Noriyuki Tamai

Oxygen tension regulates chondrocyte differentiation and function during endochondral ossification.

Cartilage functions at a lower oxygen tension than most other tissues. To determine the role of oxygen tension in chondrocyte differentiation and function, we investigated the influence of oxygen tension in the pluripotent mesenchymal cell line C3H10T1/2 and 14.5E mice embryo forelimb organ culture. 10T1/2 cells and embryo forelimbs were cultured under normoxia (20% O2) or hypoxia (5% O2) in the presence of recombinant human bone morphogenetic protein 2. To elucidate the mechanism by which oxygen tension influences chondrocyte differentiation, the Smad pathway was examined using Smad6 overexpression adenovirus and Smad6 transgenic mice embryo forelimbs. The p38 MAPK pathway was examined using dominant-negative MKK3 and FR167653, a specific p38 MAPK inhibitor. The transcriptional activities of Sox9 and Runx2 were also investigated. Hypoxia promoted bone morphogenetic protein 2-induced glycosaminoglycan production and suppressed alkaline phosphatase activity and mineralization of C3H10T1/2. Thus, hypoxia promoted chondrocytic commitment rather than osteoblastic differentiation. In the mice embryo forelimb organ culture, hypoxia increased cartilaginous matrix synthesis. These effects were primarily mediated by p38 MAPK activation, independent of Sox9. Hypoxia inhibited Col10a1 (type X collagen α1) expression via down-regulation of Runx2 activity by Smad suppression and histone deacetylase 4 activation. In conclusion, hypoxia promotes chondrocytic differentiation and cartilage matrix synthesis and suppresses terminal chondrocyte differentiation. These hypoxia-induced phenomena may act on chondrocytes to enhance and preserve their phenotype and function during chondrocyte differentiation and endochondral ossification.

Interconnected porous hydroxyapatite ceramics for bone tissue engineering

Several porous calcium hydroxyapatite (HA) ceramics have been used clinically as bone substitutes, but most of them possessed few interpore connections, resulting in pathological fracture probably due to poor bone formation within the substitute. We recently developed a fully interconnected porous HA ceramic (IP-CHA) by adopting the ‘foam-gel’ technique. The IP-CHA had a three-dimensional structure with spherical pores of uniform size (average 150 μm, porosity 75%), which were interconnected by window-like holes (average diameter 40 μm), and also demonstrated adequate compression strength (10–12 MPa). In animal experiments, the IP-CHA showed superior osteoconduction, with the majority of pores filled with newly formed bone. The interconnected porous structure facilitates bone tissue engineering by allowing the introduction of mesenchymal cells, osteotropic agents such as bone morphogenetic protein or vasculature into the pores. Clinically, we have applied the IP-CHA to treat various bony defects in orthopaedic surgery, and radiographic examinations demonstrated that grafted IP-CHA gained radiopacity more quickly than the synthetic HA in clinical use previously. We review the accumulated data on bone tissue engineering using the novel scaffold and on clinical application in the orthopaedic field.

Bone tissue engineering using novel interconnected porous hydroxyapatite ceramics combined with marrow mesenchymal cells : quantitative and three-dimensional image analysis

We developed fully opened interconnected porous calcium hydroxyapatite ceramics having two different pore sizes. One has pores with an average size of 150 μm in diameter, an average 40-μm interconnecting pore diameter, and 75% porosity (HA150). The other has pores with an average size of 300 μm in diameter, an average 60–100-μm interconnecting pore diameter, and 75% porosity (HA300). Because of its smaller pore diameter, HA150 has greater mechanical strength than that of HA300. These ceramics were combined with rat marrow mesenchymal cells and cultured for 2 weeks in the presence of dexamethasone. The cultured ceramics were then implanted into subcutaneous sites in syngeneic rats and harvested 2–8 weeks after implantation. All the implants showed bone formation inside the pore areas as evidenced by decalcified histological sections and microcomputed tomography images, which enabled three-dimensional analysis of the newly formed bone and calculation of the bone volume in the implants. The bone volume increased over time. At 8 weeks after implantation, extensive bone volume was detected not only in the surface pore areas but also in the center pore areas of the implants. A high degree of alkaline phosphatase activity with a peak at 2 weeks and a high level of osteocalcin with a gradual increase over time were detected in the implants. The levels of these biochemical parameters were higher in HA150 than in HA300. The results indicate that a combination of HA150 and mesenchymal cells could be used as an excellent bone graft substitute because of its mechanical properties and capability of inducing bone formation.

Capillary Vessel Network Integration by Inserting a Vascular Pedicle Enhances Bone Formation in Tissue-Engineered Bone Using Interconnected Porous Hydroxyapatite Ceramics

The aim of the present study was to investigate the possibility of integrating porous hydroxyapatite (HA) ceramics with a capillary vessel network via insertion of a vascular pedicle, and to determine whether this procedure enhances new bone formation in tissue engineering of bone. First, synthetic interconnected porous HA (IP-CHA) was implanted subcutaneously into rat groin with or without insertion of superficial inferior epigastric vessels. At 6 weeks, IP-CHA with vascular insertion contained thick fibrous connective tissue with a number of large blood vessels that seemed to derive from the inserted vascular bundle. Next, IP-CHA loaded with recombinant human bone morphogenetic protein 2 (BMP, 2 or 10 microg/block) was implanted with or without vascular insertion. At 3 weeks, IP-CHA/BMP (10 microg) composite with vascular insertion exhibited abundant new bone formation in the pores of the deep portion close to the inserted vessels. In contrast, IP-CHA/BMP (10 microg) without vascular insertion showed poor bone formation. Histomorphometric analysis demonstrated that vascular insertion significantly increased new bone formation. In IP-CHAs with a lower dose of BMP (2 microg), no bone formation was found, with or without vascular insertion. These results suggest that the present system of integrating a vascular network with IP-CHA is a useful technique for bone tissue engineering.

A comparative assessment of synthetic ceramic bone substitutes with different composition and microstructure in rabbit femoral condyle model

Various bone substitutes with improved biocompatibility have been developed. Because these products vary in composition and microstructure, it is difficult to understand each feature and make an appropriate selection. Three recently developed highly porous ceramic bone substitutes were evaluated, including two made of hydroxyapatite with different structures (Apaceram-AX: 85%-porosity with micropores, NEOBONE: 75%-porosity without micropores) and one composed of beta-tricalcium phosphate (OSferion: 75%-porosity with micropores) in a rabbit model. Apaceram-AX showed gradual degradation, while NEOBONE remaining intact. OSferion was almost completely degraded at 24 weeks. Numerous osteoclasts were detected in materials with micropores, whether Apaceram-AX or OSferion, but not in NEOBONE. These differences of biodegradability seemed to be related to the presence of micropores. The compressive strength of OSferion increased for several weeks and decreased at a level of cancellous bone. The strength of NEOBONE gradually increased and remained at the highest level among three. The strength of Apaceram-AX increased two to three times that of cancellous bone. Surprisingly, the strength of all materials declined during the initial 1 week, suggesting that great care should be taken in the early period after implantation. These findings may help surgeons to select an appropriate porous substitute based on understanding of their features.

Unusual skeletal metastases from myxoid liposarcoma only detectable by MR imaging.

We present two cases of skeletal metastases from myxoid liposarcoma, occurring several years after treatment of the primary tumors in the lower limb. The present two case reports have unusual radiological features only detectable by MR imaging and not by plain radiographs or bone scans. From the present two cases, we found that a negative plain radiograph of the spine or a negative bone scan could not exclude skeletal metastases from myxoid liposarcoma, and MRI was a more sensitive screening procedure for their detection, especially in T1-weighted images. Unusual radiological features of skeletal metastases from myxoid liposarcoma are not well documented and only a few cases have been previously reported. Our aim is to document two more patients exhibiting the unusual radiological features of skeletal metastases from myxoid liposarcoma to improve their early detection and management.

Novel fully interconnected porous hydroxyapatite ceramic in surgical treatment of benign bone tumor

BackgroundLarge bone defects remaining after curettage of benign bone tumors should be filled with a substitute to restore mechanical strength. In 2000, we developed a fully interconnected porous calcium hydroxyapatite ceramic (IPCHA, NEOBONE) and have utilized it as a bone substitute. IP-CHA has a finely organized, three-dimensional interconnecting pore structure. The large interconnecting channels (average diameter 40 μm) permit easy penetration of tissue into the deep pores, so IP-CHA can itself induce local bone repair processes. The purpose of this study was to evaluate the clinical outcomes with the use of IP-CHA as bone substitute after curettage of benign bone tumors.MethodsWe reviewed the results of 71 patients with benign bone tumors sequentially treated by curettage followed by implantation of IP-CHA between 2000 and 2006. There were 29 women and 42 men, with a mean age of 28 years. Assessment was based on radiography at each time point during the follow-up. Radiographic findings were classified into five stages: stage 0, no change; stage 1, slight bone formation; stage 2, moderate bone formation; stage 3, consolidation; stage 4, absorption.ResultsIn 70 of 74 operated lesions, radiographs showed that implanted IP-CHA proceeded to stage 2 or more within an average of 8 months after the surgery. In addition, 17 lesions proceeded to stage 4 within 35 months after surgery, on average. However, there were 10 local recurrences, which is similar to the recurrence rate for such tumors treated with or without implantation of CHAs and reflects the biological nature of each tumor.ConclusionsIn this study, we utilized IP-CHA as a bone substitute after curettage of benign bone tumors and demonstrated its usefulness in the clinical situation. IP-CHA comparatively exhibited excellent bone formation at an early stage although the problem of recurrence of the tumor remained. We conclude that IP-CHA is a useful bone substitute for the treatment of benign bone tumors.

The effect of simulated microgravity by three-dimensional clinostat on bone tissue engineering.

Evidence suggests that mechanical stress, including gravity, is associated with osteoblast differentiation and function. To examine effects of microgravity on bone tissue engineering, we used a three-dimensional (3D) clinostat manufactured by Mitsubishi Heavy Industries (Kobe, Japan). A 3D clinostat is a device that generates multidirectional G force. By controlled rotation on two axes, it cancels the cumulative gravity vector at the center of the device. We cultured rat marrow mesenchymal cells (MMCs) in the pores of interconnected porous calcium hydroxyapatite (IP-CHA) for 2 weeks in the presence of dexamethasone using the 3D clinostat (clinostat group). MMCs cultured using the 3D clinostat exhibited a 40% decrease in alkaline phosphatase activity (a marker of osteoblastic differentiation), compared with control static cultures (control group). SEM analysis revealed that although there was no difference between the two groups in number or distribution of cells in the pores, the clinostat group exhibited less extensive extracellular matrix formation than the control group. Cultured IP-CHA/MMC composites were then implanted into subcutaneous sites of syngeneic rats and harvested 8 weeks after implantation. All implants showed bone formation inside the pores, as indicated by decalcified histological sections and microfocus computed tomography. However, the volume of newly formed bone was significantly lower for the clinostat group than for the control group, especially in the superficial pores close to the implant surface. These results indicate that new bone formation in culture was inhibited by use of the 3D clinostat, and that this inhibition was mainly due to suppression of osteoblastic differentiation of MMCs.

Immunohistochemical detection of parathyroid hormone-related peptide, Indian hedgehog, and patched in the process of endochondral ossification in the human

Abstract. Parathyroid hormone-related peptide (PTHrP), Indian hedgehog (Ihh), and patched (Ptc; a receptor for Ihh) were immunolocalized in tissue undergoing endochondral ossification in the human. PTHrP, Ihh, and Ptc were immunolocalized in prehypertrophic and hypertrophic chondrocytes in mature cartilage matrix. PTHrP and Ptc were immunostained in proliferating chondrocytes and perichondrial cells, whereas Ihh was not. PTHrP, Ihh, and Ptc showed positive immunostaining in osteoblasts in the bone-forming area. In the bone resorption site, PTHrP was immunolocalized in osteoclasts, whereas Ihh and Ptc were not. The present findings indicated that PTHrP, Ihh, and Ptc were associated with the process of endochondral ossification, and suggested the possible involvement of Ihh and PTHrP signaling in the regulation of proliferation and hypertrophy of chondrocytes in human chondrogenesis.

Nkx3.2-induced suppression of Runx2 is a crucial mediator of hypoxia-dependent maintenance of chondrocyte phenotypes

Hypoxia is a key factor in the maintenance of chondrocyte identity. However, crucial chondrogenic transcription factors in the Sox families are not activated in this phenomenon, indicating that other pathways are involved. Nkx3.2 is a well-known chondrogenic transcription factor induced by Sonic hedgehog (Shh); it suppresses a key osteogenic transcriptional factor, Runt-related transcription factor 2 (Runx2), to maintain the chondrogenic phenotype in mesenchymal lineages. The purpose of this study was to examine the function of Nkx3.2 in hypoxia-dependent maintenance of chondrocyte identity. C3H10T1/2 pluripotent mesenchymal cells were cultured with rh-BMP2 (300 ng/ml) to induce chondrogenesis under normoxic (20% O(2)) or hypoxic (5% O(2)) conditions. Immunohistological detection of Nkx3.2 in a micromass cell culture system revealed that hypoxia promoted expression of the Nkx3.2 protein. Real-time RT-PCR analysis revealed that hypoxia promoted Nkx3.2 mRNA expression and suppressed Runx2 mRNA expression; however, Sox9 mRNA expression was not altered by oxygen conditions, as previously described. Over-expression of exogenous Nkx3.2 promoted glycosaminoglycan (GAG) production and inhibited Runx2 mRNA expression and, based on a dual luciferase assay, Runx2 promoter activity. Interestingly, downregulation of Nkx3.2 using RNAi abolished hypoxia-dependent GAG production and restored Runx2 mRNA expression and promoter activity. These results demonstrated that Nkx3.2-dependent suppression of Runx2 was a crucial factor in hypoxia-dependent maintenance of chondrocyte identity.