Hideaki Imabayashi

Combination of hTERT and bmi-1, E6, or E7 Induces Prolongation of the Life Span of Bone Marrow Stromal Cells from an Elderly Donor without Affecting Their Neurogenic Potential

ABSTRACT Murine bone marrow stromal cells differentiate not only into mesodermal derivatives, such as osteocytes, chondrocytes, adipocytes, skeletal myocytes, and cardiomyocytes, but also into neuroectodermal cells in vitro. Human bone marrow stromal cells are easy to isolate but difficult to study because of their limited life span. To overcome this problem, we attempted to prolong the life span of bone marrow stromal cells and investigated whether bone marrow stromal cells modified with bmi-1, hTERT, E6, and E7 retained their differentiated capability, or multipotency. In this study, we demonstrated that the life span of bone marrow stromal cells derived from a 91-year-old donor could be extended and that the stromal cells with an extended life span differentiated into neuronal cells in vitro. We examined the neuronally differentiated cells morphologically, physiologically, and biologically and compared the gene profiles of undifferentiated and differentiated cells. The neuronally differentiated cells exhibited characteristics similar to those of midbrain neuronal progenitors. Thus, the results of this study support the possible use of autologous-cell graft systems to treat central nervous system diseases in geriatric patients.

Can the life span of human marrow stromal cells be prolonged by bmi‐1, E6, E7, and/or telomerase without affecting cardiomyogenic differentiation?

Cell transplantation has recently been challenged to improve cardiac function of severe heart failure. Human mesenchymal stem cells (hMSCs) are multipotent cells that can be isolated from adult marrow stroma, but because of their limited life span, it is difficult to study them further. To overcome this problem, we attempted to prolong the life span of hMSCs and investigate whether the hMSCs modified with cell‐cycle‐associated genes can differentiate into cardiomyocytes in vitro.

In vivo analysis of insertional torque during pedicle screwing using cortical bone trajectory technique.

Study Design. The insertional torque of pedicle screws using the cortical bone trajectory (CBT) was measured in vivo. Objective. To investigate the effectiveness of the CBT technique by measurement of the insertional torque. Summary of Background Data. The CBT follows a mediolateral and caudocephalad directed path, engaging with cortical bone maximally from the pedicle to the vertebral body. Some biomechanical studies have demonstrated favorable characteristics of the CBT technique in cadaveric lumbar spine. However, no in vivo study has been reported on the mechanical behavior of this new trajectory. Methods. The insertional torque of pedicle screws using CBT and traditional techniques were measured intraoperatively in 48 consecutive patients. A total of 162 screws using the CBT technique and 36 screws using the traditional technique were compared. In 8 of 48 patients, the side-by-side comparison of 2 different insertional techniques for each vertebra were performed, which formed the H group. In addition, the insertional torque was correlated with bone mineral density. Results. The mean maximum insertional torque of CBT screws and traditional screws were 2.49 ± 0.99 Nm and 1.24 ± 0.54 Nm, respectively. The CBT screws showed 2.01 times higher torque and the difference was significant between the 2 techniques (P < 0.01). In the H group, the insertional torque were 2.71 ± 1.36 Nm in the CBT screws and 1.58 ± 0.44 Nm in the traditional screws. The CBT screws demonstrated 1.71 times higher torque and statistical significance was achieved (P < 0.01). Positive linear correlations between maximum insertional torque and bone mineral density were found in both technique, the correlation coefficient of traditional screws (r = 0.63, P < 0.01) was higher than that of the CBT screws (r = 0.59, P < 0.01). Conclusion. The insertional torque using the CBT technique is about 1.7 times higher than the traditional technique. Level of Evidence: 2

Redifferentiation of dedifferentiated chondrocytes and chondrogenesis of human bone marrow stromal cells via chondrosphere formation with expression profiling by large-scale cDNA analysis

Characterization of dedifferentiated chondrocytes (DECs) and mesenchymal stem cells capable of differentiating into chondrocytes is of biological and clinical interest. We isolated DECs and bone marrow stromal cells (BMSCs), H4-1 and H3-4, and demonstrated that the cells started to produce extracellular matrices, such as type II collagen and aggrecan, at an early stage of chondrosphere formation. Furthermore, cDNA sequencing of cDNA libraries constricted by the oligocapping method was performed to analyze difference in mRNA expression profiling between DECs and marrow stromal cells. Upon redifferentiation of DECs, cartilage-related extracellular matrix genes, such as those encoding leucine-rich small proteoglycans, cartilage oligomeric matrix protein, and chitinase 3-like 1 (cartilage glycoprotein-39), were highly expressed. Growth factors such as FGF7 and CTGF were detected at a high frequency in the growth stage of monolayer stromal cultures. By combining the expression profile and flow cytometry, we demonstrated that isolated stromal cells, defined by CD34(-), c-kit(-), and CD140alpha(- or low), have chondrogenic potential. The newly established human mesenchymal cells with expression profiling provide a powerful model for a study of chondrogenic differentiation and further understanding of cartilage regeneration in the means of redifferentiated DECs and BMSCs.

Nuclear Transfer of Adult Bone Marrow Mesenchymal Stem Cells: Developmental Totipotency of Tissue-Specific Stem Cells from an Adult Mammal

Abstract Recent studies have demonstrated that somatic stem cells have a flexible potential greater than previously expected when they are transplanted into different tissues. On the other hand, recent studies also have revealed that these potentials might occur because of spontaneous cell fusion with recipient cells. The nuclei of somatic cells could have been reprogrammed when they were artificially or spontaneously fused with mouse embryonic stem (ES) cells. The resultant hybrid cells acquired a developmental pluripotency that the original somatic cells did not have but that ES cells did. LaBarge and Blau (Cell 2002; 111:589–601) demonstrated that adult bone marrow-derived cells contributed to muscle tissue in a stepwise biological progression. This means that bone marrow-derived cells became satellite cells of mononucleate muscle stem cells after the first irradiation-induced damage to the mouse, and after the second irradiation-induced damage, multinucleate myofibers appeared from the bone marrow-derived cells. Considered together, the differentiation potential of the somatic stem cell nucleus itself remains unclear. Although the pluripotency of somatic stem cell populations has been evaluated, the developmental totipotency of the nuclei of somatic stem cells, whether or not they fused with other cells, has not been shown, except in only one study concerning fetal neural cells (never in adult stem cells). Here, we showed the developmental totipotency of adult bovine mesenchymal stem cells by nuclear transfer.

Morphometric measurement of cortical bone trajectory for lumbar pedicle screw insertion using computed tomography

Study Design: A morphometric measurement of cortical bone trajectory (CBT) for the lumbar pedicle screw insertion using computed tomography (CT). Objective: The aim of this study was to conduct a detailed morphometric measurement of the CBT. Summary of Background Data: The CBT is a novel lumbar pedicle screw trajectory, which follows a caudocephalad path sagittally and a laterally directed path in the transverse plane. The advantage associated with this modified technique is increased cortical bone contact, providing an enhanced screw purchase. However, little is known about the possible screw size or detailed direction of the trajectory. Methods: The CT scans of 100 adults who underwent examination for spinal problems were studied. A total of 470 lumbar vertebrae excluding spondylosis, malformation, and tumor were observed. In this trajectory, the starting point was supposed to be the junction of the center of the superior articular process and 1 mm inferior to the inferior border of the transverse process. The CT images were analyzed using 3-dimensional reconstruction software. The diameter, length, lateral angle to the vertebral sagittal plane, and cephalad angle to the vertebral horizontal plane of the trajectory were measured. Results: The mean diameter gradually increased from L1 to L5 (from 6.2 mm at L1 to 8.4 mm at L5). The mean length from L1 to L5 were 36.8, 38.2, 39.3, 39.8, and 38.3 mm, respectively. The lateral angle from L1 to L5 were 8.6, 8.5, 9.1, 9.1, and 8.8 degrees, respectively. The cephalad angle from L1 to L5 were 26.2, 25.5, 26.2, 26.0, and 25.8 degrees, respectively. Conclusions: The morphology of the pedicle, such as shape and pedicle axis angle, differed over the lumbar levels, our measurements demonstrated similar data excluding the diameter of the trajectory. There were no significant differences between each level of the lateral and cephalad angles.

Age-related changes in expression of tissue inhibitor of metalloproteinases-3 associated with transition from the notochordal nucleus pulposus to the fibrocartilaginous nucleus pulposus in rabbit intervertebral disc.

Study Design. Experimental study on age-related changes in expression of tissue inhibitor of metalloproteinases-3 (TIMP-3) associated with transition from notochordal nucleus pulposus (NP) to fibrocartilaginous NP in rabbit intervertebral disc (IVD). Objectives. To identify roles of notochordal NP in extracellular matrix (ECM) metabolism of IVD. Summary of Background Data. One of most interesting properties of TIMP-3 is to inhibit aggrecanases in addition to matrix metalloproteinases. Balance of aggrecanase/TIMP-3 is critical to maintain homeostasis of ECM metabolism. Methods. Four-week-old and 160-week-old male Japanese white rabbits were used. Age-related changes in IVDs were evaluated histologically using previously established grading system. Immunohistochemistry of TIMP-3 and semiquantitative reverse transcriptase-polymerase reaction (RT-PCR) of TIMP-3, a disintegrin and metalloproteinases with thrombospondin motifs (ADAMTS) 4, 5, and transforming growth factor-&bgr;1 (TGF-&bgr;1), were conducted. Results. Semiquantitative assessment of histologic changes indicated that 4-week-old rabbit was equivalent to fetus to 2-year-old human and 160-week-old rabbit was equivalent to 11- to 30-year-old human, particularly 11- to 16-year-old, which corresponds to transition period from notochordal to fibrocartilaginous NP. Immunohistochemistry revealed that TIMP-3 was positive in 4-week-old rabbit only. Semiquantitative RT-PCR revealed that levels of expressions of TGF-&bgr;1 and TIMP-3 mRNAs in 4-week-old were significantly higher than those in 160-week-old rabbits. There was no significant difference in expression of ADAMTS4 mRNA. ADAMTS5 mRNA was not detected or extremely low in both groups. Expression of TIMP-3 mRNA in NP was upregulated by TGF-&bgr;1 but was not affected by IL-1&bgr;. On the contrary, expression of ADAMTS4 mRNA was not upregulated by TGF-&bgr;1 but was upregulated by IL-1&bgr;. Conclusions. Levels of expression of TIMP-3 in notochordal NP were significantly lower in 160-week-old rabbits than those in 4-week-old rabbits. Decrease in expression of TIMP-3, possibly mediated in part by TGF-&bgr;1, may cause imbalance of ADAMTS4/TIMP-3 ratio at transition period from notochordal to fibrocartilaginous NP.

Cortical bone trajectory for lumbosacral fixation: penetrating S-1 endplate screw technique: technical note.

OBJECT A cortical bone trajectory (CBT) is a new pedicle screw trajectory that maximizes the thread contact with cortical bone surface, providing enhanced screw purchase. Despite the increased use of the CBT in the lumbar spine, little is known about the insertion technique for the sacral CBT. The aim of this study was to introduce a novel sacral pedicle screw trajectory. This trajectory engages with denser bone maximally by the screw penetrating the S-1 superior endplate through a more medial entry point than the traditional technique, and also has safety advantages, with the protrusion of the screw tip into the intervertebral disc space carrying no risk of neurovascular injury. METHODS In this study, the CT scans of 50 adults were studied for morphometric measurement of the new trajectory. The entry point was supposed to be the junction of the center of the superior articular process of S-1 and approximately 3 mm inferior to the most inferior border of the inferior articular process of L-5. The direction was straight forward in the axial plane without convergence, angulated cranially in the sagittal plane penetrating the middle of the sacral endplate. The cephalad angle to the sacral endplate, length of trajectory, and safety of the trajectory were investigated. Next, the insertional torque of pedicle screws using this technique was measured intraoperatively in 19 patients and compared with the traditional technique. RESULTS The mean cephalad angle in these 50 patients was 30.7° ± 5.1°, and the mean length of trajectory was 31.5 ± 3.5 mm. The CT analysis revealed that the penetrating S-1 endplate technique did not cause any neurovascular injury anteriorly in any case. The new technique demonstrated an average of 141% higher insertional torque than the traditional monocortical technique. CONCLUSIONS The penetrating S-1 endplate technique through the medial entry point is suitable for the connection of lumbar CBT, has revealed favorable stability for lumbosacral fixation, and has reduced the potential risk of neurovascular injuries.

Biomechanical evaluation of the fixation strength of lumbar pedicle screws using cortical bone trajectory: a finite element study.

OBJECT Cortical bone trajectory (CBT) maximizes thread contact with the cortical bone surface and provides increased fixation strength. Even though the superior stability of axial screw fixation has been demonstrated, little is known about the biomechanical stiffness against multidirectional loading or its characteristics within a unit construct. The purpose of the present study was to quantitatively evaluate the anchorage performance of CBT by the finite element (FE) method. METHODS Thirty FE models of L-4 vertebrae from human spines (mean age [± SD] 60.9 ± 18.7 years, 14 men and 16 women) were computationally created and pedicle screws were placed using the traditional trajectory (TT) and CBT. The TT screw was 6.5 mm in diameter and 40 mm in length, and the CBT screw was 5.5 mm in diameter and 35 mm in length. To make a valid comparison, the same shape of screw was inserted into the same pedicle in each subject. First, the fixation strength of a single pedicle screw was compared by axial pullout and multidirectional loading tests. Next, vertebral fixation strength within a construct was examined by simulating the motions of flexion, extension, lateral bending, and axial rotation. RESULTS CBT demonstrated a 26.4% greater mean pullout strength (POS; p = 0.003) than TT, and also showed a mean 27.8% stronger stiffness (p < 0.05) during cephalocaudal loading and 140.2% stronger stiffness (p < 0.001) during mediolateral loading. The CBT construct had superior resistance to flexion and extension loading and inferior resistance to lateral bending and axial rotation. The vertebral fixation strength of the construct was significantly correlated with bone mineral density of the femoral neck and the POS of a single screw. CONCLUSIONS CBT demonstrated superior fixation strength for each individual screw and sufficient stiffness in flexion and extension within a construct. The TT construct was superior to the CBT construct during lateral bending and axial rotation.

Incidence and Risk Factors of Adjacent Cranial Facet Joint Violation Following Pedicle Screw Insertion Using Cortical Bone Trajectory Technique.

Study Design. Retrospective study evaluating cranial facet joint violation (FJV) by pedicle screws. Objective. The aim of the study was to determine the incidence and risk factors of FJV following screw placement via cortical bone trajectory (CBT). Summary of Background Data. CBT is a new minimally invasive technique for lumbar pedicle screw insertion that minimizes muscle dissection. Inserting a screw from a more caudal entry point can reduce iatrogenic damage to the cranial facet joint; however, no previous reports exist describing the incidence of FJV secondary to the CBT technique. Methods. We reviewed 202 consecutive patients who underwent lumbar pedicle screw instrumentation using CBT from October 2011 to June 2015. Postoperative CT scans were obtained to determine the degree and incidence of FJV. Clinical and imaging data were analyzed to clarify the risk factors of FJV. The detailed positions of the proximal screws were also investigated and compared between the FJV and non-FJV groups. Results. The incidence of FJV by the proximal screws was 11.8% (48/404), with no occurrence of intra-articular FJV. Multiple logistic regression analysis revealed that age >70 years, vertebral slip >10%, and adjacent facet joint degeneration (Pathrias grade 2 or 3) were independent factors significantly affecting FJV. There were statistically significant differences between the FJV and non-FJV groups in the facet-screw distance (3.2 ± 1.0 vs. 8.1 ± 2.3 mm, P < 0.01), the cranial angle (25.8 ± 6.3° vs. 29.9 ± 7.6°, P < 0.01), and the lamina-screw head distance (5.6 ± 1.6 vs. 6.4 ± 1.9 mm, P < 0.01). Conclusion. Lumbar pedicle screw placement via CBT would reduce FJV; however, special care should be taken in patients with age >70 years, vertebral slip >10%, and facet degeneration. Level of Evidence: 3