Margarete K. Akens

Hierarchical Model of Fibrillar Collagen Organization for Interpreting the Second-Order Susceptibility Tensors in Biological Tissue

The second-order nonlinear polarization properties of fibrillar collagen in various rat tissues (vertebrae, tibia, tail tendon, dermis, and cornea) are investigated with polarization-dependent second-harmonic generation (P-SHG) microscopy. Three parameters are extracted: the second-order susceptibility ratio, R = [Formula: see text] ; a measure of the fibril distribution asymmetry, |A|; and the weighted-average fibril orientation, . A hierarchical organizational model of fibrillar collagen is developed to interpret the second-harmonic generation polarization properties. Highlights of the model include: collagen type (e.g., type-I, type-II), fibril internal structure (e.g., straight, constant-tilt), and fibril architecture (e.g., parallel fibers, intertwined, lamellae). Quantifiable differences in internal structure and architecture of the fibrils are observed. Occurrence histograms of R and |A| distinguished parallel from nonparallel fibril distributions. Parallel distributions possessed low parameter values and variability, whereas nonparallel distributions displayed an increase in values and variability. From the P-SHG parameters of vertebrae tissue, a three-dimensional reconstruction of lamellae of intervertebral disk is presented.

The effect of versican G3 domain on local breast cancer invasiveness and bony metastasis

IntroductionIncreased versican expression has been associated with local breast cancer invasiveness and a more aggressive tumor phenotype. The cellular mechanisms are not fully understood and this study evaluated versican G3 domain with its EGF-like motifs in influencing tumor invasion and metastasis.MethodsOne recombinant construct was synthesized (a signal peptide for product secretion and the versican G3 domain). The construct was stably transfected into human breast carcinoma MT-1 cells. Cell viability in vitro was evaluated in low serum and serum starvation conditions. In vivo study of tumor growth was evaluated in a nude mouse model. G3 effects on rodent vascular endothelial cells were evaluated in vitro on cell survival, apoptosis, migration, and vascular formation. The effects of VEGF, fibronectin, and G3 on vascular formation were examined. An intracardiac injection model of metastatic human breast carcinoma tested the effect of G3 on distant bony and soft tissue metastasis. Analysis of metastatic burden included histology, radiographs, and micro-CT quantification of osteolysis.ResultsA greater viability of cancer cells was observed in low serum and serum-free conditions in the presence of versican G3. Larger subcutaneous tumors were obtained in the G3 group following tumor cell injection into CD1 mice. G3 induced a greater degree of rodent vascular endothelial cell proliferation and migration in vitro. Simultaneous presence of fibronectin, VEGF, and G3 promoted endothelial cell migration in wound-healing assays as compared to the treatments containing none, one or two of these molecules. Systemic tumor burden to distant bony and soft tissue metastatic sites was greater in the G3 group using the intracardiac injection metastatic modelConclusionVersican G3 domain appears to be important in local and systemic tumor invasiveness of human breast cancer. Effects include enhancing cell viability, proliferation, migration and enhancing local tumor growth. Potential effects on angiogenesis include enhancing vascular endothelial proliferation, migration, and vessel formation. The interactions between tumor cells, surrounding stromal components and neo-vascularization in breast cancer may include interactions with VEGF and fibronectin. The propensity of versican G3 to influence tumor invasion to bone and the mechanisms of G3 mediated osteolysis warrants ongoing studies.

Assessment of biocompatibility and initial evaluation of genipin cross-linked elastin-like polypeptides in the treatment of an osteochondral knee defect in rabbits

Polypeptides based on the alternating hydrophobic and cross-linking domain structure of human elastin are capable of undergoing self-assembly to produce polymeric matrices with unique biological and mechanical properties. Here, we test the initial feasibility of using a genipin cross-linked elastin-based material as an acellular plug in the treatment of an osteochondral defect in the rabbit knee. Full-thickness defects in the weight-bearing surface of the medial femoral condyle in 18 New Zealand White rabbits were surgically produced and press fitted with cylindrical pads composed of genipin cross-linked elastin-like polypeptides, with identical wounds in the opposite knee left untreated as controls. The biocompatibility of the material, overall wound healing and regeneration of subchondral tissue was assessed at 2, 4 and 6weeks by histological evaluation, synovial fluid analysis and microcomputerized tomography scanning. Histological analysis revealed the regeneration of subchondral bone at the periphery of the material, with evidence of hyaline-like overgrowth across the apical surface in 11/16 cases. Pads developed tight contacts with host tissue and appeared completely biocompatible, with no evidence of localized immune response or increased inflammation compared to controls. The material was stable to 6weeks, with an aggregate elastic modulus calculated at approximately 470kPa when tested under confined compression. Further studies are required to assess material degradation over time and long-term replacement with repair tissue.

Imaging of specific activation of photodynamic molecular beacons in breast cancer vertebral metastases.

Breast cancer is the second leading cause of cancer-related death in women. Approximately 85% of patients with advanced cases will develop spinal metastases. The vertebral column is the most common site of breast cancer metastases, where overexpression of matrix metalloproteinases (MMPs) promotes the spread of cancer. Current therapies have significant limitations due to the high associated risk of damaging the spinal cord. An attractive alternative is photodynamic therapy providing noninvasive and site-selective treatment. However, current photosensitizers are limited by their nonspecific accumulation. Photodynamic molecular beacons (PP(MMP)B), activated by MMPs, offer another level of PDT selectivity and image-guidance preserving criticial tissues, specifically the spinal cord. Metastatic human breast carcinoma cells, MT-1, were used to model the metastatic behavior of spinal lesions. In vitro and in vivo evidence demonstrates MMP specific activation of PP(MMP)B in MT-1 cells. Using a clinically relevant metastatic model, fluorescent imaging establishes the specific activation of PP(MMP)B by vertebral metastases versus normal tissue (i.e., spinal cord) demonstrating the specificity of these beacons. Here, we validate that the metastasis-selective mechanism of PP(MMP)Bs can specifically image breast cancer vertebral metastases, thereby differentiating tumor and healthy tissue.

Beyond bisphosphonates: photodynamic therapy structurally augments metastatically involved vertebrae and destroys tumor tissue

Breast cancer patients commonly develop metastases in the spine, which compromises its mechanical stability and can lead to skeletal related events. The current clinical standard of treatment includes the administration of systemic bisphosphonates (BP) to reduce metastatically induced bone destruction. However, response to BPs can vary both within and between patients, which motivates the need for additional treatment options for spinal metastasis. Photodynamic therapy (PDT) has been shown to be effective at treating metastatic lesions secondary to breast cancer in an athymic rat model, and is proposed as a treatment for spinal metastasis. The objective of this study was to determine the effect of PDT, alone or in combination with previously administered systemic BPs, on the structural and mechanical integrity of both healthy and metastatically involved vertebrae. Human breast carcinoma cells (MT-1) were inoculated into athymic rats (day 0). At 14xa0days, a single PDT treatment was administered, with and without previous BP treatment at day 7. In addition to causing tumor necrosis in metastatically involved vertebrae, PDT significantly reduced bone loss, resulting in strengthening of the vertebrae compared to untreated controls. Combined treatment with BPxa0+xa0PDT further enhanced bone architecture and strength in both metastatically involved and healthy bone. Overall, the ability of PDT to both ablate malignant tissue and improve the structural integrity of vertebral bone motivates its consideration as a local minimally invasive treatment for spinal metastasis secondary to breast cancer.

Defining the therapeutic window of vertebral photodynamic therapy in a murine pre-clinical model of breast cancer metastasis using the photosensitizer BPD-MA (Verteporfin)

Breast cancer is known to cause metastatic lesions in the bone, which can lead to skeletal-related events. Currently, radiation therapy and surgery are the treatment of choice, but the success rate varies and additional adjuncts are desirable. Photodynamic therapy (PDT) has been applied successfully as a non-radiative treatment for numerous cancers. Earlier work has shown that the athymic rat model is suitable to investigate the effect of PDT on bone metastasis and benzoporphyrin-derivative monoacid ring A (BPD-MA; verteporfin) has been shown to be a selective photosensitizer. The aim of this study was to define the therapeutic window of photosensitizer with regard to drug and light dose. Human breast carcinoma cells (MT-1)—stable transfected with the luciferase gene—were injected intra-cardiacally into athymic rats. At 14xa0days, the largest vertebral lesion by bioluminescence imaging was targeted for single treatment PDT. A drug escalating-de-escalating scheme was used (starting drug dose and light energy of 0.2xa0mg/kg and 50xa0J, respectively). Outcomes included 48xa0h post-treatment bioluminescence of remaining viable tumour, histomorphometric assessment of tumour burden, and neurologic evaluation. The region of effect by bioluminescence and histology increased with increasing drug dose and light energy. A safe and effective drug-light dose combination in this model appears to be 0.5xa0mg/kg BPD-MA and applied light energy of less than 50xa0J for the thoracic spine and 1.0xa0mg/kg and 75xa0J for the lumbar spine. For translation to clinical use, it is an advantage that BPD-MA (verteporfin), a second-generation photosensitizer, is already approved to treat age-related macular degeneration. Overall, PDT represents an exciting potential new minimally-invasive local, safe and effective therapy in the management of patients with spinal metastases.

Photodynamic Therapy of Vertebral Metastases: Evaluating Tumor-to-Neural Tissue Uptake of BPD-MA and ALA-PpIX in a Murine Model of Metastatic Human Breast Carcinoma †

Photodynamic therapy has been successfully applied to numerous cancers. Its potential to treat cancer metastases in the spine has been demonstrated previously in a preclinical animal model. The aim of this study was to test two photosensitizers, benzoporphyrin‐derivative monoacid ring A (BPD‐MA) and by 5‐aminolevulinic acid (5‐ALA)‐induced protoporphyrin IX (PpIX), for their potential use to treat bony metastases. The difference in photosensitizer concentration in the spinal cord and the surrounding tumor‐bearing vertebrae was of particular interest to assess the risk of potential collateral damage to the spinal cord. Vertebral metastases in a rat model were generated by intracardiac injection of human breast cancer cells. When tumor growth was confirmed, photosensitizers were injected systemically and the animals were euthanized at different time points. The following tissues were harvested: liver, kidney, ovaries, appendicular bone, spinal cord and lumbar vertebrae. Photosensitizer tissue concentration of BPD‐MA or PpIX was determined by fluorescence spectrophotometry. In contrast to BPD‐MA, ALA‐PpIX did not demonstrate an appreciable difference in the uptake ratio in tumor‐bearing vertebrae compared to spinal cord. The highest ratio for BPD‐MA concentration was found 15u2003min after injection, which can be recommended for therapy in this model.

Quantification of the effect of osteolytic metastases on bone strain within whole vertebrae using image registration.

The vertebral column is the most frequent site of metastatic involvement of the skeleton with up to 1/3 of all cancer patients developing spinal metastases. Longer survival times for patients, particularly secondary to breast cancer, have increased the need for better understanding the impact of skeletal metastases on structural stability. This study aims to apply image registration to calculate strain distributions in metastatically involved rodent vertebrae utilizing µCT imaging. Osteolytic vertebral lesions were developed in five rnu/rnu rats 2–3 weeks post intracardiac injection with MT‐1 human breast cancer cells. An image registration algorithm was used to calculate and compare strain fields due to axial compressive loading in metastatically involved and control vertebrae. Tumor‐bearing vertebrae had greatly increased compressive strains, double the magnitude of strain compared to control vertebrae (pu2009=u20090.01). Qualitatively strain concentrated within the growth plates in both tumor bearing and control vertebrae. Most interesting was the presence of strain concentrations at the dorsal wall in metastatically involved vertebrae, suggesting structural instability. Strain distributions, quantified by image registration were consistent with known consequences of lytic involvement. Metastatically involved vertebrae had greater strain magnitude than control vertebrae. Strain concentrations at the dorsal wall in only the metastatic vertebrae, were consistent with higher incidence of burst fracture secondary to this pathology. Future use of image registration of whole vertebrae will allow focused examination of the efficacy of targeted and systemic treatments in reducing strains and the related risk of fracture in pathologic bones under simple and complex loading.

Evaluation of a bipolar-cooled radiofrequency device for ablation of bone metastases: preclinical assessment in porcine vertebrae

BACKGROUND CONTEXTnCancer spread to the spine affects bone stability and can lead to pathologic fracture and neurologic impairment. Radiofrequency ablation (RFA) recently has gained popularity in treating skeletal tumors. Conventional RFA devices use a monopolar design, which limits the ability to comprehensively treat large tumors in bony tissues and may pose risks to adjacent critical normal neurologic tissues when applied to vertebrae. New bipolar-cooled radiofrequency (BCRF) may generate larger controlled lesions without the same degree of risk to adjacent structures.nnnPURPOSEnThe purpose of this study was to evaluate the feasibility, efficacy, and safety of RFA with the use of a new bone-specific, BCRF probe in a porcine vertebral model and to evaluate the ability of magnetic resonance (MR) imaging to represent histologic outcomes of RFA treatment.nnnSTUDY DESIGNnBasic science: preclinical in vivo study.nnnMETHODSnRFA was evaluated in three noncontiguous lumbar vertebrae in six Yorkshire pigs (25-30 kg). Via a transpedicular approach for probe placement, two vertebrae received BCRF treatment and one vertebrae served as a sham control. MR imaging and neurological assessments were conducted pre- and posttreatment as well as immediately before animal sacrifice (n=3 at day 0, n=3 at day 14). MR ablation zones were compared with hematoxylin and eosin-stained histological sections.nnnRESULTSnWith BCRF, large reproducible zones of ablation were achieved, confined within the vertebrae, without damage to adjacent tissues or the spinal cord. All animals demonstrated normal consistent neurologic behavior pre- and posttreatment. External tissue temperatures around targeted vertebrae were not increased. MR imaging after 14 days was more effective in demonstrating ablation effects than images on day 0, with radiologic findings most apparent on T2-weighted sequences. Histologic analysis of samples corresponded well to the zones of ablation observed on MR images (R=0.9, p<.01).nnnCONCLUSIONSnThe study demonstrated feasibility, safety, and effectiveness of BCRF ablation of vertebral bone. This motivates ongoing preclinical evaluation in diseased models to further explore the potential for its use in clinical treatment of metastatic vertebrae.

Evaluation of a novel poly N-acetyl glucosamine (pGlcNAc) hydrogel for treatment of the degenerating intervertebral disc

AIMSnThe early stages of degenerative disc disease (DDD) primarily affect the disc nucleus pulposus (NP). Tissue-engineered strategies may enhance intervertebral disc (IVD) functionality. The aim of this study was to develop and evaluate a novel deacetylated poly-N-acetyl glucosamine (pGlcNAc) hydrogel characterizing its biochemical effect on human IVD cells as well as material biomechanical properties.nnnMAIN METHODSnA novel deacetylated derivative of a marine diatom-derived glycosaminoglycan was developed into a hydrogel formulation as a potential therapy to treat degenerating IVD NP. In vitro biochemical studies were conducted using primary human disc cell cultures to evaluate cell viability, metabolic activity, proteoglycan and extracellular matrix protein expression. The biomechanical hydration kinetics and viscoelastic behavior of the hydrogel were determined and compared with the behavior of human lumbar NP.nnnKEY FINDINGSnDisc cell viability, metabolic activity, and proteoglycan content of the treated cells were observed to be significantly greater in experimental samples when compared to untreated control groups. RT-PCR and immunohistochemical data corroborated the expression of characteristic NP disc markers, aggrecan and type II collagen in cultured cells. Rheological data demonstrated that the elastic component of the hydrogel dominated the viscous component over a frequency range of 0.1 to 15.85rad/s. Of several formulations evaluated, a sulphated, deactylated derivative of the nanofiber derived pGlcNAc hydrogel demonstrated the most robust biologic effects on cell viability, metabolic activity, and proteoglycan expression.nnnSIGNIFICANCEnThis in vitro study using human disc cells demonstrates that a sulphated deacetylated glycosaminoglycan derivative hydrogel possesses promising characteristics motivating further evaluation as a potential therapy for NP degeneration.