Hiroko Kataoka

Fibroblasts expressing Sonic hedgehog induce osteoblast differentiation and ectopic bone formation.

© 1997 Federation of European Biochemical Societies.

Sonic hedgehog is involved in osteoblast differentiation by cooperating with BMP‐2

The roles of Sonic hedgehog (Shh) and Bone morphogenetic protein‐2 (Bmp‐2) in osteoblast differentiation were investigated using in vitro cell systems. Recombinant amino‐terminal portion of SHH (rSHH‐N) dose dependently stimulated ALP activity in C3H10T1/2 and MC3T3‐E1 cells. rSHH‐N induced expression of Osteocalcin mRNA in C3H10T1/2 cells. A soluble form of the receptor for type IA BMP receptor antagonized rSHH‐N‐induced ALP activity in C3H10T1/2 and MC3T3‐E1 cells, indicating that BMPs are involved in SHH‐induced osteoblast differentiation. Simultaneous supplement with rSHH‐N and BMP‐2 synergistically induced ALP activity and expression of Osteocalcin mRNA in C3H10T1/2 cells. Pretreatment with rSHH‐N for 6 h enhanced the response to BMP‐2 by increasing ALP activity in C3H10T1/2 and MC3T3‐E1 cells. Stimulatory effects of rSHH‐N and additive effects with rSHH‐N and BMP‐2 on ALP activity were also observed in mouse primary osteoblastic cells. Transplantation of BMP‐2 (1 μg) into muscle of mice induced formation of ectopic bone, whereas transplantation of r‐SHH‐N (1–5 μg) failed to generate it. These results indicate that Shh plays important roles in osteoblast differentiation by cooperating with BMP.

Changes in Osteoblast Phenotype During Differentiation of Enzymatically Isolated Rat Calvaria Cells

Osteoblasts enzymatically isolated from newborn rat calvariae show various phenotypes including formation of mineralized bone nodules in culture. We investigated the temporal changes in osteoblast phenotype in these cells up to day 20 in culture. These cells formed unmineralized nodules by day 5. Mineralization was observed at the center of nodules by day 10, and nodules became larger on day 15. The nodules were surrounded by numerous alkaline phosphatase (ALP)-positive cells. ALP activity gradually increased by day 20. Parathyroid hormone (PTH) responsiveness increased with time in culture. Osteoblasts produced no osteocalcin by day 10, but its synthesis was detected from day 15. These cells expressed substantial levels of ALP and PTH/PTHrP receptor mRNAs as early as day 5 in culture, but very weak expression of osteocalcin mRNA on day 5. The levels of expression of these transcripts increased with time in culture. In situ hybridization demonstrated that PTH/PTHrP receptor and osteocalcin mRNAs were strongly expressed in nodules, but the former appeared much earlier than the latter. BMP-2 and BMP-4 mRNAs also appeared in the cells forming nodules. Immunohistochemical analysis demonstrated that cells expressing either BMP-2/4 or their receptors (BMPR-IA, BMPR-IB, and BMPR-II) preferentially appeared in nodules. These observations suggested that BMPs play an important role in the formation of mineralized bone nodules in an autocrine and/or paracrine fashion in these cells. The present study confirmed that osteoblasts enzymatically isolated from newborn rat calvariae are a useful tool for studying the differentiation process of osteoblasts.

Molecular cloning and heterologous expression of an α-adrenergic-like octopamine receptor from the silkworm Bombyx mori

A cDNA encoding an octopamine (OA) receptor (BmOAR1) was isolated from the nerve tissue of silkworm (Bombyx mori) larvae. Comparison of amino acid sequences showed that BmOAR1 is highly identical to OA receptors isolated from Periplaneta americana (Pa oa1), Apis mellifera (AmOA1), and Drosophila melanogaster (OAMB or DmOA1A). BmOAR1 was stably expressed in HEK‐293 cells. OA above 1 µm led to an increase in intracellular cyclic AMP concentration ([cAMP]i). The synthetic OA‐receptor agonist demethylchlordimeform also elevated [cAMP]i to the same maximal level (≈ 5‐fold over the basal level) as that induced by OA. However, other biogenic amines, tyramine and dopamine, and chlordimeform were without effects. The [cAMP]i level raised by OA was lowered by antagonists; the rank order of antagonist activity was chlorpromazine > mianserin = yohimbine. Cyproheptadine and metoclopramide had little effect. OA above 100 nm induced a transient or sustained increase in intracellular Ca2+ concentration ([Ca2+]i), depending on the concentration of OA. Sequence homology and functional analysis data indicate that BmOAR1 is an α‐adrenergic‐like OA receptor of B. mori.

Cloning and embryonic expression patterns of the zebrafish Runt domain genes, runxa and runxb.

We isolated zebrafish homologues of the Runt-related transcription factor gene family (Runx family), runxa and runxb, and analyzed their developmental expression patterns. The deduced amino acid sequence of Runxa was highly homologous to that of AML1 (also called CBFA2, PEBP2alphaB or Runx1), a critical regulator of mammalian hematopoiesis expressed in cells of the hematopoietic lineage as well as other tissues. During zebrafish development, the runxa gene was not expressed in hematopoietic tissues but in the olfactory placodes and cells attached to the otic vesicles. We identified three kinds of runxb transcripts, which encoded two types of proteins with different N-terminal regions. The Runxb proteins were highly similar to AML2 (CBFA3, PEBP2alphaC or Runx3). The expression sites of the shared region of runxb mRNAs during development were the trigeminal ganglions, dorsal neurons of the neural tube and the lateral line primordia. These findings show that expression patterns of the zebrafish Runx genes are distinct from that of the mammalian genes.

Optimum combination of monolayer and three-dimensional cultures for cartilage-like tissue engineering.

The autologous chondrocyte transplantation technique has been introduced for the repair of articular cartilage defects. The advantage of transplanting chondrocytes cultured in suspension includes the in vitro expansion of cell numbers. However, the disadvantages include the potential leakage of cells from defects, dedifferentiation of cellular phenotype, and uneven distribution of cells. Transplantation of chondrocytes cultured in collagen gel resolves those problems. However, the expansion of cells in three-dimensional culture is more difficult than in monolayer culture, and for practical reasons only limited numbers of chondrocytes can be obtained from an unloaded area of the knee. To develop a method for the production of high-quality cultured grafts, we investigated the combination of monolayer culture for cell expansion and three-dimensional culture for maintenance of cell phenotype. Articular chondrocytes from rabbits were divided into four groups, exposed to various combinations of culture conditions, and cultured for a total of 3 weeks. Each group was evaluated histologically, biochemically, and biomechanically. Our findings showed that the combination of 2 weeks of monolayer culture followed by 1 week of three-dimensional culture resulted in the highest chondroitin sulfate levels, sufficient cell numbers, and adequate stiffness of the chondrocyte-collagen composites, giving optimal graft preparation.

Role of the periosteal flap in chondrocyte transplantation: an experimental study in rabbits.

To determine the role of the periosteal flap in chondrocyte transplantation for the treatment of articular cartilage defects, a cartilage defect was created on the patellar groove of the rabbit knee. The defect was filled with chondrocytes cultured in collagen gel, and was covered with a periosteal flap the cambial layer of which was facing the patella (P group), or facing down against the bone marrow (M group). The same defect was covered with a periosteal flap that was frozen and thawed three times (F group), and an artificial collagen film (C group). At 3 and 6 months, the defects were filled with reparative tissues that showed a smooth surface and resembled hyaline cartilage in the P, M, and F groups. There were no significant differences between the reparative tissues in the three groups histologically, immunohistochemically, biochemically, and biomechanically, although the collagen film fell down into the defect and the reparative tissue had a fibrous tissue-like appearance. These results showed that the periosteal flap does not have a beneficial humoral or cellular effect on the formation of reparative tissue, suggesting that the periosteal flap might act as a mechanical barrier to prevent leakage of grafted chondrocytes.

AG-041r, a cholecystokinin-B/gastrin receptor antagonist, stimulates the repair of osteochondral defect in rabbit model

A newly synthesized compound (AG-041R), 3R-1-(2,2Diethoxyethyl)-((4methylphenyl) amino-carbonyl methyl)-3-((4methylphenyl) ureido-indoline-2-one), is a cholecystokinin-B/gastrin receptor antagonist which has stimulatory effects on the matrix synthesis of chondrocytes in vitro. In this study, we examined the effect of AG-041R on the repair of osteochondral defects (cylindrical, 4 mm diameter) in the patellar groove of the rabbit knee joint. At the time of operation, 100 microl of 1 microM of AG-041R was administered, followed by 200 microl with an osmotic pump for 14 days. Histological and biochemical evaluations were performed at 12 and 24 weeks after surgery. The histological score of the AG-041R-treated group, the quantity of glycosaminoglycan and the ratio of chondroitin sulfate in the AG-041R-treated tissue were significantly higher than in the untreated group. Moreover, the degeneration of cartilage around the defect was suppressed in the AG-041R-treated group. These findings suggest that AG-041R is effective for the repair of osteochondral defects.

Thioredoxin Gene Expression in Rat Knee Articular Cartilage After Full-Thickness Injury

To study the expression of the antioxidative protein thioredoxin (TRX) in intact and injured articular cartilage, we examined the presence of trxmRNA in rat knee joints by in situ hybridization. Our results showed that in the intact knee, most cells, including articular cartilage chondrocytes, expressed trx mRNA. We examined joints at 1, 7, 14, and 28 days after the infliction of full-thickness cartilage injuries on distal femoral condyles. At 1 day after injury, no significant changes were observed in the wound or in trx expression pattern. However, at 7 to 28 days after injury, the wound became filled with repair tissue. Also, trx expression was detected in differentiating mesenchymal cells in the deeper zones of the wound but not in fibroblast-like cells in the upper part of the repair tissue, toward the joint cavity. This lack of TRX expression in the fibroblast-like cells may underlie the susceptibility of the repair tissue fibrocartilage to oxidative stress.

Effects of different embedding gels on periostealchondrogenesis in vitro.

To develop a more useful organ culture model for periosteal chondrogenesis in vitro, we compared the effects of the embedding of explants in agarose versus collagen gels. Chondrogenic differentiation was examined by means of histological observation and in terms of the expression of mRNA encoding two cartilage markers, Type II collagen and Aggrecan. Periosteal explants were derived from the tibiae of rabbits. These explants were embedded in either agarose gel or collagen gel and cultured for 6 weeks. Histological examinations revealed that in the agarose gel, cells were neither present in the explants nor in the gel. In the collagen gel, cells migrated from the explanted tissues into the gel, and some cells were round. However, no explants showed safranin‐O staining. Only 10% of 10 surviving explants in the agarose gel expressed the Type II collagen gene and the Aggrecan gene. On the other hand, of 25 surviving explants in the collagen gel, the expression of the Type II collagen gene was detected in 18 explants (72%) and that of the Aggrecan gene was detected in 21 explants (84%). In conclusion, we demonstrated that the periosteum exerts chondrogenesis in certain circumstances and its chondrogenesis is closely related to the culture material.