Tomonori Yamaguchi

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.

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

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.MethodsA 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).ResultsPCSD1 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.ConclusionsPCSD1 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.

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.

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.

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.

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.

Abstract A43: Novel prostate cancer patient-derived xenograft models of bone metastatic castrate-resistant prostate cancer

Prostate cancer metastasis to bone occurs in 50-90% of men with advanced disease for which there is no cure. Bone metastasis leads to debilitating fractures and severe bone pain. It is associated with disease progression, therapy resistance, poor prognosis, and rapid decline. Androgen ablation therapy is standard of care for advanced prostate cancer, however, the role of androgens in bone metastatic prostate cancer is not understood. The effects of anti-androgens as seen on bone scans can also be inconsistent with the biochemical PSA response. There are few pre-clinical models to understand the interaction between the bone microenvironment and prostate cancer. It is essential to understand the unique interaction of prostate cancer with the bone environment and to develop novel therapies that target these pathways. Here we report the development of novel patient-derived intra-femoral xenograft models of prostate bone metastatic cancer. METHODS: Surgical prostate cancer bone metastasis specimens were transplanted by direct injection into the femurs of Rag2-/-γc-/- mice or sub-cutaneously into the right flank. Patient-derived xenograft (PDX) tumors that grew out were analyzed for prostate cancer biomarker expression using quantitative RT-PCR and immunohistochemistry. Bone lesion formation was measured using micro-computed tomography (μCT). RESULTS: Prostate cancer surgical bone metastasis specimens have been collected from which we have established new serially transplantable, prostate cancer bone metastasis xenograft models – PCSD1, PCSD4 and PCSD5. PCSD1 (Prostate Cancer San Diego 1) was molecularly characterized as advanced, luminal epithelial-type prostate cancer. PCSD1 intra-femoral xenografts formed mixed osteoblastic/osteolytic lesions that closely mimicked those of the patient. Treatment with the anti-androgen, bicalutamide, did not inhibit intra-femoral PCSD1 xenograft growth although there was a decrease in PSA as seen in some patients treated with anti-androgen who had discordant PSA and bone scan tests. CONCLUSION: PCSD1, PCSD4 and PCSD5 are new patient-derived prostate cancer bone metastasis-derived xenograft models. PCSD1 xenograft model closely recapitulates the mixed osteolytic/osteoblastic bone metastatic lesions seen in patients, and we are using it to develop novel therapies for inhibiting prostate cancer growth in the bone-niche. Citation Format: Christina Jamieson, Christina Wu, Amy Strasner, Jason R. Woo, Michelle Muldong, Young B. Jeong, Michael A. Liss, Omer Raheem, Tomonori Yamaguchi, Heather Leu, Deborah Marshall, Sheldon Morris, Nicholas A. Cacalano, Koichi Masuda, Catriona H.M. Jamieson, Anna A. Kulidjian, Christopher J. Kane. Novel prostate cancer patient-derived xenograft models of bone metastatic castrate-resistant prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A43.

Evaluation of Autogenous Engineered Septal Cartilage Grafts in Rabbits: A Minimally Invasive Preclinical Model

Objectives: 1) Evaluate safety of autogenous engineered septal neocartilage grafts. 2) Compare properties of implanted grafts versus in vitro controls. Methods: Constructs were fabricated from septal cartilage and serum harvested from adult rabbits. Constructs were cultured in vitro or implanted on the nasal dorsum as autogenous grafts for 30 or 60 days. Rabbits were monitored for local and systemic complications. Histological, biochemical, and biomechanical properties of implanted and in vitro constructs were evaluated and compared. Results: No systemic or serious local complications were observed. Implanted constructs contained more DNA (p=0.00, p=0.003) and a higher ratio of total collagen to GAG (p=0.015, p=0.091) when compared with in vitro controls. Confined compressive moduli were also higher in implanted constructs when compared with in vitro controls (p=0.092, p=0.122). No significant differences were detected between the 30 and 60 day cohorts. Implanted constructs displayed resorption rates of 20-45 percent. Calcium deposition in implanted constructs was observed using alizarin red histochemistry and microCT analyses. Conclusions: Autogenous engineered septal cartilage grafts were well tolerated. As seen in experiments with athymic mice, implanted constructs accumulated more DNA and had a higher ratio of total collagen to GAG when compared with in vitro controls. Confined compressive moduli were also higher in implanted constructs. Implanted constructs displayed resorption rates similar to previously published studies using autogenous implants of native cartilage. The basis for observed calcification in implanted constructs and its effect on long-term graft efficacy is unknown at this time and will be a focus of future studies.