Basic fibroblast growth factor 2 (bFGF) accelerates bone formation during fracture healing. Because the efficacy of bFGF decreases rapidly following its diffusion from fracture sites, however, repeated dosing is required to ensure a sustained therapeutic effect. We previously developed a fusion protein comprising bFGF, a polycystic kidney disease domain (PKD; s2b), and collagen-binding domain (CBD; s3) sourced from the Clostridium histolyticum class II collagenase, ColH, and reported that the combination of this fusion protein with a collagen-like peptide, poly(Pro-Hyp-Gly)10, induced mesenchymal cell proliferation and callus formation at fracture sites. In addition, C. histolyticum produces class I collagenase (ColG) with tandem CBDs (s3a and s3b) at the C-terminus. We therefore hypothesized that a bFGF fusion protein containing ColG-derived tandem CBDs (s3a and s3b) would show enhanced collagen-binding activity, leading to improved bone formation. Here, we examined the binding affinity of four collagen anchors derived from the two clostridial collagenases to H-Gly-Pro-Arg-Gly-(Pro-Hyp-Gly)12-NH2, a collagenous peptide, by surface plasmon resonance and found that tandem CBDs (s3a-s3b) have the highest affinity for the collagenous peptide. We also constructed four fusion proteins consisting of bFGF and s3 (bFGF-s3), s2b-s3b (bFGF-s2b-s3), s3b (bFGF-s3b), and s3a-s3b (bFGF-s3a-s3b) and compared their biological activities to those of a previous fusion construct (bFGF-s2b-s3) using a cell proliferation assay in vitro and a mouse femoral fracture model in vivo. Among these CB-bFGFs, bFGF-s3a-s3b showed the highest capacity to induce mesenchymal cell proliferation and callus formation in the mice fracture model. The poly(Pro-Hyp-Gly)10/bFGF-s3a-s3b construct may therefore have the potential to promote bone formation in clinical settings.

BackgroundThe infrapatellar fat pad (IFP) is implicated in knee osteoarthritis (KOA). Calcitonin gene-related peptide (CGRP), a vasoactive neuropeptide expressed in joint tissues and synovial tissues (ST), was recently found to be associated with KOA progression and pain. CGRP is expressed in the IFPs of human KOA patients; however, its regulation has not been elucidated.MethodsIFPs and STs were harvested from 138 KOA patients during total knee replacement (TKR) and analyzed for CGRP, cycloxygenase-2 (COX-2), and microsomal prostaglandin E synthase-1 (mPGES-1) expression using real-time polymerase chain reaction (PCR). To investigate CGRP regulation by prostaglandin E2 (PGE2), adipocytes (Ad) and the stromal vascular fraction (SVF) were harvested from IFPs using collagenase. Synovial cells (SYC) were also harvested from ST and stimulated with vehicle (serum-free culture medium), PGE2, or CGRP.ResultsCGRP, COX-2, and mPGES-1 expression levels were significantly higher in IFPs than STs. PGE2 stimulation increased CGRP expression in Ad, the SVF, and SYC; however, CGRP expression was significantly higher in PGE2-stimulated SVF than PGE2-stimulated SYC. CGRP stimulation had no effect on COX-2 or mPGES-1 expression.ConclusionsCGRP expression in the IFP of KOA patients is regulated by the COX-2/mPGES-1/PGE2 pathway.

We present a rare case of spinal epidural hematoma (SEH) after thoracolumbar posterior fusion without decompression surgery for a thoracic vertebral fracture. A 42-year-old man was hospitalized for a thoracic vertebral fracture caused by being sandwiched against his back on broken concrete block. Computed tomography revealed a T12 dislocation fracture of AO type B2, multiple bilateral rib fractures, and a right hemopneumothorax. Four days after the injury, in order to promote early orthostasis and to improve respiratory status, we performed thoracolumbar posterior fusion surgery without decompression; the patient had back pain but no neurological deficits. Three hours after surgery, he complained of acute pain and severe weakness of his bilateral lower extremities; with allodynia below the level of his umbilicus, postoperative SEH was diagnosed. We performed immediate revision surgery. After removal of the hematoma, his symptoms improved gradually, and he was discharged ambulatory one month after revision surgery. Through experience of this case, we should strongly consider the possibility of preexisting SEH before surgery, even in patients with no neurological deficits. We should also consider perioperative coagulopathy in patients with multiple trauma, as in this case.

Background An enzymatic crosslinking strategy using hydrogen peroxide and horseradish peroxidase is receiving increasing attention for application with in situ-formed hydrogels (IFHs). Several studies have reported the application of IFHs in cell delivery and tissue engineering. IFHs may also be ideal carrier materials for bone repair, although their potential as a carrier for bone morphogenetic protein (BMP)-2 has yet to be examined. Here, we examined the effect of an IFH made of hyaluronic acid (IFH-HA) containing BMP-2 in promoting osteogenesis in a mouse refractory fracture model. Methods Immediately following a fracture procedure, animals either received no treatment (control) or an injection of IFH-HA/PBS or IFH-HA containing 2 μg BMP-2 (IFH-HA/BMP-2) into the fracture site (n = 16, each treatment). Results Fracture sites injected with IFH-HA/BMP-2 showed significantly greater bone volume, bone mineral content, and bone union compared with sites receiving no treatment or treated with IFH-HA/PBS alone (each n = 10). Gene expression levels of osteogenic markers, Alpl, Bglap, and Osx, were significantly raised in the IFH-HA/BMP-2 group compared to the IFH-HA/PBS and control groups (each n = 6). Conclusion IFH-HA/BMP-2 may contribute to the treatment of refractory fractures.

An enzymatic crosslinking strategy using hydrogen peroxide (H 2 O 2) and horseradish peroxidase (HRP) has been receiving increasing attention for use with in situ-formed hydrogels (IFHs). Several studies have reported the application of IFHs in cell delivery and tissue engineering. IFHs may also be ideal carrier materials for bone repair, although their potential as a carrier for basic fibroblast growth factor (bFGF) has yet to be evaluated. Here, we examined the effect of an IFH made of dextran (Dex)-tyramine (TA) conjugates (IFH-Dex-TA) containing bFGF in promoting bone formation in a fracture model in mice. Immediately following a fracture procedure, animals either received no treatment (control) or an injection of IFH-Dex-TA/phosphate-buffered saline (IFH-Dex-TA/PBS) or IFH-Dex-TA containing 1 μg bFGF (IFH-Dex-TA/bFGF) into the fracture site (n=10, each treatment). Fracture sites injected with IFH-Dex-TA/bFGF showed significantly greater bone volume, mineral content, and bone union than sites receiving no treatment or treated with IFH-Dex-TA/PBS alone (each n=10). This Dex-TA gel may be an effective drug delivery system for optimizing bFGF therapy.