Preclinical animal models for segmental bone defect research and tissue engineering

Currently, well-established clinical therapeutic approaches for bone reconstruction are restricted to the transplantation of autografts and allografts, and the implantation of metal devices or ceramic-based implants to assist bone regeneration. Bone grafts possess osteoconductive and osteoinductive properties, however they are limited in access and availability and associated with donor site morbidity, haemorrhage, risk of infection, insufficient transplant integration, graft devitalisation, and subsequent resorption resulting in decreased mechanical stability. As a result, recent research focuses on the development of alternative therapeutic concepts. Analysing the tissue engineering literature it can be concluded that bone regeneration has become a focus area in the field. Hence, a considerable number of research groups and commercial entities work on the development of tissue engineered constructs for bone regeneration. However, bench to bedside translations are still infrequent as the process towards approval by regulatory bodies is protracted and costly, requiring both comprehensive in vitro and in vivo studies. In translational orthopaedic research, the utilisation of large preclinical animal models is a conditio sine qua non. Consequently, to allow comparison between different studies and their outcomes, it is essential that animal models, fixation devices, surgical procedures and methods of taking measurements are well standardized to produce reliable data pools as a base for further research directions. The following chapter reviews animal models of the weight-bearing lower extremity utilized in the field which include representations of fracture-healing, segmental bone defects, and fracture non-unions.

In vitro primary cell culture as a physiologically relevant method for preclinical testing of human oncolytic adenovirus

Ad[I/PPT-E1A] is an oncolytic adenovirus that specifically kills prostate cells via restricted replication by a prostate-specific regulatory element. Off-target replication of oncolytic adenoviruses would have serious clinical consequences. As a proposed ex vivo test, we describe the assessment of the specificity of Ad[I/PPT-E1A] viral cytotoxicity and replication in human nonprostate primary cells. Four primary nonprostate cell types were selected to mimic the effects of potential in vivo exposure to Ad[I/PPT-E1A] virus: bronchial epithelial cells, urothelial cells, vascular endothelial cells, and hepatocytes. Primary cells were analyzed for Ad[I/PPT-E1A] viral cytotoxicity in MTS assays, and viral replication was determined by hexon titer immunostaining assays to quantify viral hexon protein. The results revealed that at an extreme multiplicity of infection of 500, unlikely to be achieved in vivo, Ad[I/PPT-E1A] virus showed no significant cytotoxic effects in the nonprostate primary cell types apart from the hepatocytes. Transmission electron microscopy studies revealed high levels of Ad[I/PPT-E1A] sequestered in the cytoplasm of these cells. Adenoviral green fluorescent protein reporter studies showed no evidence for nuclear localization, suggesting that the cytotoxic effects of Ad[I/PPT-E1A] in human primary hepatocytes are related to viral sequestration. Also, hepatocytes had increased amounts of coxsackie adenovirus receptor surface protein. Active viral replication was only observed in the permissive primary prostate cells and LNCaP prostate cell line, and was not evident in any of the other nonprostate cells types tested, confirming the specificity of Ad[I/PPT-E1A]. Thus, using a relevant panel of primary human cells provides a convenient and alternative preclinical assay for examining the specificity of conditionally replicating oncolytic adenoviruses in vivo.

PSMA-targeting iron oxide magnetic nanoparticles enhance MRI of preclinical prostate cancer

Aim Evaluate potential of newly-developed, biocompatible iron oxide magnetic nanoparticles (MNPs) conjugated with J591, an antibody to an extracellular epitope of prostate specific membrane antigen (PSMA), to enhance MRI of prostate cancer (PCa). Materials & Methods Specific binding to PSMA by J591-MNP was investigated in vitro. MRI studies were performed on orthotopic tumor-bearing NOD.SCID mice 2h and 24hr after intravenous injection of J591-MNPs, or non-targeting MNPs. Results and Conclusions In vitro, MNPs did not affect PCa cell viability, and conjugation to J591 did not compromise antibody specificity and enhanced cellular iron uptake. In vivo, PSMA-targeting MNPs increased MR contrast of tumors, but not by non-targeting MNPs. This provides proof-of-concept that PSMA-targeting MNPs have potential to enhance MR detection/localization of PCa.,

Establishing prostate cancer patient derived xenografts: Lessons learned from older studies

Background Understanding the progression of prostate cancer to androgen-independence/castrate resistance and development of preclinical testing models are important for developing new prostate cancer therapies. This report describes studies performed 30 years ago, which demonstrate utility and shortfalls of xenografting to preclinical modeling. Methods We subcutaneously implanted male nude mice with small prostate cancer fragments from transurethral resection of the prostate (TURP) from 29 patients. Successful xenografts were passaged into new host mice. They were characterized using histology, immunohistochemistry for marker expression, flow cytometry for ploidy status, and in some cases by electron microscopy and response to testosterone. Two xenografts were karyotyped by G-banding. Results Tissues from 3/29 donors (10%) gave rise to xenografts that were successfully serially passaged in vivo. Two, (UCRU-PR-1, which subsequently was replaced by a mouse fibrosarcoma, and UCRU-PR-2, which combined epithelial and neuroendocrine features) have been described. UCRU-PR-4 line was a poorly differentiated prostatic adenocarcinoma derived from a patient who had undergone estrogen therapy and bilateral castration after his cancer relapsed. Histologically, this comprised diffusely infiltrating small acinar cell carcinoma with more solid aggregates of poorly differentiated adenocarcinoma. The xenografted line showed histology consistent with a poorly differentiated adenocarcinoma and stained positively for prostatic acid phosphatase (PAcP), epithelial membrane antigen (EMA) and the cytokeratin cocktail, CAM5.2, with weak staining for prostate specific antigen (PSA). The line failed to grow in female nude mice. Castration of three male nude mice after xenograft establishment resulted in cessation of growth in one, growth regression in another and transient growth in another, suggesting that some cells had retained androgen sensitivity. The karyotype (from passage 1) was 43–46, XY, dic(1;12)(p11;p11), der(3)t(3:?5)(q13;q13), -5, inv(7)(p15q35) x2, +add(7)(p13), add(8)(p22), add(11)(p14), add(13)(p11), add(20)(p12), -22, +r4[cp8]. Conclusions Xenografts provide a clinically relevant model of prostate cancer, although establishing serially transplantable prostate cancer patient derived xenografts is challenging and requires rigorous characterization and high quality starting material. Xenografting from advanced prostate cancer is more likely to succeed, as xenografting from well differentiated, localized disease has not been achieved in our experience. Strong translational correlations can be demonstrated between the clinical disease state and the xenograft model

Monitoring healing progression and characterizing the mechanical environment in preclinical models for bone tissue engineering

The treatment of large segmental bone defects remains a significant clinical challenge. Due to limitations surrounding the use of bone grafts, tissue-engineered constructs for the repair of large bone defects could offer an alternative. Before translation of any newly developed tissue engineering (TE) approach to the clinic, efficacy of the treatment must be shown in a validated preclinical large animal model. Currently, biomechanical testing, histology, and microcomputed tomography are performed to assess the quality and quantity of the regenerated bone. However, in vivo monitoring of the progression of healing is seldom performed, which could reveal important information regarding time to restoration of mechanical function and acceleration of regeneration. Furthermore, since the mechanical environment is known to influence bone regeneration, and limb loading of the animals can poorly be controlled, characterizing activity and load history could provide the ability to explain variability in the acquired data sets and potentially outliers based on abnormal loading. Many approaches have been devised to monitor the progression of healing and characterize the mechanical environment in fracture healing studies. In this article, we review previous methods and share results of recent work of our group toward developing and implementing a comprehensive biomechanical monitoring system to study bone regeneration in preclinical TE studies.

Malaria Vaccine Adjuvants: Latest Update and Challenges in Preclinical and Clinical Research

There is no malaria vaccine currently available, and the most advanced candidate has recently reported a modest 30% efficacy against clinical malaria. Although many efforts have been dedicated to achieve this goal, the research was mainly directed to identify antigenic targets. Nevertheless, the latest progresses on understanding how immune system works and the data recovered from vaccination studies have conferred to the vaccine formulation its deserved relevance. Additionally to the antigen nature, the manner in which it is presented (delivery adjuvants) as well as the immunostimulatory effect of the formulation components (immunostimulants) modulates the immune response elicited. Protective immunity against malaria requires the induction of humoral, antibody-dependent cellular inhibition (ADCI) and effector and memory cell responses. This review summarizes the status of adjuvants that have been or are being employed in the malaria vaccine development, focusing on the pharmaceutical and immunological aspects, as well as on their immunization outcomings at clinical and preclinical stages.

A LabVIEW Platform for Preclinical Imaging Using Digital Subtraction Angiography and Micro-CT.

CT and digital subtraction angiography (DSA) are ubiquitous in the clinic. Their preclinical equivalents are valuable imaging methods for studying disease models and treatment. We have developed a dual source/detector X-ray imaging system that we have used for both micro-CT and DSA studies in rodents. The control of such a complex imaging system requires substantial software development for which we use the graphical language LabVIEW (National Instruments, Austin, TX, USA). This paper focuses on a LabVIEW platform that we have developed to enable anatomical and functional imaging with micro-CT and DSA. Our LabVIEW applications integrate and control all the elements of our system including a dual source/detector X-ray system, a mechanical ventilator, a physiological monitor, and a power microinjector for the vascular delivery of X-ray contrast agents. Various applications allow cardiac- and respiratory-gated acquisitions for both DSA and micro-CT studies. Our results illustrate the application of DSA for cardiopulmonary studies and vascular imaging of the liver and coronary arteries. We also show how DSA can be used for functional imaging of the kidney. Finally, the power of 4D micro-CT imaging using both prospective and retrospective gating is shown for cardiac imaging.

Acoustic rhinometry in the dog:a novel large animal model for studies of nasal congestion

The aim of this project was to develop and pharmacologically characterize an experimental dog model of nasal congestion in which nasal patency is measured using acoustic rhinometry. Solubilized compound 48/80 (0.3-3.0%) was administered intranasally to thiopental anesthetized beagle dogs to elicit nasal congestion via localized mast cell degranulation. Compound 48/80-induced effects on parameters of nasal patency were studied in vehicle-treated animals, as well as in the same animals pretreated 2 hours earlier with oral d-pseudoephedrine or chlorpheniramine. Local mast cell degranulation caused a close-related decrease in nasal cavity volume and minimal cross-sectional area (Amin) together with a highly variable increase in nasal secretions. Maximal responses were seen at 90-120 minutes after 48/80 administration. Oral administration of the adrenergic agonist, d-pseudoephedrine (3.0 mg/kg), significantly antagonized all of the nasal effects of compound 48/80 (3.0%). In contrast, oral administration of the histamine H1 receptor antagonist chlorpheniramine (10 mg/kg) appeared to reduce the increased nasal secretions but was without effect on the compound 48/ 80-induced nasal congestion (i.e., volume and Amin). These results show the effectiveness of using acoustic rhinometry in this anesthetized dog model. The observations that compound 48/80-induced nasal congestion was prevented by d-pseudoephedrine pretreatment, but not by chlorpheniramine, suggest that this noninvasive model system may provide an effective tool with which to study the actions of decongestant drugs in preclinical investigations.

Development of human central nervous system 3D in vitro models for preclinical research

Neurological disorders are a major concern in modern societies, with increasing prevalence mainly related with the higher life expectancy. Most of the current available therapeutic options can only control and ameliorate the patients’ symptoms, often be-coming refractory over time. Therapeutic breakthroughs and advances have been hampered by the lack of accurate central nervous system (CNS) models. The develop-ment of these models allows the study of the disease onset/progression mechanisms and the preclinical evaluation of novel therapeutics. This has traditionally relied on genetically engineered animal models that often diverge considerably from the human phenotype (developmentally, anatomically and physiologically) and 2D in vitro cell models, which fail to recapitulate the characteristics of the target tissue (cell-cell and cell-matrix interactions, cell polarity). The in vitro recapitulation of CNS phenotypic and functional features requires the implementation of advanced culture strategies that enable to mimic the in vivo struc-tural and molecular complexity. Models based on differentiation of human neural stem cells (hNSC) in 3D cultures have great potential as complementary tools in preclinical research, bridging the gap between human clinical studies and animal models. This thesis aimed at the development of novel human 3D in vitro CNS models by integrat-ing agitation-based culture systems and a wide array of characterization tools. Neural differentiation of hNSC as 3D neurospheres was explored in Chapter 2. Here, it was demonstrated that human midbrain-derived neural progenitor cells from fetal origin (hmNPC) can generate complex tissue-like structures containing functional dopaminergic neurons, as well as astrocytes and oligodendrocytes. Chapter 3 focused on the development of cellular characterization assays for cell aggregates based on light-sheet fluorescence imaging systems, which resulted in increased spatial resolu-tion both for fixed samples or live imaging. The applicability of the developed human 3D cell model for preclinical research was explored in Chapter 4, evaluating the poten-tial of a viral vector candidate for gene therapy. The efficacy and safety of helper-dependent CAV-2 (hd-CAV-2) for gene delivery in human neurons was evaluated, demonstrating increased neuronal tropism, efficient transgene expression and minimal toxicity. The potential of human 3D in vitro CNS models to mimic brain functions was further addressed in Chapter 5. Exploring the use of 13C-labeled substrates and Nucle-ar Magnetic Resonance (NMR) spectroscopy tools, neural metabolic signatures were evaluated showing lineage-specific metabolic specialization and establishment of neu-ron-astrocytic shuttles upon differentiation. Chapter 6 focused on transferring the knowledge and strategies described in the previous chapters for the implementation of a scalable and robust process for the 3D differentiation of hNSC derived from human induced pluripotent stem cells (hiPSC). Here, software-controlled perfusion stirred-tank bioreactors were used as technological system to sustain cell aggregation and dif-ferentiation. The work developed in this thesis provides practical and versatile new in vitro ap-proaches to model the human brain. Furthermore, the culture strategies described herein can be further extended to other sources of neural phenotypes, including pa-tient-derived hiPSC. The combination of this 3D culture strategy with the implemented characterization methods represents a powerful complementary tool applicable in the drug discovery, toxicology and disease modeling.

A method of evaluating psychomotor tooth preparation skills in preclinical dental programs

Preclinical and clinical tooth important and significant aspect of preparation is an a dental student's education. The associated procedures rely heavily on the development of particular psychomotor skills. The most common format of instruction and evaluation in tooth preparation at many Dental Faculties, emphasizes the product (tooth preparation) and associates performance with characteristics of this product. This integrated study examines which skills should be developed and how a course of instruction can best be structured to develop the necessary skills. The skills which are identified are those necessary for tooth preparation are selected from a psychomotor taxonomy. The purpose of evaluating these skills is identified. Behavioral objectives are set for student performance and the advisability of establishing standards of performance is examined. After reviewing studies related to learning strategy for dental psychomotor the most suitable tasks as well as articles on instructor effectiveness a model is proposed. A pilot project at the University of Toronto, based on this proposed model is described. The paper concludes wi th a discussion of the implications of this proposed model.

Rapid and sensitive ultra-high-pressure liquid chromatography method for quantification of antichagasic benznidazole in plasma: application in a preclinical pharmacokinetic study

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Preclinical development of cancer vaccines

Triplex cell vaccine is a cancer immunopreventive cell vaccine that can prevent almost completely mammary tumor onset in HER-2/neu transgenic mice. A future translation of cancer immunoprevention from preclinical to clinical studies should take into account several aspects. The work reported in this thesis deals with the study of three of these aspects: vaccine schedule, activity in a therapeutic set-up and second-generation DNA vaccines. An important element in determining human acceptance and compliance of a treatment protocol is the number of vaccinations. In order to improve the vaccination schedule a minimal protocol was searched, i.e. a schedule consisting of a lower number of administrations than standard protocol but with a similar efficacy. A candidate optimal protocol was identified by the use of an in silico model, SimTriplex simulator. The in vivo test of this schedule in HER-2/neu transgenic mice only partially confirmed in silico predictions. This result shows that in silico models have the potential ability to aid in searching of optimal treatment protocols, provided that they will be further tuned on experimental data. As a further result this preclinical study highlighted that kinetic of antibody response plays a major role in determining cancer prevention, leading to the hypothesis of a threshold that must be reached rapidly and maintained lifetime. Early clinical trials would be performed in a therapeutic, rather than preventive, setting. Thus, the activity of Triplex vaccine was investigated against experimental lung metastases in HER-2/neu transgenic mice in order to evaluate if the immunopreventive Triplex vaccine could be effective also against a pre-existing tumor mass. This preclinical model of aggressive metastatic development showed that the vaccine was an efficient treatment also 4 for the cure of micrometastases. However the immune mechanisms activated against tumor mass were not antibody dependent, i.e. different from those preventing the onset of primary mammary carcinoma. DNA vaccines could be more easily used than cellular ones. A second generation of Triplex vaccine based on DNA plasmids was evaluated in an aggressive preclinical model (BALBp53neu female mice) and compared with the preventive ability of cellular Triplex vaccine. It was observed that Triplex DNA vaccine was as effective as Triplex cell vaccine, exploiting a more restricted immune stimulation.

Rag2-/-;gammac-/- immunodeficient mice, a new preclinical model to study antitumor approaches

Animal models have been relevant to study the molecular mechanisms of cancer and to develop new antitumor agents. Anyway, the huge divergence in mouse and human evolution made difficult the translation of the gained achievements in preclinical mouse based studies. The generation of clinically relevant murine models requires their humanization both concerning the creation of transgenic models and the generation of humanized mice in which to engraft a functional human immune system, and reproduce the physiological effects and molecular mechanisms of growth and metastasization of human tumors. In particular, the availability of genotypically stable immunodepressed mice able to accept tumor injection and allow human tumor growth and metastasization would be important to develop anti-tumor and anti-metastatic strategies. Recently, Rag2-/-;gammac-/- mice, double knockout for genes involved in lymphocyte differentiation, had been developed (CIEA, Central Institute for Experimental Animals, Kawasaki, Japan). Studies of human sarcoma metastasization in Rag2-/-; gammac-/- mice (lacking B, T and NK functionality) revealed their high metastatic efficiency and allowed the expression of human metastatic phenotypes not detectable in the conventionally used nude murine model. In vitro analysis to investigate the molecular mechanisms involved in the specific pattern of human sarcomas metastasization revealed the importance of liver-produced growth and motility factors, in particular the insulin-like growth factors (IGFs). The involvement of this growth factor was then demonstrated in vivo through inhibition of IGF signalling pathway. Due to the high growth and metastatic propensity of tumor cells, Rag2-/-;gammac-/- mice were used as model to investigate the metastatic behavior of rhabdomyosarcoma cells engineered to improve the differentiation. It has been recently shown that this immunodeficient model can be reconstituted with a human immune system through the injection of human cord blood progenitor cells. The work illustrated in this thesis revealed that the injection of different human progenitor cells (CD34+ or CD133+) showed peculiar engraftment and differentiation abilities. Experiments of cell vaccination were performed to investigate the functionality of the engrafted human immune system and the induction of specific human immune responses. Results from such experiments will allow to collect informations about human immune responses activated during cell vaccination and to define the best reconstitution and experimental conditions to create a humanized model in which to study, in a preclinical setting, immunological antitumor strategies.

Genetic engineering and preclinical evaluation of oncolytic herpes simplex viruses retargeted to cancer-specific receptors

Oncolytic virotherapy exploits the ability of viruses to infect and kill cells. It is suitable as treatment for tumors that are not accessible by surgery and/or respond poorly to the current therapeutic approach. HSV is a promising oncolytic agent. It has a large genome size able to accommodate large transgenes and some attenuated oncolytic HSVs (oHSV) are already in clinical trials phase I and II. The aim of this thesis was the generation of HSV-1 retargeted to tumor-specific receptors and detargeted from HSV natural receptors, HVEM and Nectin-1. The retargeting was achieved by inserting a specific single chain antibody (scFv) for the tumor receptor selected inside the HSV glycoprotein gD. In this research three tumor receptors were considered: epidermal growth factor receptor 2 (HER2) overexpressed in 25-30% of breast and ovarian cancers and gliomas, prostate specific membrane antigen (PSMA) expressed in prostate carcinomas and in neovascolature of solid tumors; and epidermal growth factor receptor variant III (EGFRvIII). In vivo studies on HER2 retargeted viruses R-LM113 and R-LM249 have demonstrated their high safety profile. For R-LM249 the antitumor efficacy has been highlighted by target-specific inhibition of the growth of human tumors in models of HER2-positive breast and ovarian cancer in nude mice. In a murine model of HER2-positive glioma in nude mice, R-LM113 was able to significantly increase the survival time of treated mice compared to control. Up to now, PSMA and EGFRvIII viruses (R-LM593 and R-LM613) are only characterized in vitro, confirming the specific retargeting to selected targets. This strategy has proved to be generally applicable to a broad spectrum of receptors for which a single chain antibody is available.

Preclinical development of anti-cancer strategies against human HER-2

HER-2 is a 185 kDa transmembrane receptor tyrosine kinase that belongs to the EGFR family. HER-2 is overexpressed in nearly 25% of human breast cancers and women with this subtype of breast cancer have a worse prognosis and frequently develop metastases. The progressive high number of HER-2-positive breast cancer patients with metastatic spread in the brain (up to half of women) has been attributed to the reduction in mortality, the effectiveness of Trastuzumab in killing metastatic cells in other organs and to its incapability to cross the blood-brain barrier. Apart from full-length-HER-2, a splice variant of HER-2 lacking exon 16 (here referred to as D16) was identified in human HER-2-positive breast cancers. Here, the contribution of HER-2 and D16 to mammary carcinogenesis was investigated in a model transgenic for both genes (F1 model). A dominant role of D16, especially in early stages of tumorigenesis, was suggested and the coexistence of heterogeneous levels of HER-2 and D16 in F1 tumors revealed the undeniable value of F1 strain as preclinical model of HER-2-positive breast cancer, closer resembling the human situation in respect to previous models. The therapeutical efficacy of anti-HER-2 agents, targeting HER-2 receptor (Trastuzumab, Lapatinib, R-LM249) or signaling effectors (Dasatinib, UO126, NVP-BKM120), was investigated in models of local or advanced HER-2-positive breast cancer. In contrast with early studies, data herein collected suggested that the presence of D16 can predict a better response to Trastuzumab and other agents targeting HER-2 receptor or Src activity. Using a multiorgan HER-2-positive metastatic model, the efficacy of NVP-BKM120 (PI3K inhibitor) in blocking the growth of brain metastases and the oncolytic ability of R-LM249 (HER-2-retargeted HSV) to reach and destroy metastatic HER-2-positive cancer cells were shown. Finally, exploiting the definition of “oncoantigen” given to HER-2, the immunopreventive activity of two vaccines on HER-2-positive mammary tumorigenesis was demonstrated.