Hajime Mishima

Gene expression analysis of major lineage‐defining factors in human bone marrow cells: Effect of aging, gender, and age‐related disorders

Adult bone marrow cells (BMCs) include two populations:;mesenchymal stem cells (MSCs), which can differentiate into bone, cartilage, and fat; and hematopoietic stem cells (HSCs), which produce all mature blood lineage. To study the effect of aging, gender, and age‐related disorders on lineage differentiation, we performed quantitative RT‐PCR to examine mRNA expression of the major factors defining BMC lineage, cbfa1 for osteoblasts, ppar‐gamma for adipocytes, sox9 for chondrocytes, and rankl for osteoclasts, in bone marrow from 80 healthy subjects and patients (14–79 years old) with two age‐related disorders: osteoarthritis (OA) and rheumatoid arthritis (RA). Two apoptosis‐related genes, bcl‐2 and drak1, were studied. RANKL and PPAR‐Gamma levels exhibited a clear positive correlation with age in female patients, but not in males, with a slight age‐related decline in CBFa1 transcripts. DRAK1 expression showed an age‐associated ascending trend with significantly greater transcripts of RANKL and DRAK1 in females (p < 0.01). Compared with age‐matched controls, RA patients exhibited increased RANKL, PPAR‐Gamma, and DRAK1 mRNA levels (p < 0.05), and OA showed the higher RANKL and PPAR‐Gamma transcripts (p < 0.05). Furthermore, SOX9 and DRAK1 expressions in the RA group were higher than in the OA group (p < 0.05). Our data indicate that aging and age‐related disorders affect gene expressions differently, suggesting that in aging, the lineage of bone marrow cells was modified with prominent changes in decreased bone marrow osteoblastogenesis, increased adipogenesis and osteoclastogenesis, while in age‐related disorders, marrow adipogenesis and the activity or number of osteoclasts may play an important role in the pathogenesis of arthritic bone loss.

Rotating three-dimensional dynamic culture of adult human bone marrow-derived cells for tissue engineering of hyaline cartilage

The method of constructing cartilage tissue from bone marrow‐derived cells in vitro is considered a valuable technique for hyaline cartilage regenerative medicine. Using a rotating wall vessel (RWV) bioreactor developed in a NASA space experiment, we attempted to efficiently construct hyaline cartilage tissue from human bone marrow‐derived cells without using a scaffold. Bone marrow aspirates were obtained from the iliac crest of nine patients during orthopedic operation. After their proliferation in monolayer culture, the adherent cells were cultured in the RWV bioreactor with chondrogenic medium for 2 weeks. Cells from the same source were cultured in pellet culture as controls. Histological and immunohistological evaluations (collagen type I and II) and quantification of glycosaminoglycan were performed on formed tissues and compared. The engineered constructs obtained using the RWV bioreactor showed strong features of hyaline cartilage in terms of their morphology as determined by histological and immunohistological evaluations. The glycosaminoglycan contents per µg DNA of the tissues were 10.01 ± 3.49 µg/µg DNA in the case of the RWV bioreactor and 6.27 ± 3.41 µg/µg DNA in the case of the pellet culture, and their difference was significant. The RWV bioreactor could provide an excellent environment for three‐dimensional cartilage tissue architecture that can promote the chondrogenic differentiation of adult human bone marrow‐derived cells.

Stable and nondisruptive in vitro/in vivo labeling of mesenchymal stem cells by internalizing quantum dots.

Progress in stem cell research has prioritized the refinement of cell-labeling techniques for in vitro and in vivo basic and therapeutic studies. Although quantum dots, because of their optical properties, are emerging as favorable nanoparticles for bioimaging, substantial refinements or modifications that would improve their biocompatibility are still required. We report here that internalizing quantum dots (i-QDs) generated by their conjugation with an internalizing antibody against a heat shock protein-70 family stress chaperone, mortalin, offered an efficient, genetically noninvasive, nontoxic, and functionally inert way to label mesenchymal stem cells (MSCs). The i-QD-labeled MSCs underwent normal adipocyte, osteocyte, and chondrocyte differentiation in vitro and in vivo, suggesting the potential application of i-QDs in in vivo diagnostics, regenerative and therapeutic medicine.

Concentration of bone marrow aspirate for osteogenic repair using simple centrifugal methods

Bone marrow transplantation can lead to osteogenic repair of intractable bone conditions. To achieve optimal clinical results, it is necessary to transplant as many cells with osteogenetic potential as possible. However, approaches involving special equipment and reagents for the extraction and purification of cells are expensive, and the complicated procedures involved are a hindrance to widespread acceptance of bone marrow transplantation for osteogenic repair. To standardize bone marrow transplantation for bone regeneration, a simple, safe, clean, and low‐cost system is required. We describe an easy‐to‐use method using a conventional manual blood bag centrifugation technique traditionally used for extracting buffy coats, for concentration of cells from bone marrow aspirates (BMAs) to obtain osteogenic progenitors.

Fate of bone marrow mesenchymal stem cells following the allogeneic transplantation of cartilaginous aggregates into osteochondral defects of rabbits.

The purpose of this study was to track mesenchymal stem cells (MSCs) labelled with internalizing quantum dots (i‐QDs) in the reparative tissues, following the allogeneic transplantation of three‐dimensional (3D) cartilaginous aggregates into the osteochondral defects of rabbits. QDs were conjugated with a unique internalizing antibody against a heat shock protein‐70 (hsp70) family stress chaperone, mortalin, which is upregulated and expressed on the surface of dividing cells. The i‐QDs were added to the culture medium for 24 h. Scaffold‐free cartilaginous aggregates formed from i‐QD‐labelled MSCs (i‐MSCs), using a 3D culture system with chondrogenic supplements for 1 week, were transplanted into osteochondral defects of rabbits. At 4, 8 and 26 weeks after the transplantation, the reparative tissues were evaluated macroscopically, histologically and fluoroscopically. At as early as 4 weeks, the defects were covered with a white tissue resembling articular cartilage. In histological appearance, the reparative tissues resembled hyaline cartilage on safranin‐O staining throughout the 26 weeks. In the deeper portion, subchondral bone and bone marrow were well remodelled. On fluoroscopic evaluation, QDs were tracked mainly in bone marrow stromata, with some signals detected in cartilage and the subchondral bone layer. We showed that the labelling of rabbit MSCs with anti‐mortalin antibody‐conjugated i‐QDs is a tolerable procedure and provides a stable fluorescence signal during the cartilage repair process for up to 26 weeks after transplantation. The results suggest that i‐MSCs did not inhibit, and indeed contributed to, the regeneration of osteochondral defects. Copyright

Midterm results of the Synergy cementless tapered stem : stress shielding and bone quality

BackgroundTapered femoral stems have been shown to produce less thigh pain and stress shielding than other cement-less stem designs. The purpose of this study was to examine the performance of this type of femoral stem in Japanese patients.MethodsA series of 40 total hip arthroplasties with a Synergy tapered femoral component were performed in 33 patients between March 1999 and February 2001 at our institution. Three hips (in three patients) were lost to follow-up, so 37 hips in 30 patients were followed for an average of 70 months. The patients’ average age at the time of surgery was 59 years (range 43–80 years). Clinical evaluation included the Japanese Orthopaedic Association (JOA) Hip Score and the incidence of thigh pain. Preoperative radiographic examination included the cortical index and Dorr’s bone type. Postoperative radiographic examination included evaluation of biological fixation, spot welds, cortical hypertrophy, and stress shielding.ResultsThe average JOA Hip Score improved significantly from 35 preoperatively to 91 postoperatively. No patient suffered thigh pain. All femoral components were classified as bony stable. Spot welds and cortical hypertrophy were commonly found in the middle to the distal portion of the component. Severe (third and fourth degree) stress shielding was observed in 24.3% of the cases. A low cortical index, a Dorr type C femur, and a large stem size were associated with severe stress shielding.ConclusionsThe midterm clinical results with the Synergy femoral component were satisfactory for Japanese patients. Although severe stress shielding was frequently observed in patients with poor bone quality, stem stability was not affected. Extent of grit blasting and stem length may be factors causing severe stress shielding in Japanese patients with poor bone quality. In conclusion, poor bone quality is a less favorable indication for the cementless tapered stem.

The Role of CCL5 in the Ability of Adipose Tissue-Derived Mesenchymal Stem Cells to Support Repair of Ischemic Regions

Mesenchymal stem cells (MSC) are multipotent and possess high proliferative activity, and thus are thought to be a reliable cell source for cell therapies. Here, we isolated MSC from adult tissues--bone marrow (BM-MSC), dental tissue (DT-MSC), and adipose tissue (AT-MSC)--to compare how autotransplantation of these MSC effectively supports the repair of bone fracture and ischemic tissue. An analysis by in vitro differentiation assays showed no significant difference among these MSC. The degree of calcification at the joint region of bone fracture was higher in mice transplanted with AT-MSC than in mice transplanted with BM-MSC or DT-MSC. To compare the abilities of MSC, characterize how those MSC affect the repair of ischemic tissue, vascular occlusion was performed by ligation of the femoral artery and vein. Of note, the blood flow in the ischemic region rapidly increased in mice injected with AT-MSC, as contrasted with mice injected with BM- or DT-MSC. The number of CD45- and F4/80-positive cells at the femoral region was higher in AT-MSC recipients than in recipients of BM-MSC or DT-MSC. We evaluated the mRNA expression of angiogenic and migration factors in MSC and found the expression of CCL5 mRNA was higher in AT-MSC than in BM-MSC or DT-MSC. Transplantation of AT-MSC with impaired expression of CCL5 clearly showed a significant delay in the recovery of blood flow compared with the control. These findings have fundamental implications for the modulation of AT-MSC in the repair of vasculature and bone fracture.

Follow-Up Results of 10–12 Years After Total Hip Arthroplasty Using Cementless Tapered Stem — Frequency of Severe Stress Shielding With Synergy Stem in Japanese Patients

Synergy stems are tapered stems featuring a proximal porous coating, grid blasting below the proximal third to the distal end. This study included 41 patients (50 hips) who underwent total hip arthroplasty with follow-ups for 10 years or more. No stem reimplantations were performed. Spot welds were observed in the distal stem in Gruen zones 3 and 5 in 35 and 32 hips, respectively. First-degree stress shielding occurred in 8 hips; 2nd-degree, 20 hips; 3rd-degree, 13 hips; and 4th-degree, 9 hips. Because of bone fixation to the distal grit-blasted section of the stem, severe stress shielding was observed in nearly half of the cases. Multiple regression analysis of stress shielding determinants revealed a correlation between stem size and short patient height, showing the cause of stress shielding to be a mismatch in size between the stem and the femoral bone.

Osseointegration of porous titanium implants with and without electrochemically deposited DCPD coating in an ovine model

BackgroundUncemented fixation of components in joint arthroplasty is achieved primarily through de novo bone formation at the bone-implant interface and establishment of a biological and mechanical interlock. In order to enhance bone-implant integration osteoconductive coatings and the methods of application thereof are continuously being developed and applied to highly porous and roughened implant substrates. In this study the effects of an electrochemically-deposited dicalcium phosphate dihydrate (DCPD) coating of a porous substrate on implant osseointegration was assessed using a standard uncemented implant fixation model in sheep.MethodsPlasma sprayed titanium implants with and without a DCPD coating were inserted into defects drilled into the cancellous and cortical sites of the femur and tibia. Cancellous implants were inserted in a press-fit scenario whilst cortical implants were inserted in a line-to-line fit. Specimens were retrieved at 1, 2, 4, 8 and 12 weeks postoperatively. Interfacial shear-strength of the cortical sites was assessed using a push-out test, whilst bone ingrowth, ongrowth and remodelling were investigated using histologic and histomorphometric endpoints.ResultsDCPD coating significantly improved cancellous bone ingrowth at 4 weeks but had no significant effect on mechanical stability in cortical bone up to 12 weeks postoperatively. Whilst a significant reduction in cancellous bone ongrowth was observed from 4 to 12 weeks for the DCPD coating, no other statistically significant differences in ongrowth or ingrowth in either the cancellous or cortical sites were observed between TiPS and DCPD groups.ConclusionThe application of a DCPD coating to porous titanium substrates may improve the extent of cancellous bone ingrowth in the early postoperative phase following uncemented arthroplasty.

Joint distraction and movement for repair of articular cartilage in a rabbit model with subsequent weight-bearing

We have previously shown that joint distraction and movement with a hinged external fixation device for 12 weeks was useful for repairing a large articular cartilage defect in a rabbit model. We have now investigated the results after six months and one year. The device was applied to 16 rabbits who underwent resection of the articular cartilage and subchondral bone from the entire tibial plateau. In group A (nine rabbits) the device was applied for six months. In group B (seven rabbits) it was in place for six months, after which it was removed and the animals were allowed to move freely for an additional six months. The cartilage remained sound in all rabbits. The areas of type II collagen-positive staining and repaired soft tissue were larger in group B than in group A. These findings provide evidence of long-term persistence of repaired cartilage with this technique and that weight-bearing has a positive effect on the quality of the cartilage.