Hiromi Nakajima

Involvement of GATA transcription factors in the regulation of endogenous bovine interferon‐Tau gene transcription

Expression of interferon‐tau (IFNT), necessary for pregnancy establishment in ruminant ungulates, is regulated in a temporal and spatial manner. However, molecular mechanisms by which IFNT gene transcription is regulated in this manner have not been firmly established. In this study, DNA microarray/RT‐PCR analysis between bovine trophoblast CT‐1 and Mardin–Darby bovine kidney (MDBK) cells was initially performed, finding that transcription factors GATA2, GATA3, and GATA6 mRNAs were specific to CT‐1 cells. These mRNAs were also found in Days 17, 20, and 22 (Day 0 = day of estrus) bovine conceptuses. In examining other bovine cell lines, ovary cumulus granulosa (oCG) and ear fibroblast (EF) cells, GATA2 and GATA3, but not GATA6, were found specific to the bovine trophoblast cells. In transient transfection analyses using the upstream region (−631 to +59 bp) of bovine IFNT gene (bIFNT, IFN‐tau‐c1), over‐expression of GATA2/GATA3 did not affect the transcription of bIFNT‐reporter construct in human choriocarcinoma JEG3 cells. Transfection of GATA2, GATA3, ETS2, and/or CDX2, however, was effective in the up‐regulation of the bIFNT construct transfected into bovine oCG and EF cells. One Point mutation studies revealed that among six potential GATA binding sites located on the upstream region of the bIFNT gene, the one next to ETS2 site exhibited reduced luciferase activity. In CT‐1 cells, endogenous bIFNT gene transcription was up‐regulated by over‐expression of GATA2 or GATA3, but down‐regulated by siRNA specific to GATA2 mRNA. These data suggest that GATA2/3 is involved in trophoblast‐specific regulation of bIFNT gene transcription. Mol. Reprod. Dev. 76: 1143–1152, 2009.

Calcium phosphate-hybridised tendon graft to reduce bone-tunnel enlargement after ACL reconstruction in goats

Bone-tunnel enlargement can have a negative impact on long-term clinical success. To solve the problem, we developed a novel technique to improve tendon-bone healing by hybridising calcium phosphate (CaP) with a tendon graft using an alternate soaking process. The objective of this study was to analyse bone-tunnel enlargement, mechanical properties and histological features, especially the number of osteoclasts at the tendon-bone interface using a CaP-hybridised tendon graft and an untreated tendon graft 6 months after anterior cruciate ligament (ACL) reconstruction in goats. The percentage of bone-tunnel enlargement for the CaP group was decreased compared with that for the control group for the femoral side (p<0.05). The failure load was not statistically different between the CaP group and the control group, and was all midsubstance rupture for both groups. In the CaP group, cartilage layer was more observed at the tendon-bone interface of the joint aperture site than in the control group (p<0.05). Many osteoclasts on the femoral side of the tendon-bone interface in the control were observed compared with that in the CaP group (p<0.05). At the femoral side, the CaP-hybridised tendon graft reduced bone-tunnel enlargement associated with tendon-bone healing 6 months after ACL reconstruction in goats. Clinically, the CaP-hybridised tendon graft for ACL reconstruction can reduce bone-tunnel enlargement.

Time dependence of changes of two cartilage layers in anterior cruciate ligament insertion after resection on chondrocyte apoptosis and decrease in glycosaminoglycan

BackgroundThe purpose of this study is to clarify the differences in time-dependent histological changes (chondrocyte apoptosis and glycosaminoglycan (GAG) layer thickness decrease) between uncalcified fibrocartilage (UF) and calcified fibrocartilage (CF) layers at the anterior cruciate ligament (ACL) insertion after ACL resection of rabbits.MethodsForty male Japanese white rabbits underwent ACL substance resection in the right knee (resection group) and same operation without resection in the left knee (sham group). Animals were sacrificed 1, 2, 4 and 6 weeks after surgery.ResultsIn the UF layer, the apoptosis rate in the resection group was significantly higher than that in the sham group at 1 and 2 weeks. The GAG layer thicknesses of the UF layer in the resection group at 1, 2, 4 and 6 weeks were lower than those in the sham group. In the CF layer, the apoptosis rate in the resection group was significantly higher than that in the sham group at 2 and 4 weeks. The GAG layer thickness of the CF layer in the resection group was lower than that in the sham group only at 6 weeks.ConclusionThe increase in chondrocyte apoptosis rate preceded the decrease in GAG layer thickness in both layers. In the UF layer, the increase in chondrocyte apoptosis rate and the decrease in GAG layer thickness preceded those in the CF layer. Using a surviving ligament and minimizing a debridement of ACL remnant during ACL reconstruction may be important to maintain cartilage layers of ACL insertion. An injured ACL should be repaired before degenerative changes of the insertion occur.

Drug-susceptibility of isolates of Brachyspira hyodysenteriae isolated from colonic mucosal specimens of pigs collected from slaughter houses in Japan in 2009.

Twenty nine isolates identified as Brachyspira hyodysenteriae were most susceptible to carbadox and metronidazole, whereas they were resistant to macrolides. The isolates showed intermediate susceptibility to tiamulin, lincomycin, penicillin G, ampicillin, chloramphenicol, tetracycline, enrofloxacin and valnemulin, with MIC50 values ranging from 0.39 to 3.13.

Effect of Calcium Phosphate–Hybridized Tendon Graft in Anatomic Single-Bundle ACL Reconstruction in Goats

Background: We previously developed a novel technique using an alternate soaking process that improves tendon-bone healing by hybridizing the tendon graft with calcium phosphate (CaP). However, the effects of the CaP-hybridized tendon graft on anatomic single-bundle anterior cruciate ligament (ACL) reconstruction remain unclear. Purpose: To determine the effects of CaP-hybridized tendon grafts compared with untreated tendon grafts 6 months after anatomic single-bundle ACL reconstruction using a goat model. Study Design: Controlled laboratory study. Methods: Animals were divided into a CaP group (n = 5 goats) and a control group (n = 5 goats), and we analyzed (1) knee kinematics and in situ forces under applied anterior tibial loads of 50 N and internal tibial torque of 2.0 N·m in the grafts at full extension and at 60° and 90° of knee flexion, (2) the mean percentage of bone tunnel enlargement using computed tomography (CT), and (3) the histology of the tendon-bone interface. Results: The in situ forces under applied anterior tibial loads of 50 N at 60° and 90° of knee flexion in the CaP group were greater than those in the control group (P < .05). The red safranin-O–stained area, indicating glycosaminoglycans in the cartilage layers at the joint aperture sites of the anterior femoral and posterior tibial bone tunnel, was greater in the CaP group than that in the control group (P < .05). The lengths of the nonbonding gap area between the anterior femoral and posterior tibial bone tunnels in the control group were greater than those in the CaP group (P < .05). No significant difference could be detected in the mean percentage of bone tunnel enlargement between the 2 groups. Conclusion: The CaP-hybridized tendon graft enhanced tendon-bone healing at the joint aperture site in both anterior femoral and posterior tibial tunnels 6 months after anatomic single-bundle ACL reconstruction in goats. The in situ forces under applied anterior tibial loads at greater flexion angles in the CaP group increased compared with controls. Clinical Relevance: Anatomic single-bundle ACL reconstruction using CaP-hybridized tendon grafts may lead to better postoperative knee function.

Influence of Knee Immobilization on Chondrocyte Apoptosis and Histological Features of the Anterior Cruciate Ligament Insertion and Articular Cartilage in Rabbits

This study examined the influence of immobilization on chondrocyte apoptosis and histological features of the anterior cruciate ligament (ACL) insertion and knee articular cartilage in rabbits. Forty-eight male Japanese white rabbits were assigned to an immobilization (n = 24) or sham (n = 24) group. Rabbits in the immobilization group underwent complete unilateral surgical knee immobilization and rabbits in the sham group underwent a sham surgery. The average thickness of the glycosaminoglycan (GAG) stained red area by safranin O staining, the chondrocyte apoptosis rate and the chondrocyte proliferation rate in the cartilage layer in the ACL insertion and the articular cartilage of the medial tibial condyle were measured at one, two, four and eight weeks in six animals from each group. In the ACL insertion, the chondrocyte apoptosis rate was higher in the immobilization group than in the sham group at two and eight weeks after surgery (p < 0.05). The chondrocyte proliferation rate gradually decreased from two weeks to eight weeks in the immobilization group. The GAG layer was thinner in the immobilization group than in the sham group at two, four and eight weeks after surgery (p < 0.05). In the articular cartilage, the chondrocyte apoptosis rate in the immobilization group was higher than in the sham group at four and eight weeks after surgery (p < 0.05). The GAG layer was significantly thinner in the immobilization group than that in the sham group at four and eight weeks after surgery (p < 0.05). Knee immobilization significantly increased chondrocyte apoptosis at two and eight weeks after surgery in the ACL insertion and at four and eight weeks after surgery in the articular cartilage of the medial tibial condyle, and decreased GAG layer thickness from two to eight weeks after surgery in the ACL insertion and from four to eight weeks after surgery in the articular cartilage.

Influence of mechanical unloading on histological changes of the patellar tendon insertion in rabbits.

BACKGROUND The purpose of this study was to clarify the influence of mechanical unloading on histological changes of the patellar tendon (PT) insertion in rabbits. MATERIALS AND METHODS The PT was completely released from stress by drawing the patella toward the tibial tubercle with a stainless steel wire installed between the patella and tibial tubercle (mechanical unloading group, n=28). The animals of the sham group underwent the same surgical procedure; however, the wire was not tightened (n=28). The average thickness of the Safranin O-stained glycosaminoglycan (GAG) area, chondrocyte apoptosis rate and chondrocyte proliferation rate of the cartilage layer at the insertion were measured at one, two, four, and six weeks. RESULTS The chondrocyte apoptosis rate in the mechanical unloading group was significantly higher than that in the sham group at one and four weeks (p<0.05). The chondrocyte proliferation rate in the mechanical unloading group was significantly lower than that in the sham group at four and six weeks (p<0.05). The average thickness of the GAG-stained area in the mechanical unloading group was significantly lower than that in the sham group at six weeks (p<0.05). CONCLUSION Mechanical unloading significantly affected the increase in the chondrocyte apoptosis rate, decrease in the chondrocyte proliferation rate, and decrease in the GAG layer thickness at the PT insertion for up to six weeks in rabbits. CLINICAL RELEVANCE We suggest that more than 6 weeks of mechanical unloading should be avoided to prevent degeneration at the PT insertion.

Influence of Gradual Elongation to the Patella Tendon Insertion in Rabbits

The purpose of this study was to examine the histological changes at the patella tendon (PT) insertion site under gradual elongation in rabbits. Gradual elongation of the PT was performed using external fixation for 4 weeks, with a lengthening speed of 0.5 mm/day (elongation group; n = 24). Rabbits in the sham group underwent the same surgical procedure without gradual elongation (sham group; n = 24). Eight animals were sacrificed 1, 2 and 4 weeks after surgery in each group, respectively. Average thicknesses of stained glycosaminoglycan (GAGs) areas by Safranin-O staining in the total cartilage layer and the uncalcified fibrocartilage layer in the elongation group were significantly higher than that in the sham group at 4 weeks (p < 0.05) and that in the intact PT group (n = 6, p < 0.05). In the elongation group, the peak in the average thicknesses of the stained GAGs areas in the total cartilage layer and the uncalcified fibrocartilage layer were observed at 4 weeks. Gradual elongation of PT insertion significantly affected the increase in the average thicknesses of the stained GAGs areas in the cartilage layer especially in the uncalcified fibrocartilage layer at 4 weeks in rabbits. Clinically, insertions of tendon and ligament can extend during gradual elongation using external fixation more than 4 weeks after the operation.

Extension of knee immobilization delays recovery of histological damages in the anterior cruciate ligament insertion and articular cartilage in rabbits

[Purpose] To investigate the influence of knee immobilization period on recovery of histological damages in the anterior cruciate ligament (ACL) insertion and articular cartilage in rabbits. This knowledge is important for determining the appropriate rehabilitation approach for patients with ligament injuries, fracture, disuse atrophy, and degenerative joint disease. [Materials and Methods] Forty-eight male Japanese white rabbits were divided equally into the remobilization and control groups. The remobilization group had the right knee surgically immobilized, and was divided equally into four subgroups according to the duration of immobilization (1, 2, 4 and 8 weeks). After the immobilization was removed, the rabbits moved freely for 8 weeks. The control group underwent sham operation and followed the same time course as the remobilization group. The chondrocyte apoptosis rate and chondrocyte proliferation rate in the ACL insertion and articular cartilage were analyzed after remobilization. [Results] In the ACL insertion, the remobilization group had a higher chondrocyte apoptosis rate than the control group after 8 weeks of immobilization, and a lower chondrocyte proliferation rate than the control group after 4 and 8 weeks of immobilization. In the articular cartilage, the remobilization group had a lower chondrocyte proliferation rate than the control group after 8 weeks of immobilization. After 8 weeks of remobilization, the ACL insertion and articular cartilage are not completely recovered after 4 and 8 weeks of immobilization, respectively. [Conclusion] Our results suggest that 8 weeks of remobilization will result in recovery of the ACL insertion after 2 weeks of knee immobilization, and recovery of the articular cartilage after 4 weeks of knee immobilization. If 8 weeks of immobilization occurs, a remobilization duration of more than 8 weeks may be necessary.

Calcium Phosphate Hybridized with Human Semitendinosus and Gracilis Tendon Grafts

We hybridized calcium phosphate (CaP) with human semitendinosus and gracilis (ST/G) tendon grafts using an alternate soaking process. To evaluate quantitatively and histologically assess the CaP hybridized human ST/G tendon grafts, we classified them into three groups according to their soaking time – number of soaking cycle: 30 sec – 20 cycles (Group A), 1 min – 15 cycles (Group B), 3 min – 5 cycles (Group C). The tendon grafts were divided into three parts: tibial end (TE), femoral end (FE) and intra-articular (IA) portion. TE was secured using the Krackow technique with No. 2 nonabsorbable sutures, and an Endobutton-CL (Smith & Nephew, USA) was passed through the looped FE, as performed clinically. Then, the IA portion was covered with the sleeve of a rubber glove to prevent CaP hybridization. More soaking cycles induced greater deposition of CaP in the tendon grafts when the total soaking time was the same. Covering the IA portion with a rubber sleeve prevented of CaP deposition. A large amount of CaP in TE was deposited because suture holes increased the total contact area with the solutions.