Hisako Fujimaki

Acceleration of bone formation during fracture healing by injectable collagen powder and human basic fibroblast growth factor containing a collagen‐binding domain from Clostridium histolyticum collagenase

Growth factor delivered with implantable biomaterials has been used to both accelerate and ensure healing of open fractures in human patients. However, a major limitation of implantable biomaterials is the requirement for open surgical placement. Here, we developed an injectable collagen material-based bone formation system consisting of injectable collagen powder with fibril morphology and collagen triple helix conformation, and basic fibroblast growth factor (bFGF) fused to the collagen-binding domain (CBD) of Clostridium histolyticum collagenase. The affinity of the CBD towards collagen was confirmed by the results of collagen-binding assays. Moreover, the combination of the collagen binding-bFGF fusion protein (CB-bFGF) with injectable collagen powder induced bone formation at protein concentrations lower than those required for bFGF alone in mice fracture models. Taken together, these properties suggest that the CB-bFGF/collagen powder composite is a promising injectable material for bone repair in the clinical setting.

Synovial macrophage-derived IL-1β regulates the calcitonin receptor in osteoarthritic mice.

Recent studies have reported that calcitonin gene‐related peptide (CGRP) contributes to joint pain. However, regulation of the CGRP/CGRP receptor signalling in osteoarthritis (OA) is not fully understood. To investigate the regulation of CGRP/CGRP receptor signalling by macrophages in the synovial tissue (ST) of OA joints, we characterized the gene expression profiles of CGRP and CGRP receptors in the ST of OA mice (STR/Ort). In addition, we examined whether macrophage depletion by the systemic injection of clodronate‐laden liposomes affected the expression of CGRP and CGRP receptors in ST. CD11c+ macrophages in the ST of STR/Ort and C57BL/6J mice were analysed by flow cytometry. Real‐time polymerase chain reaction (PCR) was used to evaluate the expression of interleukin (IL)‐1β, CGRP, calcitonin receptor‐like receptor (CLR) and receptor activity‐modifying protein 1 (RAMP1) in F4/80+ and F4/80− cells. The effects of IL‐1β on the expression of CGRP and CLR by cultured synovial cells were also examined. The percentage of CD11c+ macrophages in the ST of STR/Ort was higher than that in C57/BL6J mice. Notably, the F4/80+ cell fraction expressed IL‐1β highly, whereas the F4/80− cell fraction expressed CGRP, CLR, and RAMP1 highly. In addition, expression of the IL‐1β and CLR genes was increased in ST, but was decreased upon macrophage depletion, and the IL‐1β treatment of cultured synovial cells up‐regulated CLR. Taken together, the present findings suggest that synovial macrophages are the major producers of IL‐1β and regulators of CLR in OA mice. Therefore, macrophages and IL‐1β may be suitable therapeutic targets for treating OA pain.

Nerve Growth Factor Regulation by TNF-α and IL-1β in Synovial Macrophages and Fibroblasts in Osteoarthritic Mice

To investigate the role of macrophages as a regulator and producer of nerve growth factor (NGF) in the synovial tissue (ST) of osteoarthritis (OA) joints, the gene expression profiles of several inflammatory cytokines in the ST, including synovial macrophages and fibroblasts, of OA mice (STR/Ort) were characterized. Specifically, real-time polymerase chain reaction analysis was used to evaluate the expression of tumor necrosis factor- (TNF-) α, interleukin- (IL-) 1β, IL-6, and NGF in CD11b+ and CD11b– cells isolated from the ST of a murine OA model. The effects of TNF-α, IL-1β, and IL-6 on the expression of NGF in cultured synovial cells were also examined. The expression of TNF-α, IL-1β, IL-6, and NGF in the ST of STR/Ort was higher than that in C57/BL6J mice. Compared to the CD11b– cell fraction, higher expression levels of TNF-α, IL-1β, and IL-6 were detected in the CD11b+ cell fraction, whereas no differences in the expression of NGF were detected between the two cell fractions. Notably, TNF-α upregulated NGF expression in synovial fibroblasts and macrophages and IL-1β upregulated NGF expression in synovial fibroblasts. IL-1β and TNF-α may regulate NGF signaling in OA joints and be suitable therapeutic targets for treating OA pain.

Acceleration of callus formation during fracture healing using basic fibroblast growth factor-kidney disease domain-collagen-binding domain fusion protein combined with allogenic demineralized bone powder

BackgroundTo repair fractures with large bone defects or gaps, demineralized allogenic bone matrix (DBM) is often applied to the fracture site. However, studies have shown that the use of DBM alone has limited efficacy for repairing fractures. In the present study, we developed an allogenic demineralized bone powder (DBP) with basic fibroblast-derived growth factor containing a polycystic kidney disease (PKD) domain and collagen-binding domain (CBD) from Clostridium histolyticum collagenase (ColH) and investigated the stimulatory effects of bFGF-PKD-CBD combined with allogenic DBP on bone growth in a mouse femur fracture model.MethodsDBP mixed with either phosphate-buffered saline (PBS) (DBP/PBS), 0.58 nmol basic fibroblast growth factor (bFGF) (0.58 nmol DBP/bFGF), 0.058 nmol bFGF-PKD-CBD (0.058 nmol DBP/bFGF-PKD-CBD), or 0.58 nmol bFGF-PKD-CBD (0.58 nmol DBP/bFGF-PKD-CBD) was grafted into fracture sites.ResultsbFGF-PKD-CBD/DBP composite accelerates callus formation in a bone fracture model in mice and clearly showed that the composite also increases bone mineral density at fracture sites compared to bFGF/DBP. In addition, bFGF-PKD-CBD/DBP increased callus volume and bone mineral content to similar levels in fractures treated with a tenfold higher amount of bFGF at 4 weeks.ConclusionsOur results suggest that bFGF-PKD-CBD/DBP may be useful for promoting fracture healing in the clinical setting.

Activation of calcitonin gene-related peptide signaling through the prostaglandin E2-EP1/EP2/EP4 receptor pathway in synovium of knee osteoarthritis patients

BackgroundCalcitonin gene-related peptide (CGRP) is a 37-amino-acid vasodilatory neuropeptide that binds to receptor activity-modifying protein 1 (RAMP1) and the calcitonin receptor-like receptor (CLR). Clinical and preclinical evidence suggests that CGRP is associated with hip and knee joint pain; however, the regulation mechanisms of CGRP/CGRP receptor signaling in synovial tissue are not fully understood.MethodsSynovial tissues were harvested from 43 participants with radiographic knee osteoarthritis (OA; unilateral Kellgren/Lawrence (K/L) grades 3–4) during total knee arthroplasty. Correlationships between the mRNA expression levels of CGRP and those of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and cycloxygenase-2 (COX-2) were evaluated using real-time PCR analysis of total RNA extracted from the collected synovial tissues. To investigate the factors controlling the regulation of CGRP and CGRP receptor expression, cultured synovial cells were stimulated with TNF-α, IL-1β, IL-6, and prostaglandin E2 (PGE2) and were also treated with PGE2 receptor (EP) agonist.ResultsCGRP and COX-2 localized in the synovial lining layer. Expression of COX-2 positively correlated with CGRP mRNA expression in the synovial tissue of OA patients. The gene expression of CGRP and RAMP1 increased significantly in synovial cells exogenously treated with PGE2 compared to untreated control cells. In cultured synovial cells, CGRP gene expression increased significantly following EP4 agonist treatment, whereas RAMP1 gene expression increased significantly in the presence of exogenously added EP1 and EP2 agonists.ConclusionsPGE2 appears to regulate CGRP/CGRP receptor signaling through the EP receptor in the synovium of knee OA patients.

Acceleration of bone formation during fracture healing by poly(pro-hyp-gly)10 and basic fibroblast growth factor containing polycystic kidney disease and collagen-binding domains from Clostridium histolyticum collagenase.

Growth factor delivered in combination with animal-derived collagen materials has been used to accelerate bone fracture healing in human patients. However, the introduction of bovine proteins into humans carries the risk of zoonotic and immunologic complications. Here, we developed a collagen-like polypeptide-based bone formation system consisting of poly(Pro-Hyp-Gly)10 , which mimics the triple helical conformation of collagen, and basic fibroblast growth factor (bFGF) fused to the polycystic kidney disease (PKD) domain and collagen-binding domain (CBD) of Clostridium histolyticum collagenase. Circular dichroism spectral analysis showed that when pepsin-soluble bovine type I collagen was treated at 50°C, a positive signal corresponding to the collagen triple helix at 220 nm was not detected. In contrast, poly(Pro-Hyp-Gly)10 retained the 220-nm positive peak, even when treated at 80°C. The combination of the collagen binding-bFGF fusion protein (bFGF-PKD-CBD) with poly(Pro-Hyp-Gly)10 induced greater bone formation compared to bFGF alone in mice bone fracture models. Taken together, these properties suggest that the bFGF-PKD-CBD/poly(Pro-Hyp-Gly)10 composite is a promising material for bone repair in the clinical setting.

Bilateral carpal tunnel syndrome due to gouty tophi: conservative and surgical treatment in different hands of the same patient

Abstract Gouty tophi are an uncommon cause of carpal tunnel syndrome. We describe a case of bilateral carpal tunnel syndrome due to gouty tophi. Gouty tophi in the right wrist developed slowly, but developed acutely in flexor tendons in the left wrist. Symptoms were numbness and finger movement dysfunction in both hands. The right hand was treated surgically, while the left hand was treated by medication. Both hands improved under a well-controlled serum uremic acid level.

The cytokine expression in synovial membrane and the relationship with pain and pathological findings at hip arthroscopy

BackgroundSynovial membrane inflammation is the most common finding presenting during hip arthroscopy, and may play a role in hip pain. We sought to determine the relationships between synovial cytokine levels, hip pain, and arthroscopic findings of the hip joint.MethodsWe prospectively included 33 patients who underwent arthroscopic hip surgery (34 hips). For all patients, radiographs and severity of pain were evaluated preoperatively. During arthroscopy, we classified the chondral injury and synovitis, noted the incidence of labral tear and its instability, and a sample of the synovial membrane was harvested for quantitative PCR to determine levels of TNFα, IL1β, IL6, ADAMTS4, MMP1, and MMP3. The relationships between the levels of these cytokines, severity of hip pain, and the pathological findings during arthroscopy were examined.ResultsPain intensity and cytokine levels were not significantly different between patients with labral tear or instability and those without. By contrast, the expression of TNFα, IL1β, IL6, and MMP1 mRNA was significantly higher in patients with diffuse synovitis than in patients with focal synovitis. VAS score during rest showed significant positive correlation with IL6 (r = 0.45, p < 0.01), while VAS score on walking showed a positive correlation with TNFα (r = 0.47, p < 0.01), and ADAMTS4 (r = 0.51, p < 0.01). The modified Harris Hip pain score showed a negative correlation with TNFα (r = −0.38, p = 0.04) and IL6 (r = −0.58, p < 0.01).ConclusionsThe severity of synovitis and chondral injury are considered to be more important in the pathology of hip pain than labral tear or instability. Inflammatory cytokines, especially TNFα and IL6 might play an important role in the pathogenesis of pain in patients indicated for hip arthroscopy, possibly depending on the severity of synovitis.

Elevation of Microglial Basic Fibroblast Growth Factor Contributes to Development of Neuropathic Pain after Spinal Nerve Ligation in Rats

Study Design. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistological analysis of spinal cord and pain behavior analysis in a rat neuropathic pain model were conducted to examine the function of microglial basic fibroblast growth factor (bFGF) in the development of neuropathic pain. Objective. To investigate the role of bFGF in spinal microglia during the development of allodynia following spinal nerve ligation in rats. Summary of Background Data. Evidence suggests that the production of bFGF by spinal cord glial cells is increased in response to peripheral nerve injury. Although an association between bFGF and astrocytes has been widely reported, the relationship between bFGF and microglia, particularly with respect to the development of neuropathic pain, remains poorly understood. Methods. Spinal nerve ligation rats were used. After surgery, bFGF expression in the spinal cord was investigated using RT-PCR and immunohistochemistry. Neutralizing antibodies to bFGF were injected intrathecally into rats after spinal nerve ligaton. Spinal cords were used for RT-PCR analysis and pain behavior was analyzed using the von Frey test. Results. bFGF mRNA expression was significantly increased in the spinal cord 6 hours after spinal nerve ligation compared with untreated rats. Immunohistochemical analysis revealed that bFGF co-localized with ionized calcium-binding adaptor molecule 1, a microglial marker, and myeloperoxidase. Neutralizing antibodies to bFGF attenuated mechanical allodynia and myeloperoxidase mRNA expression. Conclusion. bFGF increased in spinal microglia during the development allodynia after spinal nerve ligation. Thus, controlling bFGF release from microglia during the acute stage of peripheral nerve injury may suppress the progression of allodynia. Level of Evidence: N/A

Hyaluronic Acid (800 kDa) Supplementation of University of Wisconsin Solution Improves Viability of Osteochondral Grafts and Reduces Matrix Metalloproteinase Expression during Cold Preservation

Osteochondral allografting is a promising option for the treatment of large cartilage defects. However, because the cell viability of osteochondral tissues (OCTs) gradually reduces during storage at 4°C, methods for maintaining the cell viability of fresh OCTs are needed to improve transplantation outcomes. Here, we evaluated whether the supplementation of preservation solution with one of three different molecular weight forms of hyaluronic acid (HA) improved the viability of rat OCTs during long-term cold storage. The supplementation of University of Wisconsin (UW) solution with 800 kDa significantly improved the cell viability of OCT after 14 days at 4°C compared to nonsupplemented UW solution. In contrast, UW solution supplemented with either 1900 or 6000 kDa HA did not markedly improve the cell viability of the OCT. Real-time PCR analysis revealed that the levels of matrix metalloproteinases 2, 3, and 9 were significantly decreased in OCT stored in UW solution supplemented with 800 kDa HA. Although further studies in human OCT are warranted, these findings demonstrate that the use of 800 kDa HA in place of serum may be a suitable approach for the long-term preservation of osteochondral allografts designated for the repair of large cartilage defects in the clinical setting.