A novel anticancer quinolone, (R)-WAC-224, has anti-leukemia activities against acute myeloid leukemia.

Approximately 60%-80% of patients who achieve complete remission eventually relapse after conventional chemotherapy and have poor prognoses despite the recent advances of novel anticancer agents. Continuing development of more effective novel treatments for acute myeloid leukemia (AML) is necessary. We developed (R)-WAC-224 (R-WAC), which is an anticancer quinolone, targeting topoisomerase II. This study evaluated the anti-leukemia potential of R-WAC or racemic WAC-224 (WAC) in vitro and in vivo. R-WAC significantly inhibited the human AML cell line proliferation (MV4-11, HL60, and KG1a), which was comparable to daunorubicin and cytarabine, not affected by P-glycoprotein overexpression. WAC did neither increase serum troponin-T nor decrease the crypt numbers in the small intestine, indicating WAC was less toxic than doxorubicin. R-WAC monotherapy demonstrated prolonged survival in the AML mice model and inhibited tumor growth in the MV4-11 xenograft mice model. Moreover, the combination of R-WAC and cytarabine demonstrated more active anti-leukemia effects than daunorubicin and cytarabine. Finally, R-WAC inhibited the colony-forming abilities using primary AML cells. These results indicate that R-WAC is a promising therapeutic agent for AML.

Specific bone region localization of osteolytic versus osteoblastic lesions in a patient-derived xenograft model of bone metastatic prostate cancer.

OBJECTIVE: Bone metastasis occurs in up to 90% of men with advanced prostate cancer and leads to fractures, severe pain and therapy-resistance. Bone metastases induce a spectrum of types of bone lesions which can respond differently to therapy even within individual prostate cancer patients. Thus, the special environment of the bone makes the disease more complicated and incurable. A model in which bone lesions are reproducibly induced that mirrors the complexity seen in patients would be invaluable for pre-clinical testing of novel treatments. The microstructural changes in the femurs of mice implanted with PCSD1, a new patient-derived xenograft from a surgical prostate cancer bone metastasis specimen, were determined. METHODS: Quantitative micro-computed tomography (micro-CT) and histological analyses were performed to evaluate the effects of direct injection of PCSD1 cells or media alone (Control) into the right femurs of Rag2-/-γc-/- male mice. RESULTS: Bone lesions formed only in femurs of mice injected with PCSD1 cells. Bone volume (BV) was significantly decreased at the proximal and distal ends of the femurs (p < 0.01) whereas BV (p < 0.05) and bone shaft diameter (p < 0.01) were significantly increased along the femur shaft. CONCLUSION: PCSD1 cells reproducibly induced bone loss leading to osteolytic lesions at the ends of the femur, and, in contrast, induced aberrant bone formation leading to osteoblastic lesions along the femur shaft. Therefore, the interaction of PCSD1 cells with different bone region-specific microenvironments specified the type of bone lesion. Our approach can be used to determine if different bone regions support more therapy resistant tumor growth, thus, requiring novel treatments.

Synthetic bone mimetic matrix-mediated in situ bone tissue formation through host cell recruitment.

Advances in tissue engineering have offered new opportunities to restore anatomically and functionally compromised tissues. Although traditional tissue engineering approaches that utilize biomaterials and cells to create tissue constructs for implantation or biomaterials as a scaffold to deliver cells are promising, strategies that can activate endogenous cells to promote tissue repair are more clinically attractive. Here, we demonstrate that an engineered injectable matrix mimicking a calcium phosphate (CaP)-rich bone-specific microenvironment can recruit endogenous cells to form bone tissues in vivo. Comparison of matrix alone with that of bone marrow-soaked or bFGF-soaked matrix demonstrates similar extent of neo-bone formation and bridging of decorticated transverse processes in a posterolateral lumbar fusion rat model. Synthetic biomaterials that stimulate endogenous cells without the need for biologics to assist tissue repair could circumvent limitations associated with conventional tissue engineering approaches, including ex vivo cell processing and laborious efforts, thereby accelerating the translational aspects of regenerative medicine.

Microstructural analysis of three-dimensional canal network in the rabbit lumbar vertebral endplate.

Insufficient nutrient supply through vertebral canal structures to the intervertebral disc (IVD) has been considered as an important contributor for disc degeneration. Despite previous canal structure characterization studies using histology, scanning electron microscopy, and angiography, among others, their three-dimensional (3D) topology inside the vertebral endplate remains poorly understood. This study aims to characterize the 3D canal structure in the rabbit lumbar vertebral endplate using micro computed tomography (µCT). Vertebral endplates were imaged using high-resolution µCT with 1.4 × 1.4 × 1.8 µm voxel size. Diameter, length, orientation, and depth starting from the vertebral endplate surface were analyzed for each canal using individual 3D canal models from the vertebral endplate scans. In the layer underneath the vertebral endplate, at a mean depth of 76.2 µm, longitudinally-oriented relatively short-length (57.6 µm) and small diameter (45.7 µm) canals were dominant. Large-scale canals with a mean diameter of 152.1 µm running parallel to the endplate surface were isolated at the depth of 224.1 µm. These canals were connected to both IVD and bone marrow spaces through vertically oriented canals.

Micro-computed tomography-based three-dimensional kinematic analysis during lateral bending for spinal fusion assessment in a rat posterolateral lumbar fusion model.

Rat posterolateral lumbar fusion (PLF) models have been used to assess the safety and effectiveness of new bone substitutes and osteoinductive growth factors using palpation, radiography, micro-computed tomography (µCT), and histology as standard methods to evaluate spinal fusion. Despite increased numbers of PLF studies involving alternative bone substitutes and growth factors, the quantitative assessment of treatment efficacy during spinal motion has been limited. The purpose of this study was to evaluate the effect of spinal fusion on lumbar spine segment stability during lateral bending using a µCT-based three-dimensional (3D) kinematic analysis in the rat PLF model. Fourteen athymic male rats underwent PLF surgery at L4/5 and received bone grafts harvested from the ilium and femurs of syngeneic rats (Isograft, n=7) or no graft (Sham, n=7). At 8 weeks after the PLF surgery, spinal fusion was assessed by manual palpation, plain radiography, µCT, and histology. To determine lumbar segmental motions at the operated level during lateral bending, 3D kinematic analysis was performed. The Isograft group, but not the Sham group, showed spinal fusion on manual palpation (6/7), solid fusion mass in radiographs (6/7), as well as bone bridging in µCT and histological images (5/7). Compared to the Sham group, the Isograft group revealed limited 3D lateral bending angular range of motion and lateral translation during lateral bending at the fused segment where disc height narrowing was observed. This µCT-based 3D kinematic analysis can provide a quantitative assessment of spinal fusion in a rat PLF model to complement current gold standard methods used for efficacy assessment of new therapeutic approaches.

Effect of scaffold microarchitecture on osteogenic differentiation of human mesenchymal stem cells.

Design of macroporous synthetic grafts that can promote infiltration of cells, their differentiation, and synthesis of bone-specific extracellular matrix is a key determinant for in vivo bone tissue regeneration and repair. In this study, we investigated the effect of the microarchitecture of the scaffold on osteogenic differentiation of human mesenchymal stem cells (hMSCs). Poly(ethylene glycol) diacrylate-co-N-acryloyl 6-aminocaproic acid cryogels were fabricated to have either a pore network consisting of cellular, randomly oriented pores (termed 'spongy') or a pore network consisting of lamellar columns (termed 'columnar'), with both cryogel types showing a similar porosity. Both spongy and columnar cryogels supported comparable levels of cell viability and proliferation of hMSCs in vitro. However, spongy cryogels promoted osteogenic differentiation to a greater extent than their columnar counterparts, as evidenced by increased alkaline phosphatase activity and osteoblastic gene expression over 21 days post culture. Leveraging upon our previous work, we further evaluated the ability of these synthetic scaffolds in conjunction with mineralisation to promote ectopic bone formation upon subcutaneous implantation in nude rats. Mineralised spongy and columnar cryogels, both in the presence and absence of exogenous hMSCs, promoted ectopic bone formation in vivo. No such bone formation was observed in acellular cryogels devoid of mineralisation, with extensive host cell infiltration and vascularisation in columnar cryogels, and negligible infiltration into spongy cryogels. Our results thus present a novel method to tune the microarchitecture of porous polymeric scaffolds, in addition to suggesting their efficacy as synthetic bone grafts.

Morphology of the cartilaginous endplates in human intervertebral disks with ultrashort echo time MR imaging.

PURPOSE: To image human disk-bone specimens by using conventional spin-echo (SE) and ultrashort echo time (TE) techniques, to describe the morphology at magnetic resonance (MR) imaging, and to identify tissue components contributing to high signal intensity near the cartilaginous endplates (CEPs). MATERIALS AND METHODS: This study was exempt from institutional review board approval, and informed consent was not required. Five cadaveric lumbar spines (mean age, 61 years ± 11) were prepared into six sample types containing different combinations of disk, uncalcified CEP, calcified CEP, and subchondral bone components and were imaged with proton density-weighted SE (repetition time msec/TE msec, 2000/15) and ultrashort TE (300/0.008, 6.6, echo-subtraction) sequences. Images were evaluated to determine the presence of intermediate-to-high signal intensity in regions excluding the bone marrow. Logistic regression was used to determine which tissue components were significant predictors of the presence of signal intensity for each MR technique. RESULTS: On ultrashort TE MR images, intact disk/uncalcified CEP/calcified CEP/bone samples exhibited bilaminar intermediate-to-high signal intensity in the region near the CEP, consistent with the histologic appearance of uncalcified and calcified CEPs. Conversely, proton density-weighted SE images exhibited low signal intensity in this region. Results of logistic regression suggested that the presence of uncalcified CEP (P = .023) and calcified CEP (P = .007) in the sample were strong predictors of the presence of signal intensity on ultrashort TE images, whereas the disk was the only predictor (P < .001) of signal intensity on proton density-weighted SE images. CONCLUSION: Ultrashort TE imaging, unlike proton density-weighted SE imaging, enabled direct visualization of the uncalcified and calcified CEP. Evaluation of the morphology and identification of sources of signal intensity at ultrashort TE MR imaging provides opportunities to potentially aid in the understanding of degenerative disk disease.

Orally available pyridinylpyrimidine derivatives as novel RANKL-induced osteoclastogenesis inhibitors.

An HTS campaign led to the identification of 4-pyrroldino-2-(pyridin-2-yl)pyrimidine compound 1 as an RANKL-induced osteoclastogenesis inhibitor. The compound 1 showed high clearance values in microsomes, however. Modification of the pyrrolidino group to a benzylamino group improved human microsomal stability with a slight loss of in vitro activity. Substitution at the ortho position of the benzyl group ameliorated in vitro activity, and further fluorination of the benzyl group improved microsomal stability in rodents. Representative members of this series, compounds 20 and 23, exhibited efficacy in RANKL-induced osteopenic mice when administered orally at 0.3 mg/kg.

The biophysical mechanisms of altered hyaluronan concentration in synovial fluid after anterior cruciate ligament transection.

OBJECTIVE: The residence time of hyaluronan (HA) in knee joint synovial fluid (SF) was investigated using a rabbit anterior cruciate ligament transection (ACLT) model. The aims of this study were to assess, at 7 and 28 days after surgery, the 1) HA concentration and molecular mass (M(r) ) distribution in the SF, 2) endogenous replenishment of HA after saline washout, 3) HA residence times in the SF, and 4) synovium and subsynovium cellularity of the knee joints of rabbits subjected to ACLT, compared to sham-operated and nonoperated control joints. METHODS: Adult NZW rabbits underwent ACLT or sham surgery on one hind limb, while each contralateral limb was the nonoperated control. On day 7 or 28 after surgery, the joints were aspirated for SF, lavaged with saline, and injected with saline or polydisperse HA, and samples were obtained for analysis at set time points up to 8 hours after injection. Joint fluid samples were analyzed for the concentration and M(r) distribution of HA to calculate the HA residence time constant. RESULTS: Analysis of HA concentrations and M(r) distributions showed 1) loss of high-M(r) HA in the SF on day 7 and a shift toward a lower-M(r) distribution on day 28, 2) endogenous replenishment of high-M(r) HA after washout, and 3) M(r) -dependent loss of HA from the knee joints after ACLT, particularly on day 7 postsurgery. The HA residence time decreased with decreasing HA M(r) (residence time ∼27 hours with an M(r) load of 7,000-2,500 kd, to ∼7 hours with an M(r) load of 250-50 kd). HA residence time also decreased (by ∼70%) in the knee joints on day 7 after ACLT. The subsynovium of the joints subjected to ACLT displayed increased cellularity and neovascularization on days 7 and 28 postsurgery. CONCLUSION: The residence time of HA in the SF is transiently decreased after ACLT, suggesting that a biophysical transport mechanism is responsible for the altered composition of the SF after joint injury or during inflammation.

Three-dimensional kinematic analysis of the cervical spine after anterior cervical decompression and fusion at an adjacent level: a preliminary report.

PURPOSE: Development of adjacent segment degeneration following anterior cervical decompression and fusion (ACDF) is still controversial, as adjacent-level kinematics is poorly understood. This study reports preliminary data from a high-accuracy 3D analysis technique developed for in vivo cervical kinematics. METHODS: From nine cervical spondylosis patients, four underwent single-level ACDF, and five underwent two-level ACDF using cylindrical titanium cage implant(s). Pre- and post-surgical CT scans were taken in flexion, neutral and extended positions, allowing us to compute segmental ranges of motion for rotation and translation, and 3D disc-height distributions. Differences in segmental motions and disc-height between fused and adjacent levels were analyzed with a Wilcoxon signed-rank test. Results are presented as mean ± SEM. RESULTS: The flexion/extension angular-ROM at the fusion level decreased after surgery (7.46 ± 1.17° vs. 3.14 ± 0.56°, p < 0.003). The flexion/extension angular-ROM at one caudal adjacent level to the fusion level (3.97 ± 1.29°) tended to be greater post-operatively (6.11 ± 1.44°, p = 0.074). Translation in the anterior-posterior direction during flexion/extension at the fusion level decreased after surgery (1.22 ± 0.20 mm vs. 0.32 ± 0.11 mm, p < 0.01). No differences were found in adjacent-level disc heights between both study time-points. CONCLUSIONS: This study showed increased segmental motion in flexion/extension angular-ROM at one level adjacent to ACDF. However, increases in the rotational angular-ROM were not statistically significant when cranial/caudal adjacent levels were analyzed separately. This preliminary study highlighted the capabilities of a 3D-kinematic analysis method to detect subtle changes in kinematics and disc height at the adjacent levels to ACDF. Thus, reliable evidence related to ACDF's influence on adjacent-level cervical kinematics can be collected.

A novel patient-derived intra-femoral xenograft model of bone metastatic prostate cancer that recapitulates mixed osteolytic and osteoblastic lesions.

UNLABELLED: Prostate cancer metastasizes to bone in the majority of patients with advanced disease leading to painfully debilitating fractures, spinal compression and rapid decline. In addition, prostate cancer bone metastases often become resistant to standard therapies including androgen deprivation, radiation and chemotherapy. There are currently few models to elucidate mechanisms of interaction between the bone microenvironment and prostate cancer. It is, thus, essential to develop new patient-derived, orthotopic models. Here we report the development and characterization of PCSD1 (Prostate Cancer San Diego 1), a novel patient-derived intra-femoral xenograft model of prostate bone metastatic cancer that recapitulates mixed osteolytic and osteoblastic lesions. METHODS: A femoral bone metastasis of prostate cancer was removed during hemiarthroplasty and transplanted into Rag2(-/-);γc(-/-) mice either intra-femorally or sub-cutaneously. Xenograft tumors that developed were analyzed for prostate cancer biomarker expression using RT-PCR and immunohistochemistry. Osteoblastic, osteolytic and mixed lesion formation was measured using micro-computed tomography (microCT). RESULTS: PCSD1 cells isolated directly from the patient formed tumors in all mice that were transplanted intra-femorally or sub-cutaneously into Rag2(-/-);γc(-/-) mice. Xenograft tumors expressed human prostate specific antigen (PSA) in RT-PCR and immunohistochemical analyses. PCSD1 tumors also expressed AR, NKX3.1, Keratins 8 and 18, and AMACR. Histologic and microCT analyses revealed that intra-femoral PCSD1 xenograft tumors formed mixed osteolytic and osteoblastic lesions. PCSD1 tumors have been serially passaged in mice as xenografts intra-femorally or sub-cutaneously as well as grown in culture. CONCLUSIONS: PCSD1 xenografts tumors were characterized as advanced, luminal epithelial prostate cancer from a bone metastasis using RT-PCR and immunohistochemical biomarker analyses. PCSD1 intra-femoral xenografts formed mixed osteoblastic/osteolytic lesions that closely resembled the bone lesions in the patient. PCSD1 is a new primary prostate cancer bone metastasis-derived xenograft model to study metastatic disease in the bone and to develop novel therapies for inhibiting prostate cancer growth in the bone-niche.