Toshihiro Izumi

A local application of recombinant human fibroblast growth factor 2 for tibial shaft fractures: A randomized, placebo‐controlled trial

Fibroblast growth factor 2 (FGF‐2) is a potent mitogen for mesenchymal cells, and a local application of recombinant human FGF‐2 (rhFGF‐2) in a gelatin hydrogel has been reported to accelerate bone union in our animal studies and preparatory dose‐escalation trial on patients with surgical osteotomy. We have performed a randomized, double‐blind, placebo‐controlled trial in which patients with fresh tibial shaft fractures of transverse or short oblique type were randomly assigned to three groups receiving a single injection of the gelatin hydrogel containing either placebo or 0.8 mg (low‐dosage group) or 2.4 mg (high‐dosage group) of rhFGF‐2 into the fracture gap at the end of an intramedullary nailing surgery. Of 194 consecutive patients over 2 years, 85 met the eligibility criteria, and 70 (24 in the placebo group and 23 each in low‐ and high‐dosage groups) completed the 24‐week study. The cumulative percentages of patients with radiographic bone union were higher in the rhFGF‐2‐treated groups (p = .031 and .009 in low‐ and high‐dosage group, respectively) compared with the placebo group, although there was no significant difference between low‐ and high‐dosage groups (p = .776). At 24 weeks, 4, 1, and 0 patients in the placebo, low‐dosage, and high‐dosage groups, respectively, continued to show delayed union. No patient underwent a secondary intervention, and the time to full weight bearing without pain was not significantly different among the three groups (p = .567). There also was no significant difference in the profiles of adverse events among the groups. In conclusion, a local application of the rhFGF‐2 hydrogel accelerated healing of tibial shaft fractures with a safety profile.

Localization and quantification of proliferating cells during rat fracture repair: detection of proliferating cell nuclear antigen by immunohistochemistry.

Bilateral femurs of 12‐week‐old female Sprague‐Dawley rats were fractured, and the fractured femurs were harvested 36 h, 3, 7, 10, and 14 days after the fracture. Localization of cell proliferation in the fracture calluses was investigated using immunohistochemistry with antiproliferating cell nuclear antigen (PCNA) monoclonal antibodies. Thirty‐six hours after the fracture, many PCNA‐positive cells were observed in the whole callus. The change was not limited to mesenchymal cells at the fracture site where the inflammatory reaction had occurred, but extended in the periosteum along almost the entire femoral diaphysis where intramembranous ossification was initiated. On day 3, periosteal cells or premature osteoblasts in the newly formed trabecular bone during intramembranous ossification still displayed intense staining. On day 7, many premature chondrocytes and proliferating chondrocytes were PCNA positive. Endochondral ossification appeared on days 10 and 14, and the premature osteoblasts and endothelial cells in the endochondral ossification front were stained with anti‐PCNA antibodies. Quantification of PCNA‐positive cells was carried out using an image analysis computer system, obtaining a PCNA score for each cellular event. The highest score was observed in the periosteum early after the fracture near the fracture site. Immunohistochemistry using anti‐PCNA antibodies showed that the distribution of proliferating cells and the degree of cell proliferation varied according to the time lag after the fracture, suggesting the existence of local regulatory factors such as growth factors, and that significant cell proliferation was observed at the beginning of each cellular event.

Serum 1α,25-Dihydroxyvitamin D3 Accumulates into the Fracture Callus during Rat Femoral Fracture Healing

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is thought to be an important systemic factor in the fracture repair process, but the mechanism of action of 1,25(OH)2D3 has not been clearly defined. In this study, the role of 1,25(OH)2D3 in the fracture repair process was analyzed in a rat closed femoral fracture model. The plasma concentration of 1,25(OH)2D3 rapidly decreased on day 3 and continued to decrease to 10 days after fracture. We assessed whether this decrease was based on the accelerated degradation or retardation of the synthesis rate of 1,25(OH)2D3, from 25(OH)D3. After radiolabeled 3H-1,25(OH)2D3 or 3H-25(OH)D3 was injected i.v. into fractured or control (unfractured) rats, the concentrations of 25(OH)D3 and 1,25(OH)2D3 metabolites were measured by HPLC. The plasma concentrations of these radiolabeled metabolites in fractured group were similar to those in control rats early after operation. However, radioactivity in the femurs of fractured rats was higher than that of the control group. Furthermore, the radioactivity was concentrated in the callus of the fractured group analyzed by autoradiography. 1,25(OH)2D3 receptor gene expression was detected early after fracture and, additionally, both in the soft and hard callus on days 7 and 13 after fracture. These results showed that the rapid disappearance of 1,25(OH)2D3 in the early stages after fracture was not due to either increased degradation or decreased synthesis of 1,25(OH)2D3, but rather to increased consumption. Further, these results suggest the possibility that plasma 1,25(OH)2D3 becomes localized in the callus and may regulate cellular events in the process of fracture healing.

Administration of growth hormone modulates the gene expression of basic fibroblast growth factor in rat costal cartilage, both in vivo and in vitro

We examined the effect of growth hormone on local growth factor mRNA expression in male Sprague-Dawley rats. Repetitive systemic administration of growth hormone (0.4 IU every 4 h) increased the expression of IGF-I mRNA up to 2.8-fold in costal cartilage tissue compared with controls. Basic FGF (bFGF) mRNA expression gradually increased up to 15.5-fold compared with pre-injection samples, where the mRNA expression was 5.3-times greater than vehicle-injected controls. TGF-beta mRNA showed little changes. Moreover, one microgram/ml of growth hormone enhanced the expression of bFGF mRNA in costal chondrocytes in culture. We conclude that growth hormone increased the local expression of bFGF, as well as that of IGF-I, in cartilage, and suggest that bFGF is directly regulated by growth hormone within a local area.

Upside-down etching; a simple method for reducing unetchable sediment during deep-etching of fresh untreated tissues

A simple new method of deep‐etching, ‘upside‐down etching’, has been developed in an attempt to obtain clearer images or replicas, especially of fresh, untreated biological tissues. During etching, specimens were kept facing downwards and shaken periodically so that non‐volatile materials in the subliming ice could fall out of the frozen tissue. This improvement leads to a considerable reduction of sedimented debris of unetchable materials on ‘true’ membrane surfaces, as well as in the cytoplasm exposed by deep‐etching.