Grape seed extract supplement increases bone callus formation and mechanical strength: an animal study.

BACKGROUND: The positive effects of grape seed proanthocyanidin extract (GSPE) on bone health, which is a potent antioxidant, are known but its effects on fracture healing are not sufficiently covered in the literature. This study aims to investigate the effects of GSPE on fracture healing and biomechanics of healing bone. MATERIALS AND METHODS: Sixty-four adult Wistar-Albino male rats were divided into 8 groups of 8 animals in each group. Osteotomy was performed to the right femurs of all groups except the negative control (G1) and positive control (G2) groups, and intramedullary Kirchner wire was used for fixation. GSPE was given to half of the rats (G2-G4-G6-G8) 100 mg/kg/day by oral gavage. The rats were sacrificed on the tenth (G3-G4), twentieth (G5-G6), and thirtieth (G1-G2-G7-G8) days, respectively, and histopathological, radiological, and biomechanical examinations were performed. RESULTS: Histopathological examination of the specimens from the callus tissues revealed that bone healing was more prominent in the groups supplemented with GSPE (G4, G6, G8). There was a statistically significant improvement in radiological recovery scores and callus volumes in groups with GSPE. When biomechanical strengths were evaluated, it was found that GSPE increased bone strength not only in fracture groups but also in the positive control group (G2). CONCLUSIONS: As a result, this study showed that GSPE, a potent anti-oxidant, had a positive effect on bone healing and improved mechanical strength of the healing bone.

Evaluation of composition and mineral structure of callus tissue in rat femoral fracture.

Callus formation is a critical step for successful fracture healing. Little is known about the molecular composition and mineral structure of the newly formed tissue in the callus. The aim was to evaluate the feasibility of small angle x-ray scattering (SAXS) to assess mineral structure of callus and cortical bone and if it could provide complementary information with the compositional analyses from Fourier transform infrared (FTIR) microspectroscopy. Femurs of 12 male Sprague-Dawley rats at 9 weeks of age were fractured and fixed with an intramedullary 1.1 mm K-wire. Fractures were treated with the combinations of bone morphogenetic protein-7 and/or zoledronate. Rats were sacrificed after 6 weeks and both femurs were prepared for FTIR and SAXS analysis. Significant differences were found in the molecular composition and mineral structure between the fracture callus, fracture cortex, and control cortex. The degree of mineralization, collagen maturity, and degree of orientation of the mineral plates were lower in the callus tissue than in the cortices. The results indicate the feasibility of SAXS in the investigation of mineral structure of bone fracture callus and provide complementary information with the composition analyzed with FTIR. Moreover, this study contributes to the limited FTIR and SAXS data in the field.

The study of ascorbate peroxidase, catalase and peroxidase during in vitro regeneration of Argyrolobium roseum.

Here, we demonstrate the micropropagation protocol of Argyrolobium roseum (Camb.), an endangered herb exhibiting anti-diabetic and immune-suppressant properties, and antioxidant enzymes pattern is evaluated. Maximum callogenic response (60 %) was observed from leaf explant at 1.0 mg L(-1) 1-nephthalene acetic acid (NAA) and 0.5 mg L(-1) 6-benzyl aminopurine (BA) in Murashige and Skoog (MS) medium using hypocotyl and root explants (48 % each). Addition of AgNO3 and PVP in the culture medium led to an increase in callogenic response up to 86 % from leaf explant and 72 % from hypocotyl and root explants. The best shooting response was observed in the presence of NAA, while maximum shoot length and number of shoots were achieved based on BA-supplemented MS medium. The regenerated shoots were rooted and successfully acclimatized under greenhouse conditions. Catalase and peroxidase enzymes showed ascending pattern during in vitro plant development from seed while ascorbate peroxidase showed descending pattern. Totally reverse response of these enzymes was observed during callus induction from three different explants. During shoot induction, catalase and peroxidase increased at high rate while there was a mild reduction in ascorbate peroxidase activity. Catalase and peroxidase continuously increased; on the other hand, ascorbate peroxidase activity decreased during root development and acclimatization states. The protocol described here can be employed for the mass propagation and genetic transformation of this rare herb. This study also highlights the importance and role of ascorbate peroxidase, catalase, and peroxidase in the establishment of A. roseum in vitro culture through callogenesis and organogenesis.

Callus formation enhanced by BMP-7 ex vivo gene therapy during distraction osteogenesis in rats.

This study was designed to observe the effects of bone morphogenetic protein-7 (BMP-7) ex vivo gene therapy on callus formation during rat mandibular distraction osteogenesis (DO). Fifty-four Sprague-Dawley rats underwent osteodistraction of the right mandible and were then randomly divided into three groups. Immediately after distraction, autologous bone marrow mesenchymal stem cells (MSCs) transfected with BMP-7, MSCs untransfected with BMP-7, and physiological saline were injected into the distraction gaps of the mandibles in groups A, B, and C, respectively. Nine animals from each group were euthanized at 2 and 6 weeks after completion of distraction. The distracted mandibles were removed and processed for radiographic, histological, immunohistochemical, and scanning electron microscopic examinations as well as Ca/P ratio analysis. Group A animals showed greater bone formation and earlier mineralization in the distracted callus when compared with group B. Similarly increased callus formation was found in group B than group C. Positive immunostaining of BMP-7 was observed in the distracted callus in all groups. However, BMP-7 expression was much stronger in group A compared with groups B and C. The results of this study suggest that BMP-7-mediated ex vivo gene transfer based on MSCs may accelerate callus formation in distraction osteogenesis and facilitate consolidation. Local gene therapy may ultimately be an alternative or supplemental approach to DO enhancement, especially for patients whose osteogenic potentials are compromised by diseases such as osteoporosis, severe trauma, and postoncologic irradiation.

Callusmassage. A new treatment modality for non-unions of the irradiated mandible.

Recent reports on orthopaedic surgery focus on mechanical stimulation of the regenerate during distraction therapy of non-unions in long-bone-surgery. In the field of maxillofacial surgery, callus stimulating techniques are rarely reported. The case of a 65-year-old man with a radiogenic mandibular non-union after ablative tumour therapy and pre-operative radiation therapy presented with a non-union. Vertical distraction in combination with subsequent repeated, stepwise compression and distraction (=massage) had a positive effect on the consolidation of the regenerate.

Bone mineral density changes in distracted callus stimulated by pulsed direct electrical current.

To evaluate the effect of application of pulsed direct current electrical stimulation to callus tissue, a 1-cm bone-lengthening model using an external lengthener was applied to rabbit tibia. Twenty-microampere pulsed direct current was applied 12 hours daily from the day of osteotomy until 40 days after the completion of lengthening. The area, bone mineral content, and bone mineral density of the distracted callus and of the proximal and distal segments of the tibia were evaluated using dual-energy xray absorptiometry. The absolute and relative values of bone mineral density of the electrically stimulated callus were significantly increased as compared with those in the control group. Pulsed direct current electrical stimulation may be indicated in bone lengthening to stimulate the poorly mineralized callus, and may shorten the overall time course of leg lengthening.

Assessment of bone formation during osteoneogenesis: a canine model.

Distraction osteoneogenesis, callotasis, has been demonstrated to be an effective means of lengthening long bones. A variation of Ilizarov's technique produces a transport disk from one cut surface of bone within a defect and advances the disk to the opposite surface to close the defect. This process, previously described by Costantino et al. (Arch Otolaryngol Head Neck Surg 1990; 116:535-45), demonstrated bone formation within the distraction site. The precise mechanism of bone formation has not yet been described for the mandible. Four conditioned beagles were studied, with one control dog maintained in neutral fixation and three dogs distracted at 0.25 mm every 8 hours. A two-cm defect was closed, and dogs were kept in fixation for 1 week after closure, after which they were killed. Three sites were evaluated: (1) the distraction seam, (2) the interface of the cortical and distracted bone, and (3) the cortexes at the closed defect. Each site was bisected, and one half was decalcified for immunohistochemical and hematoxylin and eosin pathologic evaluation. The vascular basement membrane was labeled for laminin and type IV collagen. Both of these substances demonstrate the differentiation of the vascular matrix component predisposing primary bone formation. Labels were intense at the distraction seam where intense angiogenesis occurred. No hyalin cartilage was observed at the distraction site, which indicates that the fixation was stable and that ossification occurred primarily without intermediate callous formation. This model demonstrated that osteoclasts within the canine model produce bone through primary bone formation within an angiogenic matrix rich in basement membrane laminin and type IV collagen. Likewise, bone is species specific in mineral composition for dog mandible. Understanding the formation and composition of distracted bone is essential for understanding application of this technique within the clinical setting.

Electrical stimulation of bone nonunion with the presence of a gap.

A total of 22 established nonunions was treated with a capacitively-coupled electrical signal. A gap of 0.5 cm or more between the fragments was present in all of these nonunions. After an average of 26 weeks of treatment with capacitive coupling, radiographic assessment showed solid bone union in 72.7% of the cases. The results were better when the fracture site was metaphyseal. When the site was diaphyseal, bone healing was mainly achieved by bone trabeculae invading the gap. When the site was metaphyseal, healing occurred by the formation of a peripheral callus. The results were not affected by the presence of infection. In 8 of the cases osteomyelitis occurred, but all healed.

Experimental and clinical experience with distraction osteogenesis.

G.A. Ilizarov's clinical insights and experimental biology have developed into what is known today as distraction osteogenesis. Initially used for the treatment of fractures and nonunions, his methods have proven successful for limb lengthening and bone transportation. A brief history of the development of distraction osteogenesis, definition of terms, methods, and monitoring techniques are described. The primary mechanism, intramembranous ossification, is direct bone formation in uniform gradients of mineralization from a central fibrous interzone. New bone is produced from the local host surfaces and quickly remodels to the equivalent macro and microstructure. Blood supply at the focus of distraction, dependency on a latency period, different rates, and rhythms of distraction are all tested and discussed. Data from experimental tibial lengthening in more than 125 animals (dogs, rabbits, and rats) show that distraction osteogenesis provides unlimited new bone formation that remodels at daily rates ranging from 200 to 400 microns. In over 100 clinical cases, patients ranging in age from 18 months to 49 years have regenerated bone at an average rate of 213 microns in adults and 385 microns in children. Approximately 10% of these cases required supplemental bone grafts. The article proposes that distraction osteogenesis might be successfully applied in craniofacial surgery.

[The relationship between electrical callus formation and the amount of electricity].

Electrical stimulation to enhance callus formation has been in use for some time now. This experiment was undertaken to find the relationship between electrical callus formation and the amount of electricity. In this experiment, the long bones of canines were stimulated by direct current and observed microscopically for callus formation. Moreover, distribution patterns of electric potential and current density were calculated theoretically by finite element method. The results are summarized as follows: Electrical callus formation was observed in the medullary canal with 8.7-20 microA direct current. Electrical callus is fibrous ossification and the peak of callus formation is from fourteen to twenty one days. There is no difference in volume and/or speed of callus formation between the simple and the constant direct current. Using platinum electrodes, the amount of callus formed around the cathode and anode is the same. To prevent electrolysis of the tissues, distance between electrodes must be kept at a minimum. On the other hand, the surface area of the electrodes must be widen to keep the electric potential at the minimum level. The area of callus formation is related to 5-10 microA/cm2 of electric current density.

Modification of fracture repair with selected pulsing electromagnetic fields.

We assayed different pulsing electromagnetic fields for their effects on the mechanical and histological repair properties of an osteotomy of the radius of the rat fourteen days postoperatively. Highly significant differences were found in the control and experimental initial load values and their decay as a function of time. These results correlate well with the histological pattern in the bridging callus. A pulse that produces an increase (above the control level) in initial load by a factor of 2.4 and a slower decay was characterized by more extensive calcification of fibrocartilage and its replacement by fibrous bone at this early, but important, stage in fracture-healing.

Osteogenetic stimulation by externally applied dc current.

A new, simple, safe and noninvasive technique for the electrical stimulation of fracture healing is introduced. The safety and the simplicity of the technique makes it possible to apply it almost immediately to clinical experimentation. Electrodes were applied externally to the fractured site producing current across the limb. It was observed that the current density changes the volume of callus and affects the direction of the trabecular orientation. When the trabecular orientation is completely changed from longitudinal to transverse, the larger volume of callus does not compensate for the loss of strength as compared with the callus on the control bone.

The electrical induction of callus formation and external skeletal fixation using methyl methacrylate for delayed union of open tibial fracture with segmental loss.

A case of delayed union of an open tibial fracture with segmental loss is presented in which electricity was applied to the bone defect through two screws that were connected to a methy methacrylate skeletal fixation device. The fracture developed electrically induced callus formation around the cathode, callus extended to the anode, and solid bony union ensued. External skeletal fixation using methyl methacrylate is convenient and is a suitable adjunct to electrical stimulation of fracture healing.

The electrical enhancement of periosteal proliferation in normal and delayed fracture healing.

Normal fracture healing in long bones is a poorly defined process which to a great extent is dependent on periosteal response to injury. When this normally effective mechanism fails, electrical enhancement may prove an effective adjunct and less morbid solution than standard bone grafting procedures for periosteal reactivation.