In situ-formed hyaluronan gel/BMP-2/hydroxyapatite composite promotes bone union in refractory fracture model mice.

BACKGROUND: A combination of synthetic porous materials and BMP-2 has been used to promote fracture healing. For bone healing to be successful, it is important to use growth factor delivery systems that enable continuous release of BMP-2 at the fracture site. We previously reported that in situ-formed gels (IFGs) consisting of hyaluronan (HyA)-tyramine (TA), horseradish peroxidase and hydrogen peroxide enhance the bone formation ability of hydroxyapatite (Hap)/BMP-2 composites in a posterior lumbar fusion model. OBJECTIVE: We examined the effectiveness of IFGs-HyA/Hap/BMP-2 composites for facilitating osteogenesis in refractory fracture model mice. METHODS: After establishing the refractory fracture model, animals were either treated at the site of fracture with Hap harboring BMP-2 (Hap/BMP-2) or IFGs-HyA with Hap harboring BMP-2 (IFGs-HyA/Hap/BMP-2) (n = 10 each). Animals that underwent the fracture surgery but did not receive any treatment were considered the control group (n = 10). We determined the extent of bone formation at the fracture site according to findings on micro-computed tomography and histological studies four weeks following treatment. RESULTS: Animals treated with IFGs-HyA/Hap/BMP-2 demonstrated significantly greater bone volume, bone mineral content and bone union than those treated with vehicle or IFG-HyA/Hap alone. CONCLUSIONS: IFGs-HyA/Hap/BMP-2 could be an effective treatment option for refractory fractures.

Effect of spheroid size on gene expression profiles of a mouse mesenchymal stem cell line in spheroid culture.

BACKGROUND: Mesenchymal stem cell (MSC)-based therapies offer potential for bone repair. MSC spheroid cultures may harbor enhanced therapeutic potential over MSC monolayers through increased secretion of trophic factors. However, the impact of spheroid size on trophic factor expression is unclear. OBJECTIVE: We investigated the effect of spheroid size on trophic factor-related gene expression. METHODS: KUM10, a murine MSC line was used. RNA-seq was used to screen the transcriptional profiles of MSC monolayer and spheroid cultures. Differentially expressed genes identified in RNA-seq were evaluated by q-PCR in cultures of 5 × 104 (S group), 5 × 105 (M group), 5 × 106 (L group) cells/well. RESULTS: Comparison of expression levels between KUM10 monolayer and spheroid cultures identified 2140 differentially expressed genes, of which 1047 were upregulated and 1093 were downregulated in KUM10 spheroids. Among these, 12 upregulated genes (Bmp2, Fgf9, Fgf18, Ngf, Pdgfa, Pdgfb, Tgfb1, Vegfa, Vegfc, Wnt4, Wnt5a, Wnt10a) were associated with secretory growth factors. Of these, expression of Fgf9, Fgf18, Vegfa and Vegfc was elevated in the L group, and Pdgfb and Tgfb1 was elevated in the S group. CONCLUSIONS: Spheroid size may impact trophic factor expression. Our results will be useful for future studies assessing the utility of MSC spheroids for treating bone injury.

Effect of Bone Morphogenetic Protein-2 (BMP-2)/Hydroxyapatite/In Situ-Formed Hyaluronan Hydrogel Composites on Bone Formation in a Murine Model of Posterolateral Lumbar Fusion.

Several types of calcium phosphate (CaP) biomaterial carriers have been designed to deliver bone morphogenetic protein-2 (BMP-2) to augment spinal fusion in spinal surgery. Here, we evaluated an in situ-formed hydrogel (IFH) constructed from hyaluronan (IFH-HA) combined with a BMP2/hydroxyapatite (HAP) composite in bone formation in a murine model of posterolateral lumbar fusion (PLF). HAP was submerged in HA-tyramine (TA) polymer solution containing horseradish peroxidase (HRP) and 2 µg BMP-2 (BMP2/HA-TA/HRP solution). H2O2 was added to initiate the curing reaction (BMP-2/IFH-HA). phosphate-buffered saline (PBS) was added to the BMP2/HA-TA/HRP solution (BMP-2/HA-TA) instead of H2O2 to evaluate the effectiveness of the curing reaction. HAP immersed in PBS was used as a control. PLF model mice were randomly assigned to receive one these composites (n = 10 each). X-ray images were taken to assess the bone fusion, and microcomputed tomography analysis was conducted to examine new bone formation at the graft site four weeks following surgery. No evidence of fusion was observed four weeks after surgery in the Control or BMP2/HA-TA group. In contrast, the BMP2/IFH-HA group exhibited newly formed bone between the transverse processes and bone union in coronal sections. Relative to the Control and BMP2/HA-TA groups, the BMP2/IFH-HA group showed significantly greater bone volume. The BMP2/IFH-HA group also showed significantly elevated bone mineral content relative to the BMP2/HA-TA group. A composite comprising BMP2/HAP and IFH-HA, thus, enhanced the new bone formation in a murine model of PLF, suggesting its promise for augmenting spinal fusion.

Expression of nerve growth factor in the callus during fracture healing in a fracture model in aged mice.

BACKGROUND: Impaired fracture healing results in extensive and prolonged disability and long-term pain. Previous studies reported that nerve growth factor (NGF) was expressed during fracture healing and that anti-NGF antibody improves physical activity associate with facture pain. However, NGF expression levels in delayed or non-union are not fully understood. OBJECTIVE: We compared chronological changes in NGF in the callus of young mice after femur fracture with those in aged mice after femur fracture as a model of bone fracture in the elderly. METHODS: We used young (age 8 weeks) and aged (age 10 months) male C57BL/6J mice. A fracture was generated in the femur. At 5, 7, 10, 14, 17, and 21 days after creation of a fracture, mRNA expression levels of Col2a1, Col10a1, NGF were evaluated using quantitative PCR. We examined NGF protein expression levels and localization in the callus at day 14 using ELISA and immunohistochemistry, respectively. RESULTS: Expression of NGF in the callus after femur fracture in aged mice was significantly greater than that in young mice at days 5, 7, 10, 17, and 21 days. NGF protein levels in the callus of aged mice were also significantly higher than that in young mice. Immunohistochemical staining showed that NGF was heavily expressed in hypertrophic chondrocytes in the callus in aged mice. CONCLUSIONS: It is suggested that delayed Col2a1 and Col10a1 expression reflects delayed chondrocyte formation and delayed chondrocyte maturation in aged mice and that higher NGF expression in aged mice at day 14 may be associated with the presence of remaining hypertrophic chondrocytes in callus with delaying endochondral ossification.

Increase in CD5L expression in the synovial membrane of knee osteoarthritis patients with obesity.

INTRODUCTION: Obesity appears to be a powerful risk factor for the development of knee osteoarthritis (KOA), but the mechanisms of this are not fully understood. CD5L is expressed in tissue macrophages and is increased in obese mice. We hypothesized that CD5L expression is increased in the synovial membrane (SM) of obese KOA patients. Here, we investigated CD5L expression in the SM of these patients. MATERIAL AND METHODS: Ninety KOA patients (26 males, 64 females) were allocated to one of three groups based on body mass index (BMI): normal weight (NW, < 25 kg/m2), overweight (OW, 25-29.99 kg/m2) and obese (OB, ≥ 30 kg/m2), according to the World Health Organization BMI classification (each n = 30). Expression of CD5L in SM among the groups was compared using real-time polymerase chain reaction. To investigate CD5L-expressing cells in SM, CD14+ (macrophage fraction) and CD14- (fibroblast fraction) cells were separated from the SM. RESULTS: CD5L expression was significantly higher in the OB group than in the NW and OW groups (p < 0.001). CD5L expression was observed in the CD14+ fraction but not in the CD14- fraction. CONCLUSIONS: CD5L is highly expressed in the SM of KOA patients with obesity. Further investigation is required to identify the role of CD5L in the relationship between KOA pathology and obesity.

Acceleration of bone formation using in situ-formed hyaluronan-hydrogel containing bone morphogenetic protein-2 in a mouse critical size bone defect model.

BACKGROUND: An enzymatic crosslinking strategy using hydrogen peroxide and horseradish peroxidase is receiving increasing attention for application with in situ-formed hydrogels (IFHGs). IFHGs may also be ideal carrier materials for bone repair, although their ability to carry bone morphogenetic protein-2 (BMP2) has yet to be examined. OBJECTIVE: We examined the effectiveness of an IFHG made of hyaluronan (IFHG-HA) containing BMP2 for promoting bone formation in a mouse critical size bone defect model. METHODS: C57/BL6J mice received a 2-mm femoral critical-sized bone defect before being randomly assigned to one of the following treatment groups (n = 6): control (no treatment), IFHG-HA only, PBS with BMP2, and IFHG-HA with BMP2. X-ray radiographs were utilized to track new bone formation, and micro-computed tomography and histological examination were performed on new bone formed at the bone defect site two weeks after surgery. RESULTS: Mice treated with PBS with BMP2 and IFHG-HA with BMP2 had greater bone volume (BV) and bone mineral content (BMC) than those receiving control, and successfully achieved consolidation. Mice treated with IFHG-HA with BMP2 had significantly higher BV and BMC than those treated with PBS with BMP2. CONCLUSIONS: IFHG-HA may be an effective carrier for BMP2 to enable delivery for bone defect repair.

Acceleration of Bone Healing by In Situ-Forming Dextran-Tyramine Conjugates Containing Basic Fibroblast Growth Factor in Mice.

An enzymatic crosslinking strategy using hydrogen peroxide (H2O2) 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.

Acceleration of bone union by in situ-formed hydrogel containing bone morphogenetic protein-2 in a mouse refractory fracture model.

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.

Vascular Endothelial Growth Factor Is Regulated by the Canonical and Noncanonical Transforming Growth Factor-ß Pathway in Synovial Fibroblasts Derived from Osteoarthritis Patients.

BACKGROUND: Previous studies suggest the presence of an association of vascular endothelial growth factor (VEGF) with osteoarthritis (OA) severity and pain in patients with knee OA. VEGF expression in human synovial fibroblasts (SFs) is induced by transforming growth factor-beta (TGFß). However, the signaling pathway governing TGFß-mediated regulation of VEGF in SFs has not been identified. METHODS: OA patients who underwent total knee arthroplasty had their synovial tissue (SYT) extracted and the constituent SFs cultured. The cells were stimulated with culture medium (control), human recombinant TGFß (hrTGFß), hrTGFß + ALK5 inhibitor SB505124, hrTGFß + transforming growth factor activating kinase 1 (TAK1) inhibitor (5Z)-7-oxozeaenol, or hrTGFß + p38 inhibitor SB203580 for 6 h. VEGF mRNA expression in SFs was examined using real-time polymerase chain reaction and VEGF protein production in the cell supernatant was examined using enzyme-linked immunosorbent assay. Additionally, phosphorylated levels of SMAD2 and p38 were examined using western blotting. RESULTS: ALK5 (SB505124) and TAK1 (5Z-oxozeaenol) inhibitors completely suppressed TGFß-induced VEGF mRNA expression and VEGF protein production. Both SB505124 and 5Z-oxozeaenol also suppressed SMAD2 and p38 phosphorylation. The p38 inhibitor (SB203580) partially inhibited TGFß-mediated VEGF mRNA and VEGF protein production. CONCLUSION: TGFß-mediated regulation of VEGF expression and VEGF protein production in the SYT of OA patients occurs through both the canonical and noncanonical pathway.

Osteoarthritis patients with high haemoglobin A1c have increased Toll-like receptor 4 and matrix metalloprotease-13 expression in the synovium.

PURPOSE: While research has identified diabetes mellitus (DM) as a risk factor for knee osteoarthritis (KOA), the underlying mechanisms are not fully understood. Studies suggest that Toll-like receptor 4 (TLR4) expression is elevated in osteoarthritic lesions of OA patients and in target tissues of insulin resistance such as adipose tissue and skeletal muscle in patients with DM. TLR4 is associated with inflammation and catabolic response via regulation of matrix metalloproteases (MMPs). We hypothesized that TLR4 and MMP expression may be increased in the synovial tissue (SYN) of KOA patients with diabetic pathology. We therefore investigated TLR and MMP expression in the SYN of KOA patients with and without high haemoglobin A1c concentrations. PATIENTS AND METHODS: A total of 171 patients radiographically diagnosed with KOA were grouped based on their HbA1c concentration (HbA1c ≥6.5 and HbA1c <6.5). We used real-time polymerase chain reaction to compare the expression of TLRs (TLR2, TLR4) and MMPs (MMP2, MMP3, MMP9 and MMP13) in patients' SYN between the two groups. MMP13 regulation by the TLR4 ligand, lipopolysaccharide (LPS), in SYN cells was examined in culture by stimulating SYN cells with LPS or vehicle (culture medium) for 24 h. RESULTS: The expression of TLR4 and MMP13 in the HbA1c ≥6.5 group was significantly elevated compared to that in the HbA1c <6.5 group. In contrast, TLR2, MMP2, MMP3 and MMP9 expression levels were similar between the groups. MMP13 mRNA and MMP13 protein levels in SYN cells were significantly higher following stimulation with LPS compared to vehicle. CONCLUSIONS: TLR4 and MMP13 expression were elevated in the synovium of osteoarthritis patients with high HbA1c concentrations. Our results may provide insight into the pathology of OA patients with DM.

Regulation of calcitonin gene-related peptide expression through the COX-2/mPGES-1/PGE2 pathway in the infrapatellar fat pad in knee osteoarthritis.

BACKGROUND: The 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. METHODS: IFPs 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. RESULTS: CGRP, 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. CONCLUSIONS: CGRP expression in the IFP of KOA patients is regulated by the COX-2/mPGES-1/PGE2 pathway.

Basic Fibroblast Growth Factor Fused with Tandem Collagen-Binding Domains from Clostridium histolyticum Collagenase ColG Increases Bone Formation.

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.

Spinal Epidural Hematoma after Thoracolumbar Posterior Fusion Surgery without Decompression for Thoracic Vertebral Fracture.

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.