Co-evolution of breast-to-brain metastasis and neural progenitor cells.

Brain colonization by metastatic tumor cells offers a unique opportunity to investigate microenvironmental influences on the neoplastic process. The bi-directional interplay of breast cancer cells (mesodermal origin) and brain cells (neuroectodermal origin) is poorly understood and rarely investigated. In our patients undergoing neurosurgical resection of breast-to-brain metastases, specimens from the tumor/brain interface exhibited increased active gliosis as previously described. In addition, our histological characterization revealed infiltration of neural progenitor cells (NPCs) both outside and inside the tumor margin, leading us to investigate the cellular and molecular interactions between NPCs and metastases. Since signaling by the TGF-ß superfamily is involved in both developmental neurobiology and breast cancer pathogenesis, we examined the role of these proteins in the context of brain metastases. The brain-metastatic breast cancer cell line MDA-MB-231Br (231Br) expressed BMP-2 at significantly higher levels compared to its matched primary breast cancer cell line MDA-MB-231 (231). Co-culturing was used to examine bi-directional cellular effects and the relevance of BMP-2 overexpression. When co-cultured with NPCs, 231 (primary) tumor cells failed to proliferate over 15 days. However, 231Br (brain metastatic) tumor cells co-cultured with NPCs escaped growth inhibition after day 5 and proliferated, occurring in parallel with NPC differentiation into astrocytes. Using shRNA and gene knock-in, we then demonstrated BMP-2 secreted by 231Br cells mediated NPC differentiation into astrocytes and concomitant tumor cell proliferation in vitro. In xenografts, overexpression of BMP-2 in primary breast cancer cells significantly enhanced their ability to engraft and colonize the brain, thereby creating a metastatic phenotype. Conversely, BMP-2 knockdown in metastatic breast cancer cells significantly diminished engraftment and colonization. The results suggest metastatic tumor cells create a permissive neural niche by steering NPC differentiation toward astrocytes through paracrine BMP-2 signaling.

Lineage mapping and characterization of the native progenitor population in cellular allograft.

BACKGROUND CONTEXT: The gold standard for bone grafting remains the autograft. However, the attractiveness of autograft is counterbalanced by donor site morbidity. To mimic autograft-and its fundamental properties of osteoconductivity, osteoinductivity, and osteogenicity-novel bone grafting materials such as cellular allograft (Osteocel Plus) are composed of allograft in which the progenitor cells are preserved. However, the true identity of these cells remains obscure largely due to the lack of specific bona fide antigenic markers for stem versus progenitor cells. PURPOSE: To characterize the stem and progenitor population in cellular allograft, Osteocel Plus. STUDY DESIGN: To determine whether cells endogenous to a cellular allograft undergo extensive self-renewal (a functional hallmark of stem cells), we employed a novel use of lineage mapping using a modern and refined replication incompetent lentiviral library with high complexity to uniquely label single cells with indelible genetic tags faithfully passed on to all progeny, allowing identification of highly proliferative clones. We used genetic and proteomic profiling as well as functional assays to show that these cells are capable of multipotential differentiation (the second functional hallmark of stem cells). Use of these two functional hallmarks enabled us to establish the existence of a stem and progenitor cell population in cellular allografts. METHODS: Specifically, we employed (1) cellular dissociation and (2) in vitro expansion and differentiation capacity of cells released from cellular allograft. We determined differential gene expression profiling of a bona fide human mesenchymal stem cell line and cells from cellular allograft using focused PCR arrays mesenchymal stem cell (MSC) and osteogenesis associated. Proteomic profiling of cells from cellular allograft was performed using (1) immunofluorescence for BMP-2, Runx2 SMADs, CD44, Stro-1, Collagen, RANKL, Osterix Osteocalcin, and Ki67; (2) flow cytometry for Ki67, CD44, Stro-1, Thy1, CD146, and Osteocalcin; and (3) enzyme-linked immunosorbent assays (ELISA) for BMP-2, Osteocalcin, RANKL, Osteoprotegrin, and Osteocalcin. Clonal analysis of cells from cellular allograft was performed utilizing advance lentivirus lineage mapping techniques and massive parallel sequencing. Alizarin Red, Alcian Blue, and Oil red O staining assessed tripotential differentiation capacity. RESULTS: Serial trypsinization of allograft cellular bone matrix yielded approximately 1×105 cells per mL with viability greater than 90%. Cells expressed a panel of 84 MSC-associated genes in a pattern similar to but not identical to pure MSCs; specifically, 59 of 84 genes showed less than a 2.5-fold change in both cell types. Protein analysis showed that cellular allograft -derived cells maintained in nondifferentiation media expressed the early osteo-progenitor markers BMP-2, SMADs, and Runx2. Corresponding flow cytometry data for MSC markers revealed the presence of Stro-1 (49%), CD44 (99%), CD90 (42%), and CD146 (97%). Lineage mapping indicated that 62% of clones persisted and generated progeny through 10 passages, strongly suggesting the presence of bona fide stem cells. Passage 10 clones also exhibited tri-lineage differentiation capacity into osteogenic (Alizarin Red with H&E counterstain), chondrogenic (Alcian Blue), and adipogenic (Oil red O). Cells that did not proliferate through 10 passages presumably differentiated along an osteo-progenitor lineage. CONCLUSION: These data indicate that cellular allograft (Osteocel Plus) contains a heterogeneous population of cells with most cells demonstrating the capacity for extensive self-renewal and multipotential differentiation, which are hallmarks of stem cells. Whether stem cell-enriched allografts function comparably to autograft will require further studies, and their efficacy in facilitating arthrodesis will depend on randomized clinical studies.

Pediatric neurosurgery outreach: sustainability appraisal of a targeted teaching model in Kiev, Ukraine.

PURPOSE: This study evaluates the efficacy of operative skill transfer in the context of targeted pediatric outreach missions completed in Kiev, Ukraine. In addition the ability to create sustainable surgical care improvement is investigated as an efficient method to improve global surgical care. METHODS: Three 1-week targeted neurosurgical missions were performed (2005-2007) to teach neuroendoscopy, which included donation of the necessary surgical equipment, so the host team can deliver newly acquired surgical skills to their citizens after the visiting mission team departs. The neuroendoscopy data for the 4 years after the final mission in 2007 was obtained. RESULTS: After performing pediatric neurosurgery missions in 2005-2007, with a focus on teaching neuroendoscopy, the host team demonstrated the sustainability of our educational efforts in the subsequent 4 years by performing cases independently for their citizens. Since the last targeted mission of 2007, neuroendoscopic procedures have continued to be performed by the trained host surgeons. In 2008, 33 cases were performed. In 2009 and 2010, 29 and 22 cases were completed, respectively. In 2011, local neurosurgeons accomplished 27 cases. To date, a total of 111 operations have been performed over the past 4 years independent of any visiting team, illustrating the sustainability of educational efforts of the missions in 2005-2007. CONCLUSIONS: Effective operative skill transfer to host neurosurgeons can be accomplished with limited international team visits using a targeted approach that minimizes expenditures on personnel and capital. With the priority being teaching of an operative technique, as opposed to perennially performing operations by a visiting mission team, sustainable surgical care was achieved and perpetuated after missions officially concluded.

Targeted neurosurgical outreach: 5-year follow-up of operative skill transfer and sustainable care in Lima, Peru.

PURPOSE: This study evaluates the efficacy of operative skill transfer in the context of targeted pediatric outreach missions. In addition, the ability to implement surgical care improvements that are sustainable is investigated. METHODS: Three 1-week targeted neurosurgical missions were performed (2004-2006) to teach neuroendoscopy, which included donation of the necessary equipment so newly acquired surgical skills could be performed by local neurosurgeons in between and after the departure of the mission team. After the targeted missions were completed, 5 years of neuroendoscopy case follow-up data were obtained. RESULTS: After performing pediatric neurosurgery missions in 2004-2006, with a focus on teaching neuroendoscopy, the host team demonstrated the sustainability of our didactic efforts in the subsequent 5 years by performing cases independently for their citizens. To date, a total of 196 operations have been performed in the past 5 years independent of any visiting team. CONCLUSIONS: Effective operative skill transfer to host neurosurgeons can be accomplished with limited international team visits utilizing a targeted approach that minimizes expenditures on personnel and capital. With the priority being teaching of an operative technique, as opposed to perennially performing operations by the mission team, sustainable surgical care was achieved after missions officially concluded.

Progenitor cells: role and usage in bone tissue engineering approaches for spinal fusion.

Advancement of in vitro osteogenesis, or the production of bone, is a complex process that has significant clinical implications. Surgical intervention of several spinal disorders entails decompression of the spinal cord and nerves which can lead to subsequent biomechanical instability of the spine. Spinal arthrodesis (fusion) is often required to correct this instability and necessary to eliminate the resulting pathological motion of vertebral segments. Therefore, the achievement of proper spinal fusion, is a critical determinant of treatment efficacy. This chapter focuses on the molecular and cellular components that are involved in bone growth and healing. Mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) are the precursor cells essential for the formation of the five different types of bone cells: osteoprogenitor cells, osteoblasts, osteoclasts, osteocytes and lining cells. Similarly, endothelial progenitor cells (EPCs) differentiate into endothelial cells, which are essential in angiogenesis and neovascularization. MSCs tri-lineage potential (osteogenic, chondrogenic and adipogenic lineages) have made them the focus of most experimental approaches. Here, we describe their individual roles, as well as pose novel concepts on how their collective role may be the optimal strategy to improve upon in vitro osteogenesis and whether this could also be translated to improved bone formation in vivo. Further, we discuss the various molecular markers that are available for cell identification and the tissue engineering strategies that could replicate the osteoinductive, osteoconductive and osteoproductive milieuthat is available in autograft. Finally, we present a broad primer on the possible integration of cellular, molecular and tissue engineering strategies to improve osteogenesis and the future trends that may bring the promise seen in the laboratory to fruition in preclinical animal models.

Animal models of neurological disease.

The use of animal models to study human pathology has proved valuable in a number of fields. Animal models of neurological disease have successfully and accurately recreated many aspects of human illness allowing for in-depth study ofneuropathophysiology. These models have been the source of a plethora of information, such as the importance of certain molecular mechanisms and genetic contributions in neurological disease. Additionally, animal models have been utilized in the discovery and testing of possible therapeutic treatments. Although most neurological diseases are still not yet completely understood and reliable treatment is lacking, animal models provide a major step in the right direction.

Biological horizons for targeting brain malignancy.

Though currently available clinical treatments and therapies have clearly extended the survival of patients with brain tumors, many of these advances are short lived, particularly with respect to high grade gliomas such as glioblastoma multiforme. The missing link to an efficacious treatment of high grade gliomas is a more complete understanding of the basic molecular and cellular origin of brain tumors. However, new discoveries of stem cell and developmental neurobiology have now borne the cancer stem cell hypothesis, drawing off of intriguing similarities between benign and malignant cells within the central nervous system. Investigation of cancer stem cell hypothesis and brain tumor propagation is the current frontier of stem cell and cancer biology. Neurosurgery is also watching closely this promising new area of focus. "Molecular neurosurgery", glioma treatments involving biologics using neural stem cells to target the cancer at the level of individual migratory cell, is a rapidly evolving field. This coming progression of applied cancer stem cell research, coupled with current modalities, promises more comprehensive brain cancer interventions.

Ballistics for the neurosurgeon.

Craniocerebral injuries from ballistic projectiles are qualitatively different from injuries in unconfined soft tissue with similar impact. Penetrating and nonpenetrating ballistic injuries are influenced not only by the physical properties of the projectile, but also by its ballistics. Ballistics provides information on the motion of projectiles while in the gun barrel, the trajectory of the projectile in air, and the behavior of the projectile on reaching its target. This basic knowledge can be applied to better understand the ultimate craniocerebral consequences of ballistic head injuries.