In-vivo imaging of the fracture healing in medaka revealed two types of osteoclasts before and after the callus formation by osteoblasts.

The fracture healing research, which has been performed in mammalian models not only for clinical application but also for bone metabolism, revealed that generally osteoblasts are induced to enter the fracture site before the induction of osteoclasts for bone remodeling. However, it remains unknown how and where osteoclasts and osteoblasts are induced, because it is difficult to observe osteoclasts and osteoblasts in a living animal. To answer these questions, we developed a new fracture healing model by using medaka. We fractured one side of lepidotrichia in a caudal fin ray without injuring the other soft tissues including blood vessels. Using the transgenic medaka in which osteoclasts and osteoblasts were visualized by GFP and DsRed, respectively, we found that two different types of functional osteoclasts were induced before and after osteoblast callus formation. The early-induced osteoclasts resorbed the bone fragments and the late-induced osteoclasts remodeled the callus. Both types of osteoclasts were induced near the surface on the blood vessels, while osteoblasts migrated from adjacent fin ray. Transmission electron microscopy revealed that no significant ruffled border and clear zone were observed in early-induced osteoclasts, whereas the late-induced osteoclasts had clear zones but did not have the typical ruffled border. In the remodeling of the callus, the expression of cox2 mRNA was up-regulated at the fracture site around vessels, and the inhibition of Cox2 impaired the induction of the late-induced osteoclasts, resulting in abnormal fracture healing. Finally, our developed medaka fracture healing model brings a new insight into the molecular mechanism for controlling cellular behaviors during the fracture healing.

Ultrastructural characterization of the implant interface response to loading.

Dynamic loading can affect the bone surrounding implants. For ultrastructural exploration of the peri-implant tissue response to dynamic loading, titanium implants were installed in rat tibiae, in which one implant was loaded while the contralateral served as the unloaded control. The loaded implants received stimulation either within 24 hrs after implantation (immediate loading) or after a 28-day healing period (delayed loading) for 4, 7, 14, 21, or 28 days. The samples were processed for histology and gene expression quantification. Compared with the unloaded control, bone-to-implant contact increased significantly by immediate loading for 28 days (p < .05), but not in case of delayed loading. No effect of loading was observed on the bone formation in the implant thread areas, on the blood vessel area, and on endosteal callus formation. Loading during healing (immediate) for 7 days induced, relative to the unloaded control, a 2.3-fold increase of Runx2 in peri-implant cortical bone (p < .01) without a change in the RANKL/Opg ratio. Loading after healing (delayed) for 7 days up-regulated Runx2 (4.3-fold, p < .01) as well as Opg (22.3-fold, p < .05) compared with the unloaded control, resulting in a significantly decreased RANKL/Opg ratio. These results indicate a stimulating effect of dynamic loading on implant osseointegration when applied during the healing phase. In addition, gene expression analyses revealed molecular adaptations favoring bone formation and, at the same time, affecting bone remodeling.

Locally administrated perindopril improves healing in an ovariectomized rat tibial osteotomy model.

Angiotensin-converting enzyme inhibitors are widely prescribed to regulate blood pressure. High doses of orally administered perindopril have previously been shown to improve fracture healing in a mouse femur fracture model. In this study, perindopril was administered directly to the fracture area with the goal of stimulating fracture repair. Three months after being ovariectomized (OVX), tibial fractures were produced in Sprague-Dawley rats and subsequently stabilized with intramedullary wires. Perindopril (0.4 mg/kg/day) was injected locally at the fractured site for a treatment period of 7 days. Vehicle reagent was used as a control. Callus quality was evaluated at 2 and 4 weeks post-fracture. Compared with the vehicle group, perindopril treatment significantly increased bone formation, increased biomechanical strength, and improved microstructural parameters of the callus. Newly woven bone was arranged more tightly and regularly at 4 weeks post-fracture. The ultimate load increased by 66.1 and 76.9% (p<0.01), and the bone volume over total volume (BV/TV) increased by 29.9% and 24.3% (p<0.01) at 2 and 4 weeks post-fracture, respectively. These findings suggest that local treatment with perindopril could promote fracture healing in ovariectomized rats.

Roles of the endoplasmic reticulum stress transducer OASIS in fracture healing.

A new type of endoplasmic reticulum (ER) stress transducer, Old Astrocyte Specifically Induced Substance (OASIS), which is induced by bone morphogenetic protein-2 (BMP2), has been reported to activate the transcription of type I collagen and contribute to the secretion of bone matrix proteins in osteoblasts. Here, we examined the role of OASIS in fracture healing using the fracture models in wild-type (WT) and OASIS(-/-) mice. We found that the expression of OASIS mRNA was induced after fracture. Micro-computed tomography indicated that the callus density of OASIS(-/-) mice was less than that of WT mice, and the newly formed bone in OASIS(-/-) mice exhibited a decrease of the bone volume by bone morphometric analysis. Biomechanically, the callus bone strength of OASIS(-/-) mice was inferior to that of WT mice. Based on RT-PCR, in situ hybridization, immunohistochemical, and electrophoretic analyses, it was clarified that the synthesis of type I collagen was impaired in OASIS(-/-) mice. Electron microscopic analysis revealed that OASIS(-/-) osteoblasts in the fracture callus contained the abnormal expansion of the ERs, similar to OASIS(-/-) osteoblasts in the normal skeletal development. Thus, OASIS may play a role in bone formation through the expression of type I collagen and the secretion of bone matrix proteins in fracture healing.

Vertebral cancellous bone turn-over: microcallus and bridges in backscatter electron microscopy.

Backscatter electron microscopy (BSE) is a powerful technique for investigating cancellous bone structure. Its main function is to offer information regarding the degree of mineralization of the tissue within individual trabeculae. To illustrate the qualitative information that can be drawn from BSE imaging technique, we present a study on human vertebral cancellous bone. This tissue is continuously remodeled through osteoclastic resorption and osteoblastic new bone apposition. It is thought that osteoclastic resorption pits are especially deleterious for vertebral bone architecture since they often perforate the thin trabeculae; the osteoblasts being unable to repair the gap. In addition, excessive stress may also disrupt the architecture in case of trabecular fracture or damage accumulation. Waves of new bone formation were easy to identify in BSE. Often these waves were connecting both edges of a perforation and called bridges. Additionally, we present a few images of microcallus formations. A microcallus is described as a small mass of woven bone that generally repairs a trabecula. The microstructural aspects of different microcalluses are presented and discussed. Both bridges and microcallus should be considered as examples of the repair porcess since they obviously preserve the connectivity of the trabeculae. However, bridges were much more frequent than microcallus (396 vs 15). Both mechanisms probably illustrate the normal response to different local stimuli.

Microstructural investigations of strain-related collagen mineralization.

Distraction osteogenesis in rabbit mandibles after osteotomy can be used as an experimental model to study the microstructural features of mineralization of callus under defined mechanical loads. Our aim was to study the relation between the micromotions in the gap and the resulting features of mineralization of the matrix. We found that assembly of collagen and formation of crystals depended on the magnitude of the mechanical stress applied. At physiological bone strains (2000 microstrains), the callus had collagen type I in a mature bone-like extracellular arrangement, whereas at 20000 microstrains bundles were orientated predominantly towards the tension vector. Maximum loads (200000 microstrains) resulted in disorganized assembly of the collagen. Quantitative energy-dispersive analysis by X-rays confirmed that high strains were associated with substantially lower concentrations of calcium and phosphate. In contrast to bone-like apatitic formation of crystals at physiological strains, significantly fewer but larger crystals were detected by electron diffraction analysis in samples exposed to high strains. We suggest that mechanical stress regulates the assembly and mineralization of collagen during distraction osteogenesis.

Stress-relaxation plates and the remodeling of callus and cortex under the plate in rabbits.

OBJECTIVE: To study the influence of a stress-relaxation plate on the remodeling of callus and cortex under the plate. METHODS: The bilateral tibia diaphysis of New Zealand rabbit were osteotomized and fixed with stress-relaxation plate (SRP) and rigid plate (RP), respectively. Polarized light microscopy and transmission electron microscopy (TEM) were used to study the remodeling of callus and the cortex under the plate from 4 to 24 weeks postoperatively. RESULTS: Polarized light microscopy: the structural changes of callus and cortex beneath the plate are similar in the SRP and RP groups at the early postoperative stage, manifesting an alignment disorder of collagen fibers with a weak birefringence in the callus and absorption cavities in the cortex under the plate. After the twelfth postoperative week, the SRP group showed callus starting to transform to lamellar bone and absorption cavities in the cortex under the plate becoming smaller. By contrast in the RP group the absorption cavities in the callus and cortex under the plate became larger and the whole layer of cortex was cancellated. TEM: the active osteoclasts appeared in both SRP and RP groups in the period from 4 to 8 weeks postoperatively. In the SRP group, many functionally active osteoblasts could be seen on the surface of the bone, while in the RP group, the osteoblasts were not very active. By 24 weeks postoperatively, the shape of osteocytes were normal but the number of the osteoclasts were small in the SRP group. In the RP group, the osteoclasts became more active and osteocytic osteolysis was manifested. CONCLUSIONS: Fixation with SRP not only enhanced callus remodeling, but also abated the degree of osteoporosis in the cortex under the plate. This approach may lead to an improved osteosynthetic apparatus.

Early histologic and ultrastructural changes in microvessels of periosteal callus.

OBJECTIVE: To document early histological and ultrastructural changes in periosteal fracture callus blood vessels. DESIGN: Rabbit control and fractured ribs, after healing for three, six, and twelve hours and daily for seven days, were evaluated by light and electron microscopy. RESULTS: Control periosteal microvessels were formed mainly by endothelial cells and occasionally by pericytes. Only these cells displayed basal lamina within the periosteum. Three to twelve hours postfracture, periosteal microvessels were little changed. By two days postfracture, dramatic increases in size and population of microvessel cells resulted in a smaller lumen and thicker wall. Microvessel cells, while retaining their basal lamina, had transformed to mesenchymal cells. Transformed pericytes, as evidenced by their basal lamina, had extravasated. Three to four days postfracture, additional transformed pericytes had extravasated. Within the distal periosteal callus, a close spatial relationship among transformed microvessels, extravascular mesenchymal cells (some with basal lamina), and osteoblasts was present. Four to five days postfracture, within the proximal periosteal callus, a close spatial relationship among transformed microvessels (rapidly disappearing because of continued extravasation), extravascular mesenchymal cells (some with basal lamina), and chondroblasts (some with basal lamina) was present. CONCLUSIONS: New evidence showed that after fracture, periosteal microvessel endothelial cells and pericytes increased in population and transformed to mesenchymal cells. These changes, their subsequent extravasation as mesenchymal cells, and their development into chondroblasts were verified by basal lamina evidence. New evidence also suggested that continued extravasation of transformed microvessel cells rendered the fracture callus cartilage avascular.

Effects of single high-dose vitamin D3 on fracture healing. An ultrastructural study in healthy guinea pigs.

The benefits of vitamin D3 on fracture healing have been commonly demonstrated in vitamin-D3-depleted animal models. The aim of this study was to examine the effects of a single high dose of vitamin D3 on fracture healing in a healthy animal model, which has not been previously reported. Twenty healthy young adult guinea pigs were randomly divided into groups as 'control' and 'vitamin D', and their right tibias were fractured with digital manipulation. Guinea pigs in vitamin D group were injected intramuscularly with 50,000 i.u./kg of vitamin D3. The animals were killed at 7, 14, 21 and 28 days following fracture. Ultrastructural analysis of the harvested tibias revealed that a single high dose of vitamin D3 stimulated fracture healing. The observed effects at the fracture zone in a healthy animal model included advancement of blood supply, acceleration of synthesis and organization of collagen fibres, acceleration of the proliferation and differentiation of osteoprogenitor cells, and activation of the mineralization of the matrix.

Structural heterogeneity within the axis: the main cause in the etiology of dens fractures. A histomorphometric analysis of 37 normal and osteoporotic autopsy cases.

Fractures of the odontoid process are potentially serious injuries; Type II and III fractures, as described by Anderson and D'Alonzo, are seen in the emergency room especially in young adolescents and individuals over 60 years of age. The etiology of these fractures is still controversial. Malunion and nonunion in both types of fractures are presumed to be due to insufficient external or internal fixation, but this theory has not been fully explained. To examine these issues, the authors expanded their prior studies of the anatomy of the axis. For histomorphometric analysis of cancellous and cortical bone, the axis was removed in 37 autopsies (26 normal and 11 osteoporotic cases) and sectioned in the sagittal plane to a thickness of 1 mm using a surface-stained block-grinding technique. The base of the dens is the region of least resistance for fractures because of its reduced trabecular bone volume, a poorer trabecular interconnection, and a cortical thickness one-third that of the axis. In all cases, trabeculae were disconnected from the trabecular lattice, and in 30%, microcallus formations were demonstrated in the base of the dens. A special filigree type of trabeculae in the base of the dens is often seen in patients with osteoporosis; microarchitectural differences of cancellous bone between the base of the dens and the other regions of the axis are also markedly increased. The authors infer from the data that the bone structure of the axis is responsible for the location, distribution, and frequency of fractures of the odontoid process in normal healthy bone and this frequency is greatly increased in individuals with osteoporosis. The deficiency of bone mass within the base also suggests a new explanation for the occurrence of nonunions, even after treatment of fractures of the base of the dens.

Microstructural investigation of the early external callus after diaphyseal fractures of human long bone.

Microstructures of the early external callus after diaphyseal fractures of human long bone were investigated by using scanning electron microscopy, X-ray diffraction, and transmission electron microscopy. It was found that the main structural framework of the human early callus consists of disordered, mineralized collagen fibrils with a small fraction of regions of ordered collagen fibrils. X-ray diffraction analyses show that hydroxyapatite containing some carbonate impurity has been the dominant crystalline phase in the human early callus. In addition, a small amount of brushite phase was detected. Selected area diffraction analyses indicated that hydroxyapatite microcrystals were embedded in microfibrils with a diameter of 4.5 nm and well-banded fibrils, whereas brushite particles of 15-20 nm in an irregular shape were located in the noncollagenous organic matter around the nonmineralized, ordered collagen fibrils. The spatial distribution of the brushite particles in the human early callus was for the first time determined. The brushite particles probably serve as the reservoir of calcium and phosphate ions for subsequent mineralized periods rather than the precursor of hydroxyapatite.

Early fracture callus in the diaphysis of human long bones. Histologic and ultrastructural study.

The medullary callus and the periosteal callus of fractured long bones were studied in 26 adults undergoing open reduction and internal fixation of closed diaphyseal fractures that occurred 1 to 21 days before surgery. In the 1st week after fracture, a progressive increase was observed in the number of polymorphic mesenchymal cells in the medullary callus and of fibroblast-like cells in the periosteum, where the first calcification foci were seen 7 days after injury. In the 2nd week after fracture, the medullary callus presented numerous mesenchymal cells, fibroblasts, and newly formed capillaries, whereas the inner periosteal layer showed many osteoblast-like cells. New bone trabeculae were first seen in the periosteal callus 12 days after injury. In the 3rd week after fracture, new trabecular bone appeared in the medullary callus. Cartilage also became apparent in the medullary and periosteal callus but remained limited in amount. Calcification within cartilage was first observed in the periosteum 18 days after fracture. The process of fracture healing in long bones in humans is similar, though not identical, to that described for long bones in laboratory animals.

Evaluation of distraction osteogenesis by scanning electron microscopy.

A model of bifocal distraction osteogenesis in the canine model was used to assess and quantitate the mineral content of the newly forming bone within the canine mandible. A 2-cm defect was created in the body of the mandible, and after a posterior osteotomy, the transport disk was advanced at 0.25 mm per 8 hours for 21 days and then held in rigid fixation for an additional week. As a control for this study, three additional dogs underwent the same procedure with the exception that the transport disk was not advanced. Electron dispersive spectroscopy analysis was performed on the newly formed regenerate bone and compared with areas of existing cortical bone of both the transport disk and the mandible. In the control model, special note was made of the pericortical callus at the osteotomy site as well as of the regenerative bone that filled the 2-cm defect in the body of the mandible. Calcium/phosphorous ratios were used to assess the composition of the mineralized regions of the mandible. The regenerate bone that filled the defect and the mineralized callus surrounding the site of osteoclasis in the control mandible were significantly different in composition when compared with the regenerate bone that formed during distraction osteogenesis. This suggests that distraction osteogenesis may effect an initial matrix production that is more similar in composition to the mature cortical bone from which it was derived than does periosteal regeneration and filling of an osseous defect.

Mechanical properties of microcallus in human cancellous bone.

Until now, the mechanical properties of the microcalluses that form in human cancellous bone have been unexplained. We measured the microhardnesses of microcalluses in cancellous bone, of the trabeculae within the microcalluses, of the trabeculae adjacent to microcalluses, and of trabeculae lacking microcalluses in a human tibia and femur. We observed no important differences between materials at the four different sites. Because the microhardness of bone is very closely related to its stiffness, this finding indicates that microcalluses are likely to stiffen the trabeculae in which they are formed, even though they may surround unhealed fractures of the cancellous trabeculae.

Histochemical localization of calcium in the fracture callus with potassium pyroantimonate. Possible role of chondrocyte mitochondrial calcium in callus calcification.

Potassium pyroantimonate was employed as a histochemical stain for calcium at the ultrastructural level in the cartilaginous fracture callus in the rat rib. In areas of the callus showing no matrix mineralization, the electron-dense precipitate of the antimony-calcium complex was heavily deposited in chondrocyte mitochondria, lipid, and cell membrane. In areas showing early mineralization the mitochondria, lipid, and cell membrane showed a smaller amount of antimony-calcium complex, and in areas of more advanced matrix mineralization the mitochondria, lipid, and cell membrane were completely void of any stain. In the matrix, the initial site of mineralization was associated with matrix vesicles located in areas of early matrix mineralization. These findings suggest the hypothesis that mitochondria play an important role in matrix calcification in cartilaginous fracture callus.

Effect of CO2 laser irradiation on experimental fracture healing: a transmission electron microscopic study.

The healing of standardized fracture of rabbit radius was expedited as a result of treatment with low-power CO2 laser irradiation. Observation with transmission electron microscope revealed the following favorable effects of CO2 laser irradiation: The red blood cells were induced to disintegrate, thus promoting the absorption of the hematoma. The macrophages emerged early and increased in number so that debridement of the necrotic tissues was enhanced. The fibroblasts were more active in producing the fibrous callus. The chondrocytes were unusually active in forming bone tissues. The early and sustained appearance of osteoclasts favored the bone remodeling process. Increased capillary formation endowed the fracture healing with rich blood supply. Deposition of calcium salts took place early.

Histological studies of electrically induced intramedullary callus.

The author studied the early changes of electrically stimulated bone marrow and carried out histological examinations. A constant current of 10 microAmp was delivered to the cathode which was placed in the humerus of each rabbit. On the 5th, 14th and 21 st day, the rabbits were killed, and the intramedullary tissues around the tip of the cathode were processed for light and electron microscopic observations. In the early stages after electrical stimulation, the leakage of erythrocytes, membranocystic degeneration of lipid cells, and proliferation of undifferentiated mesenchymal cells with moderately developed rER and collagen bundles were recognized. These findings indicate that the oxygen tension around the cathode was diminished in advance of the electrically stimulated callus formation. After prolonged electrical stimulation, both membranous and endochondral ossifications were more active than those in the control group.

[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.