Preclinical Large Animal Model Research Articles

Translation of an injectable triple-interpenetrating-network hydrogel for intervertebral disc regeneration in a goat model.

The results of this work demonstrate that a radiopaque hydrogel is capable of normalizing the mechanical function of the degenerative disc, is supportive of disc cell and mesenchymal stem cell viability and matrix production, and can be maintained in the disc space without extrusion following intradiscal delivery in a preclinical large animal model. These results support evaluation of this hydrogel as a minimally invasive disc therapeutic in long-term preclinical studies as a precursor to future clinical application in patients with disc degeneration and low back pain.

Electrospun vascular scaffold for cellularized small diameter blood vessels: A preclinical large animal study.

This study demonstrates the utility of tissue engineering to provide platform technologies for rehabilitation of patients recovering from severe, devastating cardiovascular diseases. The long-term goal is to provide alternatives to vascular grafting using bioengineered blood vessels derived from an autologous cell source with a functionalized vascular scaffold. This novel bilayered vascular construct for engineering blood vessels is designed to offer "off-the-shelf" availability for clinical translation.

Development of Transplantable Engineered Graft by Organ Derived Acellular Frame

Background: Stem cell technology has accelerated regenerative medicine approaches targeting the liver. However, some technical difficulties in scale-up or delivery of large amount of these cells still remain as a barrier of clinical application. To overcome these limitations, we have developed a tissue engineering approach using “de/recellularized organ frame”. The aim of this study was to generate native liver derived organ frame, which was filled with porcine hepatocytes or human induced pluripotent stem (iPS) cells. Its function and clinical utility was assessed by preclinical large animal model. Methods: The decellularization protocol for porcine liver was re-optimized. Acellular liver frame was then filled with 1x109 of hepatocytes isolated from porcine liver or 5x108 of hepatocyte like cells derived from human iPS cells, co-seeding with porcine or human endothelial cells. The engineered grafts were transplanted into normal liver pigs or pigs with liver injury induced by retrorsine (RS) administration with 60% hepatectomy. Functional outcomes of the graft was assessed by angiography, H-E stein, Azan stein, SEM, immunostain, Bilirubin stein, blood test, and CYP gene expression profiling. 60% hepatectomized pigs without RS was designed as a control group.

Induction of Immune Tolerance to Foreign Protein via Adeno-Associated Viral Vector Gene Transfer in Mid-Gestation Fetal Sheep.

A major limitation to adeno-associated virus (AAV) gene therapy is the generation of host immune responses to viral vector antigens and the transgene product. The ability to induce immune tolerance to foreign protein has the potential to overcome this host immunity. Acquisition and maintenance of tolerance to viral vector antigens and transgene products may also permit repeat administration thereby enhancing therapeutic efficacy. In utero gene transfer (IUGT) takes advantage of the immunologic immaturity of the fetus to induce immune tolerance to foreign antigens. In this large animal study, in utero administration of AAV6.2, AAV8 and AAV9 expressing green fluorescent protein (GFP) to ~60 day fetal sheep (term: ~150 days) was performed. Transgene expression and postnatal immune tolerance to GFP and viral antigens were assessed. We demonstrate 1) hepatic expression of GFP 1 month following in utero administration of AAV6.2.GFP and AAV8.GFP, 2) in utero recipients of either AAV6.2.GFP or AAV8.GFP fail to mount an anti-GFP antibody response following postnatal GFP challenge and lack inflammatory cellular infiltrates at the intramuscular site of immunization, 3) a serotype specific anti-AAV neutralizing antibody response is elicited following postnatal challenge of in utero recipients of AAV6.2 or AAV8 with the corresponding AAV serotype, and 4) durable hepatic GFP expression was observed up to 6 months after birth in recipients of AAV8.GFP but expression was lost between 1 and 6 months of age in recipients of AAV6.2.GFP. The current study demonstrates, in a preclinical large animal model, the potential of IUGT to achieve host immune tolerance to the viral vector transgene product but also suggests that a single exposure to the vector capsid proteins at the time of IUGT is inadequate to induce tolerance to viral vector antigens.

Intra-articular Recombinant Human Proteoglycan 4 Mitigates Cartilage Damage After Destabilization of the Medial Meniscus in the Yucatan Minipig

Background: Lubricin, or proteoglycan 4 (PRG4), is a glycoprotein responsible for joint boundary lubrication. PRG4 has been shown previously to be down-regulated after traumatic joint injury such as a meniscal tear. Preliminary evidence suggests that intra-articular injection of PRG4 after injury will reduce cartilage damage in rat models of surgically induced posttraumatic osteoarthritis. Objective: To determine the efficacy of intra-articular injection of full-length recombinant human lubricin (rhPRG4) for reducing cartilage damage after medial meniscal destabilization (DMM) in a preclinical large animal model. Study Design: Controlled laboratory study. Methods: Unilateral DMM was performed in 29 Yucatan minipigs. One week after DMM, animals received 3 weekly intra-articular injections (3 mL per injection): (1) rhPRG4 (1.3 mg/mL; n = 10); (2) rhPRG4+hyaluronan (1.3 mg/mL rhPRG4 and 3 mg/mL hyaluronan [~950 kDA]; n = 10); and (3) phosphate-buffered saline (PBS; n = 9). Hindlimbs were harvested 26 weeks after surgery. Cartilage integrity was evaluated by use of macroscopic (India ink) and microscopic (safranin O–fast green and hematoxylin and eosin) scoring systems. Secondary outcomes evaluated via enzyme-linked immunosorbent assay (ELISA) included PRG4 levels in synovial fluid, carboxy-terminal telepeptide of type II collagen (CTX-II) concentrations in urine and serum, and interleukin 1β (IL-1β) levels in synovial fluid and serum. Results: The rhPRG4 group had significantly less macroscopic cartilage damage in the medial tibial plateau compared with the PBS group (P = .002). No difference was found between the rhPRG4+hyaluronan and PBS groups (P = .23). However, no differences in microscopic damage scores were observed between the 3 groups (P = .70). PRG4 production was elevated in the rhPRG4 group synovial fluid compared with the PBS group (P = .033). The rhPRG4 group presented significantly lower urinary CTX-II levels, but not serum levels, when compared with the PBS (P = .013) and rhPRG4+hyaluronan (P = .011) groups. In serum and synovial fluid, both rhPRG4 (P = .006; P = .017) and rhPRG4+hyaluronan groups (P = .009; P = .03) presented decreased IL-1β levels. Conclusion: All groups exhibited significant cartilage degeneration after DMM surgery. However, animals treated with rhPRG4 had the least amount of cartilage damage and less inflammation, providing evidence that intra-articular injections of rhPRG4 may slow the progression of posttraumatic osteoarthritis. Clinical Relevance: Patients with meniscal trauma are at high risk for posttraumatic osteoarthritis. This study demonstrates that an intra-articular injection regimen of rhPRG4 may attenuate cartilage damage after meniscal injury.

Next-generation nanotech meds: Diagnostic and therapeutic applications of non-organic nanoparticles are making their way into clinical use.

Nanoparticles have been used for more than a decade in medicine, one of the more prominent applications being silver particles in wound dressing to prevent infection and local inflammation. Most nanoparticles though are used for drug delivery, a substantial and fast growing market, worth about US$41 billion in 2014 and projected to reach US$118 billion by 2023 (http://www.transparencymarketresearch.com/nanotechnology-drug-delivery.html). In most cases, these particles are made of organic molecules to encapsulate therapeutic compounds or to home in on specific cell types such cancer cells. Recently, though, research has taken a growing interest in inorganic nanoparticles and early clinical trials are underway or about to begin soon. The potential applications of inorganic nanoparticles include diagnosis, therapy, or a combination of both for a wide range of degenerative, oncological, infectious and auto‐immune or inflammatory diseases. > … Nanoparticles often have properties that are different from the same elements or compounds at larger scale… Nanoparticles are usually defined as anything in size between 1 and 100 nm, which is just the right size for targeted delivery and diagnostics at the level of biomolecules. Moreover, nanoparticles often have properties that are different from the same elements or compounds at larger scale, while their large surface to volume ratio increases chemical reactivity and thus potential as catalysts. By the same token, this increases potential for deleterious side effects, which has so far held back the use of nanomaterials in the clinic. The most widely researched nanoparticles, particularly for applications in biology and medicine, are carbon nanotubes, gold nanoparticles, and cadmium selenide quantum dots. To a large extent, the properties that make these particles attractive for industrial applications are equally relevant in biology. Gold nanoparticles, for example, are highly flexible in terms of size, shape, surface chemistry, and aggregation state. Quantum dots can deliver high doses of energy …

Cardiac Gene Delivery in Large Animal Models: Antegrade Techniques.

Percutaneous antegrade coronary injection is among the least invasive cardiac selective gene delivery methods. However, transduction efficiency is quite low with a simple bolus antegrade injection. In order to improve the transduction efficiency using antegrade delivery, several additional approaches have been proposed.In this chapter, we briefly discuss important elements associated with intracoronary delivery methods and present protocols for three different catheter-based antegrade delivery techniques in a preclinical large animal model. Despite the lower transduction efficacy relative to more invasive delivery techniques, antegrade techniques have the advantage of being clinically well established and having safer profiles which is important when treating patients with cardiac disease.

Cell-Based Measurement of Neutralizing Antibodies Against Adeno-Associated Virus (AAV).

In recent years gene therapy using adeno-associated viral (AAV) vectors to treat cardiac disease has seen an unprecedented surge, owing to its safety, low immunogenicity relative to other vectors and high and long-term transduction efficiency. This field has also been hampered by the presence of preexisting neutralizing antibodies, not only in patients participating in clinical trials but also in preclinical large animal models. These conflicting circumstances have generated the need for a simple, efficient, and fast assay to screen subjects for the presence of neutralizing antibodies, or lack thereof, in order for them to be included in gene therapy trials.

Surgical technique: establishing a pre-clinical large animal model to test aortic valve leaflet substitute.

To overcome current limitations of valve substitutes and tissue substitutes the technology of tissue engineering (TE) continues to offer new perspectives in congenital cardiac surgery. We report our experiences and results implanting a decellularized TE patch in nine sheep in orthotropic position as aortic valve leaflet substitute. Establishing the animal model, feasibility, cardiopulmonary bypass issues and operative technique are highlighted.

Pim1 Kinase Overexpression Enhances ckit+ Cardiac Stem Cell Cardiac Repair Following Myocardial Infarction in Swine

BackgroundPim1 kinase plays an important role in cell division, survival, and commitment of precursor cells towards a myocardial lineage, and overexpression of Pim1 in ckit+ cardiac stem cells (CSCs) enhances their cardioreparative properties. ObjectivesThe authors sought to validate the effect of Pim1-modified CSCs in a translationally relevant large animal preclinical model of myocardial infarction (MI). MethodsHuman cardiac stem cells (hCSCs, n = 10), hckit+ CSCs overexpressing Pim1 (Pim1+; n = 9), or placebo (n = 10) were delivered by intramyocardial injection to immunosuppressed Yorkshire swine (n = 29) 2 weeks after MI. Cardiac magnetic resonance and pressure volume loops were obtained before and after cell administration. ResultsWhereas both hCSCs reduced MI size compared to placebo, Pim1+ cells produced a ∼3-fold greater decrease in scar mass at 8 weeks post-injection compared to hCSCs (−29.2 ± 2.7% vs. −8.4 ± 0.7%; p < 0.003). Pim1+ hCSCs also produced a 2-fold increase of viable mass compared to hCSCs at 8 weeks (113.7 ± 7.2% vs. 65.6 ± 6.8%; p <0.003), and a greater increase in regional contractility in both infarct and border zones (both p < 0.05). Both CSC types significantly increased ejection fraction at 4 weeks but this was only sustained in the Pim1+ group at 8 weeks compared to placebo. Both hCSC and Pim1+ hCSC treatment reduced afterload (p = 0.02 and p = 0.004, respectively). Mechanoenergetic recoupling was significantly greater in the Pim1+ hCSC group (p = 0.005). ConclusionsPim1 overexpression enhanced the effect of intramyocardial delivery of CSCs to infarcted porcine hearts. These findings provide a rationale for genetic modification of stem cells and consequent translation to clinical trials.

Collection of hematopoietic CD34 stem cells in rhesus macaques using Spectra Optia.

Nonhuman primates, particularly rhesus macaques, are ideal preclinical large animal models to investigate organ tolerance induction protocols using donor hematopoietic stem cells (HSCs) to induce chimerism. Their relatively small size poses some challenges for the safe and effective collection of peripheral blood HSCs through apheresis procedures. We describe our experiences using the Spectra Optia apheresis unit to successfully obtain HSCs from mobilized peripheral blood of rhesus macaques. Mobilization of peripheral blood HSCs was induced using granulocyte stimulating factor (G-CSF) and Mozobil. The Spectra Optia unit was used in 18 apheresis procedures in 13 animals (4.9-10 kg). Animal health was carefully monitored during and after the procedure. Changes in peripheral blood cells before, during and after procedure were determined by complete blood count and flow cytometry. The automatic settings of the Spectra Optia unit were applied successfully to the procedures on the rhesus macaque. All animals tolerated the procedure well with no mortality. Mobilization of HSCs were most consistently achieved using 50 μg/kg of G-CSF for 5 days and a single dose of Mozobil on the 5th day, followed by collection of cells 3 h after Mozobil injection. The final apheresis product contained an average of 23 billion total nucleated cells with 47% granulocytes, 3,871 million total CD3 cells and 77 million CD34 cells which resulted in an average of 10 million CD34+ cells/kg of donor weight. Apheresis of peripheral blood mobilized HSCs in rhesus macaques using Spectra Optia is a safe and effective procedure.

Biocompatibility Assessment of Detonation Nanodiamond in Non-Human Primates and Rats Using Histological, Hematologic, and Urine Analysis.

Detonation nanodiamonds (DNDs) have been widely explored for biomedical applications ranging from cancer therapy to magnetic resonance imaging due to several promising properties. These include faceted surfaces that mediate potent drug binding and water coordination that have resulted in marked enhancements to the efficacy and safety of drug delivery and imaging. In addition, scalable processing of DNDs yields uniform particles. Furthermore, a broad spectrum of biocompatibility studies has shown that DNDs appear to be well-tolerated. Prior to the clinical translation of DNDs for indications that are addressed via intravenous administration, comprehensive assessment of DND safety in both small and large animal preclinical models is needed. This article reports the results of a DND biocompatibility study in both non-human primates and rats. The rat study was performed as a multiple dose subacute investigation in two cohorts that lasted for 2 weeks and included histological, serum, and urine analysis. The non-human primate study was performed as a dual gender, multiple dose, and long-term investigation in both standard/clinically relevant and elevated dosing cohorts that lasted for 6 months and included comprehensive serum, urine, histological, and body weight analysis. The results from these studies indicate that NDs are well-tolerated at clinically relevant doses. Examination of dose-dependent changes in biomarker levels provides important guidance for the downstream in-human validation of DNDs for clinical drug delivery and imaging.

Non-immunosuppressive FTY720-derivative OSU-2S mediates reactive oxygen species-mediated cytotoxicity in canine B-cell lymphoma.

OSU-2S is a FTY720 (Fingolimod) derivative that lacks immunosuppressive properties but exhibits strong anti-tumour activity in several haematological and solid tumour models. We have recently shown OSU-2S to mediate potent cytotoxicity in human mantle cell lymphoma cell lines and primary cells. We report here the pre-clinical activity of OSU-2S in spontaneous B-cell lymphoma of dogs which shares many characteristics of human lymphoma. OSU-2S mediated apoptosis in canine B-cell lines and primary B-cell lymphoma cells obtained from spontaneous lymphoma bearing dogs. OSU-2S induced reactive oxygen species (ROS) in canine lymphoma cells and inhibition of ROS partially rescued OSU-2S-mediated cell death. These studies provide a rational basis for the use of spontaneous lymphoma in pet dogs as a preclinical large animal model for the development of OSU-2S as small molecule for treating people and dogs with lymphoma.

724. A Comprehensive Map of CNS Transduction by Eight Recombinant Adeno-Associated Virus Serotypes upon Cerebrospinal Fluid Administration in Pigs

Gene transfer of recombinant adeno-associated virus (rAAVs) holds promises to treat neurological disorders. Studies on different recombinant vector serotypes and modes of delivery to the central nervous system (CNS) showed that the combination of both rAAV serotype used and delivery routes play a key role in CNS transduction properties and thus in disease phenotype rescue outcome. However, one of the major hurdles to developing an effective clinical protocol for neurological disorders is the efficiency of vectors to reach the specific cell types in disease-specific CNS subdomains. An attractive vehicle to reach the CNS is represented by the cerebral spinal fluid (CSF) and different injection techniques (intrathecal cisterna magna, intra-ventricular and intrathecal lumbar injections) actually allow the central nervous system targeting by using the CSF flux. Along these years different AAV serotypes have been tested by intrathecal injections and some of these displayed a very specific tropism for the central nervous system. However, a quantitative and qualitative analysis of transduction patterns of the most promising rAAV serotypes for brain targeting in large animal models is missing. Here, we characterize distribution, transduction efficiency and cellular targeting of rAAV serotypes 1, 2, 5, 7, 9, rh.10, rh.39 and rh.43 delivered into the cisterna magna of wild type pigs. Despite the rAAV9 showed the highest transduction efficiency and the widest distribution capability among the vectors tested, the other serotypes showed a specific distribution pattern from the rostral to the caudal part of the CNS. rAAV9 robustly transduced both glia and neurons, including the motor neurons of the spinal cord. Relevant cell transduction specificity of the glia was observed after rAAV1 and rAAV7 delivery. rAAV7 also displayed a specific tropism to Purkinje cells. Evaluation of biochemical and hematological markers suggested that all rAAV serotypes tested were well tolerated. This study provides a comprehensive CNS transduction map in a useful pre-clinical large animal model enabling the selection of potentially clinically transferable rAAV serotypes based on disease specificity. Therefore, our data are instrumental for the clinical evaluation of these rAAV vectors in human neurodegenerative diseases.

(373) - MHC-Inbred CLAWN Miniature Swine as Preclinical Large Animal Model for Lung Transplantation

(373) - MHC-Inbred CLAWN Miniature Swine as Preclinical Large Animal Model for Lung Transplantation

Quo Vadis Breast Tissue Engineering?

Quo Vadis Breast Tissue Engineering?

Chimeric Allografts Induced by Short-Term Treatment With Stem Cell Mobilizing Agents Result in Long-Term Kidney Transplant Survival Without Immunosuppression: II, Study in Miniature Swine.

Transplantation is now lifesaving therapy for patients with end-stage organ failure but requires lifelong immunosuppression with resultant morbidity. Current immunosuppressive strategies inhibit T cell activation and prevent donor-recipient engagement. Therefore, it is not surprising that few host cells are demonstrated in donor grafts. However, our recent small animal studies found large numbers of recipient stem cells present after transplantation and pharmacological mobilization, resulting in a chimeric, repopulated organ. We now confirm these findings in a well-characterized large animal preclinical model. Here, we show that AMD3100 and FK506 mobilization of endogenous stem cells immediately post kidney transplantation combined with repeat therapy at 1, 2, and 3 months led to drug-free long-term survival in maximally immunologically mismatched swine. Three long-term recipients have stable chimeric transplants, preserved antidonor skin graft responses, and normal serum creatinine levels despite withdrawal of all medication for 3 years.

Establishment of a preclinical ovine screening model for the investigation of bone tissue engineering strategies in cancellous and cortical bone defects.

BackgroundNew tissue engineering strategies for bone regeneration need to be investigated in a relevant preclinical large animal model before making the translation into human patients. Therefore, our interdisciplinary group established a simplified large animal screening model for intramembranous bone defect regeneration in cancellous and cortical bone.MethodsRelated to a well-established model of cancellous drill hole defect regeneration in sheep, both the proximal and distal epimetaphyseal regions of the femur and the humerus were used bilaterally for eight drill hole cancellous defects (Ø 6 mm, 15 mm depth). Several improvements of the surgical procedure and equipment for an easier harvest of samples were invented. For the inclusion of cortical defect regeneration, a total of eight unicortical diaphyseal drill holes (6 mm Ø) were placed in the proximal-lateral and distal-medial parts of the metacarpal (MC) and metatarsal (MT) diaphyseal bone bilaterally. Acting moments within a normal gait cycle in the musculoskeletal lower limb model were compared with the results of the biomechanical in vitro torsion test until failure to ensure a low accidental fracture risk of utilized bones (ANOVA, p < 0.05). The model was tested in vivo, using thirteen adult, female, black-face sheep (Ø 66 kg; ± 5 kg; age ≥ 2.5 years). In a two-step surgical procedure 16 drill holes were performed for the investigation of two different time points within one animal. Defects were left empty, augmented with autologous cancellous bone or soft bone graft substitutes.ResultsThe in vitro tests confirmed this model a high comparability between drilled MC and MT bones and a high safety margin until fracture. The exclusion of one animal from the in vivo study, due to a spiral fracture of the left MC bone led to a tolerable failure rate of 8 %.ConclusionsAs a screening tool, promising biomaterials can be tested in this cancellous and cortical bone defect model prior to the application in a more complex treatment site.

Human Mesenchymal Stromal Cells Improve Cardiac Perfusion in an Ovine Immunocompetent Animal Model

ABSTRACTBackground: Mesenchymal stromal cells (MSCs) hold considerable promise in the treatment of ischemic heart disease. Most preclinical studies of MSCs for acute myocardial infarction (AMI) have been performed either in syngeneic animal models or with human cells in xenogeneic immunodeficient animals. A preferable pre-clinical model, however, would involve human MSCs in an immunocompetent animal. Methods: AMI was generated in adult sheep by inducing ischemia reperfusion of the second diagonal branch. Sheep (n = 10) were randomized to receive an intravenous injection of human MSCs (1 × 106 cells/kg) or phosphate buffered saline. Cardiac function and remodeling were evaluated with echocardiography. Perfusion scintigraphy was used to identify sustained myocardial ischemia. Interaction between human MSCs and ovine lymphocytes was assessed by a mixed lymphocyte response (MLR). Results: Sheep receiving human MSCs showed significant improvement in myocardial perfusion at 1 month compared with baseline measurements. There was no change in ventricular dimensions in either group after 1 month of AMI. No adverse events or symptoms were observed in the sheep receiving human MSCs. The MLR was negative. Conclusion: The immunocompetent ovine AMI model demonstrates the clinical safety and efficacy of human MSCs. The human cells do not appear to be immunogenic, further suggesting that immunocompetent sheep may serve as a suitable pre-clinical large animal model for testing human MSCs.

Endometrial mesenchymal stem cells as a cell based therapy for pelvic organ prolapse.

Pelvic organ prolapse (POP) occurs when the pelvic organs (bladder, bowel or uterus) herniate into the vagina, causing incontinence, voiding, bowel and sexual dysfunction, negatively impacting upon a woman's quality of life. POP affects 25% of all women and results from childbirth injury. For 19% of all women, surgical reconstructive surgery is required for treatment, often augmented with surgical mesh. The surgical treatment fails in up to 30% of cases or results in adverse effects, such as pain and mesh erosion into the bladder, bowel or vagina. Due to these complications the Food and Drug Administration cautioned against the use of vaginal mesh and several major brands have been recently been withdrawn from market. In this review we will discuss new cell-based approaches being developed for the treatment of POP. Several cell types have been investigated in animal models, including a new source of mesenchymal stem/stromal cells (MSC) derived from human endometrium. The unique characteristics of endometrial MSC, methods for their isolation and purification and steps towards their development for good manufacturing practice production will be described. Animal models that could be used to examine the potential for this approach will also be discussed as will a rodent model showing promise in developing an endometrial MSC-based therapy for POP. The development of a preclinical large animal model for assessing tissue engineering constructs for treating POP will also be mentioned.