of 99mTc-pyrophosphate was determined in the lower extremities of rabbits (normal, abacterial and bacterial affected soft tissues), in osteoarthritis of the hip joint (capsule and muscle) as well as in knee joint effusions. Compared with the 85Sr-concentration, reflecting the calcification capacity, concentrations of 99mTc-pyrophosphate in soft tissues were found to be lower 2 hours p.i., but were up to elevenfold higher 24 hours p.i. These findings should be due to a fixation of 99mTc-pyrophosphate in collagen containing tissues as in the soft tissue tumors (myosarcoma, synvialioma, breast cancer) presented. A mechanism of delayed equilibration could explain augmented uptake in lymph-edema, ascites and effusions in florid osteoarthritis of the knee joint. The possible dependence of 99mTc-pyrophosphate concentration in bone and soft tissue on collagenous contents is discussed.