A staining method for bone canaliculi

The modified Bodian method with protargol (silver protein) is ordinarily used to detect nerve fibers. With this technique, applied to decalcified rat bone sections, the bone canaliculi were clearly stained black with good contrast to the bone matrix in both lamellar and woven bone. In addition, the connections between the bone canaliculi and other canaliculi, osteoblasts, osteoclasts, and chondrocytes were easily detectable. We found that the bone canaliculi of woven bone were fewer in number and ran more irregularly than those of lamellar bone.

We believe that this staining method for bone canaliculi in decalcified bone is superior to previously reported methods and may be useful in studies on bone pathology.     

Staining of bone canaliculi using decalcified bone tissues]

Recently we were able to stain bone canaliculi using the decalcified bone tissues, according to a modified method of the Bodian staining method, which is commonly used for staining the nerve fibers with silver impregnation of protargol. The bone tissues from normal rabbits, rats, and humans were decalcified with buffered EDTA solution after 10% formalin fixation. The tissues were embedded in paraffin and were sliced at 4 microns then the sections were attached to the gelatin-coated glass slides. These sections were treated first with potassium dichromate followed by 2% protargol solution containing copper. After gold chrolide treatment, they were immersed in oxalic acid. Both the lacunae and canaliculi of osteocytes were stained black in sharp contrast to bone matrix of red purple. Using the present staining method, the bone canaliculi were clearly detectable in fine details without producing apparent artificial damage of soft tissues. The canaliculi showed also markedly complex fine structures. By combining the present staining method with eosin or toluidine blue staining, we were able to recognize even immature osteocytes in enchondral ossification at growth cartilage with fine bone canaliculi. From these results, we conclude that the present method is very useful in histopathological studies of the lacuna-canalicular system of osteocytes in the decalcified bone tissues.     

Cortical bone repair. The relationship of the lacunar-canalicular system and intercellular gap junctions to the repair process

Repair of cortical bone was studied in 2.4-millimeter-diameter mid-diaphyseal femoral and tibial defects in young New Zealand White rabbits using light microscopy, transmission electron microscopy, and histomorphometry. The initial source of repair tissue is the marrow. Vessels grow into the defect, accompanied by undifferentiated mesenchymal cells. Woven bone is synthesized initially at the periphery of the defect on pre-existing cortex. Differentiating mesenchymal osteoblasts surround themselves with osteoid in a woven conformation. Once a scaffold has formed, surface osteoblasts align themselves in a regular array on the woven matrix surface and synthesize osteoid in a lamellar conformation. The long axes of the repair vessels, lamellae, and osteocyte lacunae are perpendicular to the long axis of the bone. Polarized-light microscopy showed maintenance of this pattern at six, eight, and twelve weeks, even when the defect was filled with lamellar bone. Remodeling is performed slowly by osteoclast cutting cones over a period of several months. The lacunar-canalicular system is clearly demonstrated in plastic-embedded, toluidine blue-stained sections. A canaliculus passes into or away from a lacuna every 1.9 micrometers over the entire osteocyte perimeter. Undifferentiated mesenchymal cells have no processes, as seen by transmission electron microscopy, but soon sprout a florid array of processes as differentiation to early mesenchymal osteoblasts proceeds. Osteoblast and osteocyte cell processes are packed with intermediate filaments that are continuous with those in the cell bodies. Intercellular gap junctions are seen between surface osteoblasts, between osteoblasts and underlying osteocytes, and between osteocyte cell processes in the canaliculi.     

Osteocyte density in woven bone

Woven bone forms rapidly during tissue growth, following injury and in response to certain anabolic stimuli. Functional differences between woven and lamellar bone may be due, in part, to differences in osteocyte density (cells per unit tissue). Woven bone has been estimated to contain four to eight times more osteocytes than lamellar bone, although primary data to support this assertion are limited. Given recent findings implicating osteocytes as regulators of bone remodeling, bone formation and bone volume, such large differences in osteocyte density between woven and lamellar bone may have important consequences. In this study, we compared the density of osteocyte lacunae (lacunae/mm(2) tissue) in rat lamellar bone with that in woven bone formed under several different circumstances. We found that the lacunar density of lamellar cortical bone in the rat (834+/-83 cells/mm2, mean+/-SD) did not differ significantly from that of periosteal woven bone formed via intramembranous osteogenesis, either in response to mechanical loading (921+/-204 cells/mm2) or in the periosteal buttressing region of the fracture callus (1138+/-168 cells/mm2). In contrast, lacunar density of endochondrally derived woven bone in the center (gap) region of fracture callus was nearly 100% greater (1875+/-270 cells/mm2) than in lamellar cortical bone while lacunar density of primary spongiosa of the growth plate was 40% greater (1674+/-228 cells/mm2) than that in lamellar cancellous bone (1189+/-164). These findings demonstrate that lacunar density in woven bone varies depending on skeletal site and developmental history and appears to be elevated in endochondrally derived woven bone adjacent to marrow space. Given the considerable evidence supporting osteocytes as local initiators of bone remodeling, we suggest that woven bone with increased lacunar density may undergo remodeling at an accelerated rate.     

Filling the bone defect with osteogenic material

In this experimental study with bone defects, we focussed on the one hand on external and internal osteogenic callus formation after filling the defect and on the other on the osteochondrogenic differentiation capacity of 4-day-old fibrous-like callus grafts and 12-day-old woven bone grafts in an osteogenic environment. A standard cortical bone defect of the femur was created in 95 young rats. The defect was filled with a cortical bone graft and 4- and 12-day-old callus grafts. The grafts were transplanted as such or in Nucleopore chambers. Follow-up was done at 1, 2, 3 and 6 weeks. The osteochondrogenic tissue formed was studied histologically and histomorphometrically. The results suggest that the filling of the bone defect had no influence on the primary external and internal osteogenic callus formation at 1 and 2 weeks. At 3 and 6 weeks in the chamber groups the persisting internal bridging woven bone was converted into more compact lamellar bone whereas periosteal callus remained at the edges of the defect. In the other groups at 3 and 6 weeks the normal shape of the cortex was reconstituting. Four-day-old fibrous-like callus formed bone in the Nucleopore chamber, indicating that fibrous-like callus tissue at 4 days contains osteogenic cells. Twelve-day-old callus consisting of woven bone was partially differentiated to cartilage, showing that woven bone contains cells capable of chondrogenic differentiation.     

Immunolocalization and expression of bone morphogenetic proteins 2 and 4 in fracture healing

Recently, it has become increasingly evident that fracture healing involves a complex interaction of many local and systemic regulatory factors. The roles of some of these growth factors have been described; however, little is understood about the presence of the bone morphogenetic proteins in fracture repair, despite the fact that they are the most potent osteoinductive proteins known. This study defines and characterizes the physiologic presence, localization, and chronology of the bone morphogenetic proteins in fracture healing with an established rat fracture healing model. With use of a recently developed monoclonal antibody against bone morphogenetic proteins 2 and 4 developed with standard avidin-biotin complex/immunoperoxidase protocols, frozen undecalcified fracture calluses were analyzed semiquantitatively for the percentage of various types of fracture cells staining positively. During the early stages of fracture healing, only a minimum number of primitive cells stained positively in the fracture callus. As the process of endochondral ossification proceeded, the presence of bone morphogenetic proteins 2 and 4 increased dramatically, especially in the primitive mesenchymal and chondrocytic cells. While the cartilaginous component of the callus matured with a concomitant decrease in the number of primitive cells, there was a concomitant decrease in both the intensity and the number of positively staining cells. As osteoblasts started to lay down woven bone on the chondroid matrix, these osteoblastic cells exhibited strong positive staining. The intensity of this staining decreased, however, as lamellar bone replaced the primitive woven bone. A similar observation was noted for the areas of the callus undergoing intramembranous ossification. Initially, within several days after the fracture, periosteal cells and osteoblasts exhibited intense staining for bone morphogenetic proteins 2 and 4. As the woven bone was replaced with mature lamellar bone, this staining decreased. These data, and the awareness of the strong osteoinductive capacities of bone morphogenetic protein, suggest that bone morphogenetic proteins 2 and 4 are important regulators of cell differentiation during fracture repair.     

Osteocyte-directed bone demineralization along canaliculi

The mammalian skeleton stores calcium and phosphate ions in bone matrix. Osteocytes in osteocyte lacunae extend numerous dendrites into canaliculi less than a micron in diameter and which are distributed throughout bone matrix. Although osteoclasts are the primary bone-resorbing cells, osteocytes also reportedly dissolve hydroxyapatite at peri-lacunar bone matrix. However, robust three-dimensional evidence for peri-canalicular bone mineral dissolution has been lacking. Here we applied a previously reported Talbot-defocus multiscan tomography method for synchrotron X-ray microscopy and analyzed the degree of bone mineralization in mouse cortical bone around the lacuno–canalicular network, which is connected both to blood vessels and the peri- and endosteum. We detected cylindrical low mineral density regions spreading around canaliculi derived from a subset of osteocytes. Transmission electron microscopy revealed both intact and demineralized bone matrix around the canaliculus. Peri-canalicular low mineral density regions were also observed in osteopetrotic mice lacking osteoclasts, indicating that osteoclasts are dispensable for peri-canalicular demineralization. These data suggest demineralization can occur from within bone through the canalicular system, and that peri-canalicular demineralization occurs not uniformly but directed by individual osteocytes. Blockade of peri-canalicular demineralization may be a therapeutic strategy to increase bone mass and quality.     

Bisphosphonate (YM529) delays the repair of cortical bone defect after drill-hole injury by reducing terminal differentiation of osteoblasts in the mouse femur

We evaluated the effects of YM529, a nitrogen-containing bisphosphonate, on the repair of cortical bone after drill-hole injury at the tissue-, cell- and gene-levels in the femur of mice. Eight-week-old male C57BL/6N mice were treated with an intravenous injection of 0.01, 0.05 and 0.1 mg/kg body weight (BW) of YM529, or the vehicle (VC) once a week from 8 weeks of age until sacrifice. At 10 weeks of age (day 0), a drill-hole was made in the diaphysis of bilateral femurs. Femoral specimens were obtained at days 3, 5, 7, 10, 14, 21 and 28 after surgery. Histology and histomorphometry confirmed the early woven bone formation in 7 days after injury in all four groups, but the following lamellar bone repair in the cortical tissue area delayed only in the YM529-treated groups. Since the findings were not dose-dependent, following evaluations were performed in VC and YM529 0.1 mg/kg BW dose groups. Calcein-labeled surface of regenerated bone decreased at day 21 in the YM529 group. At day 0, CFU-f number and mineralized nodule area that developed from marrow cells were significantly smaller in YM529 group than in VC group. At day 5, however, these values increased to levels similar to those in VC group. The mRNA expression levels of BMP-2, cbfa1, osterix, type I collagen, and osteocalcin in the injured bone and marrow cells at days 3 and 5 were similar in the two groups, but were higher in YM529 group at day 7 compared with that in the VC group. At day 14, the levels of these mRNAs were still high, while that of osteocalcin was significantly reduced compared to the VC group. These data indicate that the action of YM529 on bone formation is bimodal, stimulatory on the developments of osteogenic cells for the woven bone regeneration and inhibitory on the terminal differentiation of osteoblasts for the later remodeling, consequently leading to a delay in the lamellar bone healing in the cortical tissue area.     

Bone structure in aseptic necrosis of the femoral head by light microscopy, contact microradiography, and fluorescent microscopy --comparing with osteoarthritis and rapidly destructive coxarthrosis

In order to obtain further information of the pathological changes and bone structure of aseptic necrosis (AN), the present author carried out histological investigation on 20 femoral heads removed by prosthesis or total hip replacement from 18 patients. Both decalcified and undecalcified sections were investigated by microscopy, contact microradiography (CMR) and fluorescent microscopy. Schemata of individual cases were drawn to know the relationship between femoral head deformity and bone reactivity. Moreover, 19 femoral heads with secondary osteoarthritis (OA) removed from 18 patients and 7 with rapidly destructive coxarthrosis (RDC) from 6 patients were similarly investigated to compare with AN. The following results were obtained; Histologically, there are four different areas in AN. From the top of the femoral head, the lesion consisted of (1) subchondral necrosis, (2) granulation tissue, (3) an area of variable bone formation, and (4) normal bone tissue. As the advancement of the head deformity, fibrous component in the granulation increases and seems to divide the femoral head into necrotic and living areas, which by fibrous as well as enchondral ossifications become more prominent in the edge of the demarcation. As for the bone formation, four different types of bone tissues are discernible by CMR; (1) low calcified appositional bone and its absorption, (2) low calcified, appositional bone on wide trabeculae, (3) low calcified appositional bone on narrow trabeculae, and (4) irregularly calcified woven bone. Although during the comparatively early stage, the bone formation is seen in almost all of the living area, as the advancement of the head deformity, its activity decreases and results in overt head deformity to resemble that of OA which would be caused by mechanical factor. Increase of fibrous component in the granulation along with decrease of the bone formation would reduce the repairing activity of the femoral head. In the femoral heads with OA, a wedge-shaped area of bone formation is formed of which peak is located on the medial thickened cortex. In the femoral heads with RDC, a large amount of woven bone with irregular calcification is found in the bone formation area, in the upper part of a femoral head.     

Sclerostin and the regulation of bone formation: Effects in hip osteoarthritis and femoral neck fracture

Remodeling imbalance in the elderly femoral neck can result in thin cortices and porosity predisposing to hip fracture. Hip osteoarthritis protects against intracapsular hip fracture. By secreting sclerostin, osteocytes may inhibit Wnt signaling and reduce bone formation by osteoblasts. We hypothesised that differences in osteocytic sclerostin expression might account for differences in osteonal bone‐formation activity between controls and subjects with hip fracture or hip osteoarthritis. Using specific antibody staining, we determined the osteocytic expression of sclerostin within osteons of the femoral neck cortex in bone removed from subjects undergoing surgery for hip osteoarthritis (hOA: 5 males, 5 females, 49 to 92 years of age) or hip fracture fixation (FNF: 5 males, 5 females, 73 to 87 years of age) and controls (C: 5 males, 6 females, 61 to 90 years of age). Sclerostin expression and distances of each osteocyte to the canal surface and cement line were assessed for all osteonal osteocytes in 636 unremodeled osteons chosen from fields (～0.5 mm in diameter) with at least one canal staining for alkaline phosphatase (ALP), a marker of bone formation. In adjacent sections, ALP staining was used to classify basic multicellular unit (BMUs) as quiescent or actively forming bone (ALP⁺). The areal densities of scl^? and scl⁺ osteocytes (number of cells per unit area) in the BMU were inversely correlated and were strong determinants of ALP status in the BMU. In controls and hip fracture patients only, sclerostin‐negative osteocytes were closer to osteonal surfaces than positively stained cells. Osteon maturity (progress to closure) was strongly associated with the proportion of osteonal osteocytes expressing sclerostin, and sclerostin expression was the chief determinant of ALP status. hOA patients had 18% fewer osteocytes per unit bone area than controls, fewer osteocytes expressed sclerostin on average than in controls, but wide variation was seen between subjects. Thus, in most hOA patients, there was increased osteonal ALP staining and reduced sclerostin staining of osteocytes. In FNF patients, newly forming osteons were similar in this respect to hOA osteons, but with closure, there was a much sharper reduction in ALP staining that was only partly accounted for by the increased proportions of osteonal osteocytes staining positive for sclerostin. There was no evidence for a greater effect on ALP expression by osteocytes near the osteonal canal. In line with data from blocking antibody experiments, osteonal sclerostin appears to be a strong determinant of whether osteoblasts actively produce bone. In hOA, reduced sclerostin expression likely mediates increased osteoblastic activity in the intracapsular cortex. In FNF, full osteonal closure is postponed, with increased porosity, in part because the proportion of osteocytes expressing sclerostin increases sharply with osteonal maturation. © 2010 American Society for Bone and Mineral Research     

Pathology of femoral head collapse following transtrochanteric rotational osteotomy for osteonecrosis

We investigated the pathology of femoral head collapse following transtrochanteric anterior rotational osteotomy. Six femoral heads were obtained during total hip arthroplasty some 2–12 years after osteotomy. In all cases, the preoperatively necrotic lesions exhibited mostly osteonecrosis with accumulation of bone marrow cell debris and trabecular bone with empty lacunae, although repair tissue such as granulation tissue and appositional bone formation were observed in limited areas in some cases. In the transposed intact articular surface of the femoral head, osteoarthritic changes such as fissure penetration to the subchondral bone and osteophyte formation were commonly observed. In newly created subchondral areas at weight-bearing sites, trabecular thickness and the number of trabecular bones had decreased, with few osteoblasts, osteoclasts, and osteocytes being present, resulting in a coarse lamellar structure of the trabecular bone. These findings suggest that transposed areas in cases of failure consist mostly of low-turnover osteoporotic lesions which could cause collapse of the femoral head. Received: 5 October 1999     

管型与非管型脱钙骨基质支架材料成骨活性差别的实验研究

目的探讨管型与非管型骨缺损修复材料成骨活性的差别。方法以兔同种异体尺桡骨脱钙骨基质为原料,制成管型(A组)及非管型(B组)修复材料各30只,随机植入30只兔双侧桡骨中上段1.5 cm骨缺损中,分别于术后1、2、4、8、12周随机处死6只取材进行X线、组织学切片、大体观察、单位湿重Ca值等检查,结果进行统计学处理。结果早期(术后2周)A组具有更多的骨痂生成(P<0.05)、更高的单位湿重Ca值(P<0.05);中期(术后4周)A组编织骨具有更高的孔隙率(P<0.05);后期(术后8、12周)A组表现为更薄的板层骨、更好的髓腔再通率、更好的塑形、更成熟的骨髓形成(P<0.05)、更高的单位湿重Ca值(P<0.05)。结论实验显示管型脱钙骨基质较非管型具有更好的节段性骨缺损修复能力,提示管型人工骨治疗节段性骨缺损具有美好的前景。     

Organization of apatite crystals in human woven bone

The organization of collagen fibrils differs in woven bone and lamellar bone, and it reflects certain aspects of the nature of the mineral crystals associated with them. In order to investigate the morphology and distribution of apatite crystals in woven bone, mineralized collagen fibrils and isolated crystals from the mid-diaphyses of human fetal femurs were observed with scanning and transmission electron microscopy and high-resolution electron microscopy. A number of features of woven bone were observed for the first time by these means. Similar to mature crystals from lamellar bone, the apatite crystals in woven bone are also platelet-shaped. However, most likely because of a high rate of old bone resorption and new bone formation in woven material, the average crystal dimensions are considerably smaller than those of mature crystals in lamellar bone. Apatite crystals were noted on the surface of collagen fibrils in woven bone. In densely packed woven bone, the periodicity of mineral deposited on individual fibrils is in registration over many fibrils. In addition to their association with collagen surfaces, crystals also appear distributed in both extrafibrillar and intrafibrillar collagen regions. In both cases, the minerals are crystalline and defect-free. These characteristics provide insight into the spatial and temporal relation between collagen and mineral that is the basis for the structure and organization of the mineral comprising human woven bone.     

Use of cryopreserved bone in spinal surgery

One hundred fifty-two consecutive spinal fusions were performed over a 4-year period in 143 patients. Autogenous bone was used in 62 patients and frozen cryopreserved bone in 90. A variety of anterior and posterior procedures with and without instrumentation were performed. The percentage of successful arthrodesis was 87 in those who received autogenous bone, and 86.6 in those who received allograft bone. Thirty-four spinal fusions were surgically explored. Histologic evaluation of the bone taken at the time of surgical exploration showed viable osteocytes laying down osteoid, woven and lamellar bone, and no inflammatory or foreign body reaction. The authors conclude that cryopreserved bone, harvested and processed as described, is advantageous, safe, and results in a rate of bone union comparable to that of autogenous bone.     

Stimulation of bone formation in vivo by transforming growth factor-beta: remodeling of woven bone and lack of inhibition by indomethacin

Local effects of transforming growth factor-beta (TGF beta) on bone have been investigated in growing mice. The influence of indomethacin on TGF beta effects was also examined. Five daily injections of TGF beta-1 or -2 were administered subcutaneously over the frontal and parietal bones of seven-week-old mice. In animals treated with TGF beta alone or TGF beta and indomethacin, then killed on day 19 (day 1 = day of first TGF beta injection), the combined frontal and parietal bones were heavier and the frontal bones were thicker than in controls. On day 4, multiple layers of differentiating osteoblasts were visible in the periosteum of calvariae from TGF beta-treated animals. On day 8, highly vascularised periosteal woven bone was seen in the calvariae of TGF beta-treated animals. On day 19 (14 days after the last injection) the woven bone had been partially remodeled into lamellar bone containing bone marrow, and was still actively being remodeled. The histological appearance of bones from animals treated concurrently with TGF beta and indomethacin was identical to that of animals treated with TGF beta alone, and there appeared to be no difference between TGF beta-1 and TGF beta-2. Thus TGF beta-stimulated bone formation in vivo is a long term effect that appears to be independent of prostaglandins. Osteoclast counts in sections of TGF beta-treated animals were not elevated on day 4, although at this stage effects on osteoblastic cells were already morphologically apparent. Osteoclast counts in TGF beta-treated bones were significantly increased over control values on days 8 and 19.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early anchoring collagen fibers at the bone-tendon interface are conducted by woven bone formation: light microscope and scanning electron microscope observation using a canine model.

To clarify the early process of recovery at the bone-tendon interface, we used light microscopy and SEM to examine the process of anchoring of collagen fibers to bone in a canine model. At two weeks, tendon, scar tissue, woven bone and lamellar bone were present at the insertion site. SEM revealed anchoring of collagen fibril bundles of the scar to the woven bone. By 4 weeks, the number of anchoring fibers had increased and a parallel arrangement of fibers was observed. SEM demonstrated deep penetration of fibers into the woven bone layer. In addition, the fibers were observed to project into and intermingle with the scar tissue. By 6 weeks, the anchoring fibers had developed fully and were distributed densely over the interface. SEM also revealed that the collagen fibril bundles in the scar tissue had connected with the collagen fibrils of the woven bone by way of the anchoring bundles. The woven bone was identifiable throughout the early stages of recovery as the interface between soft tissue and hard tissue. Throughout all experimental periods, no staining was observed at the interface of the tendon and bone by Saffranin-O. The formation of woven bone was important during early recovery of the tendon-bone interface prior to the completion of fibrocartilage-mediated insertion.     

<Emphasis Type="Italic">In Vivo</Emphasis> Mechanical Loading Modulates Insulin-Like Growth Factor Binding Protein-2 Gene Expression in Rat Osteocytes

Mechanical stimulation is essential for maintaining skeletal integrity. Mechanosensitive osteocytes are important during the osteogenic response. The growth hormone-insulin-like growth factor (GH-IGF) axis plays a key role during regulation of bone formation and remodeling. Insulin-like growth factor binding proteins (IGFBPs) are able to modulate IGF activity. The aim of this study was to characterize the role of IGFBP-2 in the translation of mechanical stimuli into bone formation locally in rat tibiae. Female Wistar rats were assigned to three groups (n = 5): load, sham, and control. The four-point bending model was used to induce a single period of mechanical loading on the tibial shaft. The effect on IGFBP-2 mRNA expression 6 hours after stimulation was determined with nonradioactive in situ hybridization on decalcified tibial sections. Endogenous IGFBP-2 mRNA was expressed in trabecular and cortical osteoblasts, some trabecular and subendocortical osteocytes, intracortical endothelial cells of blood vessels, and periosteum. Megakaryocytes, macrophages, and myeloid cells also expressed IGFBP-2 mRNA. Loading and sham loading did not affect IGFBP-2 mRNA expression in osteoblasts, bone marrow cells, and chondrocytes. An increase of IGFBP-2 mRNA-positive osteocytes was shown in loaded (1.68-fold) and sham-loaded (1.35-fold) endocortical tibial shaft. In conclusion, 6 hours after a single loading session, the number of IGFBP-2 mRNA-expressing osteocytes at the endosteal side of the shaft and inner lamellae was increased in squeezed and bended tibiae. Mechanical stimulation modulates IGFBP-2 mRNA expression in endocortical osteocytes. We suggest that IGFBP-2 plays a role in the lamellar bone formation process.     

The treatment of osteomyelitis with gentamicin-reconstituted bone xenograft-composite

We have developed a new drug-delivery system using reconstituted bone xenograft to treat chronic osteomyelitis. This material, which has the capabilities of osteoinduction and osteoconduction, was supplemented with up to 2000 times the minimum inhibitory concentration of gentamicin against Staphylococcus aureus to prepare a gentamicin-reconstituted bone xenograft-composite (G-RBX-C). In a rabbit model, we evaluated the release of gentamicin from this composite in vivo, its capability for induction of ectopic bone and the repair of segmental defects of the radius. There was a high level of concentration of antibiotics, which was sustained for at least ten days. In the study of induction of ectopic bone, there was abundant woven bone in the G-RBX-C group two weeks after operation. At 16 weeks after implantation of G-RBX-C the radial defects had been repaired, with the formation of lamellar bone and recanalisation of the marrow cavity. Our findings suggest that G-RBX-C may be useful in the treatment of chronic osteomyelitis.     

Cancellous bone of aged rats maintains its capacity to respond vigorously to the anabolic effects of prostaglandin E2 by modeling-dependent bone gain

The present study examined the early effects of prostaglandin (PG)E2 on proximal tibial metaphyses of 20-month-old Wistar male rats. PGE, was given to intact rats for 10 and 30 days at 3mg/kg/day. After multiple in vivo fluorochrome labeling, undecalcified longitudinal sections were subjected to analysis of bone histomorphometry and classification of the contour of the cement line in bone formation units. The latter was used to classify bone formation units into modeling, remodeling and uncertain units. After 10 days of treatment, there was a 2% increase in woven bone formation with the appearance of osteoprogenitor cells and increases in the number of osteoblasts (649%) and osteoid (375%) surfaces. Remodeling and modeling units increased by 56% and 429%. respectively. After 30 days of treatment, there was an increase of 212% of total trabecular bone mass, 60% of which was woven bone. In addition, there were increases in labeling surface (147%), mineral apposition rate (760%), bone formation rates tissue area (BFR/T.Ar, 1920%; BFR/B.Pm, 343%), and bone turnover (BFR/B.Ar, 426%). Osteoblasts and osteoid production at 30 days were 29% and 58% less than at 10 days post-treatment. Modeling and remodeling activity did not differ from that seen at 10 days. In addition, PGE2 treatment tended to stimulate the closing of growth plates and decrease the fatty marrow area. We conclude that the aged skeleton was able to respond vigorously to PGE2 treatment. Massive osteoprogenitors cells, and osteoid and osteoblast formations were observed within 10 days. and dramatic woven and lamellar bone formation was seen at 30 days post-treatment. The anabolic effects were driven mainly by modeling.