Bioengineered vascular grafts with a pathogenic TGFBR1 variant model aneurysm formation in vivo and reveal underlying collagen defects.

Thoracic aortic aneurysm (TAA) is a life-threatening vascular disease frequently associated with underlying genetic causes. An inadequate understanding of human TAA pathogenesis highlights the need for better disease models. Here, we established a functional human TAA model in an animal host by combining human induced pluripotent stem cells (hiPSCs), bioengineered vascular grafts (BVGs), and gene editing. We generated BVGs from isogenic control hiPSC-derived vascular smooth muscle cells (SMCs) and mutant SMCs gene-edited to carry a Loeys-Dietz syndrome (LDS)-associated pathogenic variant (TGFBR1A230T). We also generated hiPSC-derived BVGs using cells from a patient with LDS (PatientA230T/+) and using genetically corrected cells (Patient+/+). Control and experimental BVGs were then implanted into the common carotid arteries of nude rats. The TGFBR1A230T variant led to impaired mechanical properties of BVGs, resulting in lower burst pressure and suture retention strength. BVGs carrying the variant dilated over time in vivo, resembling human TAA formation. Spatial transcriptomics profiling revealed defective expression of extracellular matrix (ECM) formation genes in PatientA230T/+ BVGs compared with Patient+/+ BVGs. Histological analysis and protein assays validated quantitative and qualitative ECM defects in PatientA230T/+ BVGs and patient tissue, including decreased collagen hydroxylation. SMC organization was also impaired in PatientA230T/+ BVGs as confirmed by vascular contraction testing. Silencing of collagen-modifying enzymes with small interfering RNAs reduced collagen proline hydroxylation in SMC-derived tissue constructs. These studies demonstrated the utility of BVGs to model human TAA formation in an animal host and highlighted the role of reduced collagen modifying enzyme activity in human TAA formation.

The role of T cell stimulated agonistic autoantibodies to the angiotensin II type I receptor (AT1-AA) in mediating multiorgan dysfunction in IL-17 induced hypertension during pregnancy.

PROBLEM: Preeclampsia (PE), new-onset hypertension during pregnancy accompanied by organ dysfunction, is associated with chronic inflammation including elevated IL-17, CD4+ T cells, B cells and natural killer (NK) cells. IL-17 can serve as a signal for either the adaptive or innate immune activation. We have previously shown that IL-17 contributes to increased blood pressure in association with elevated TH17 cells, NK cells and B cells secreting angiotensin II type 1 receptor agonistic autoantibodies (AT1-AA) during pregnancy. Moreover, we have shown an important role for CD4+T cells and AT1-AA in multiorgan dysfunction as measured by mitochondrial oxidative stress (mt ROS). However, we do not know the role of adaptive immune cells such as T cells or B cells secreting AT1-AA in mediating the PE phenotype in response to elevated IL-17. METHOD OF STUDY: In order to answer this question, we infused IL-17 (150 pg/day i.p.) into either Sprague Dawley (SD) or athymic nude rats via mini-osmotic pump from gestational day (GD) 14-19 of pregnancy. On GD 19, blood pressure was determined and NK cells, mtROS and respiration and AT1-AA production from B cells were measured. RESULTS: Infusion of IL-17 increased blood pressure in the presence or absence of T cells. Mean arterial pressure (MAP) increased with IL-17 from 98 ± 2 mm Hg (n = 12) to 114 ± 2 (n = 12) in SD rats and from 99 ± 4 mm Hg (n = 7) versus 115 ± 2 mm Hg (n = 7) in athymic nude rats. Similar trends were seen in NK cells and placental mt ROS. Knowing that IL-17 stimulates AT1-AA in SD pregnant rats, we included a group of SD and athymic nude pregnant rats infused with IL-17 and the AT1-AA inhibitor peptide ('n7AAc'). The inhibitor attenuated blood pressure (104.9 ± 3.2, p = .0001) and normalized NK cells and mt function in SD pregnant rats. Importantly, the AT1-AA was not produced in pregnant nude IL-17 treated rats, nor did 'n7AAc' effect MAP, in nude athymic rats. CONCLUSION: These findings suggest two conclusions; one is that IL-17 causes hypertension and multiorgan dysfunction in the absence of T cells and AT1-AA, possibly through its activation of innate cells and secondly, in the presence of T cells, blockade of the AT1-AA attenuates the effect of IL-17. This study indicates the critical effects of elevated IL-17 during pregnancy and suggest treatment modalities to consider for PE women.

Longitudinal Magnetic Resonance Imaging Tracking of Transplanted Neural Progenitor Cells in the Spinal Cord Utilizing the Bright-Ferritin Mechanism.

Human neural progenitor cells (hNPCs) hold promise for treating spinal cord injury. Studies to date have focused on improving their regenerative potential and therapeutic effect. Equally important is ensuring successful delivery and engraftment of hNPCs at the injury site. Unfortunately, no current imaging solution for cell tracking is compatible with long-term monitoring in vivo. The objective of this study was to apply a novel bright-ferritin magnetic resonance imaging (MRI) mechanism to track hNPC transplants longitudinally and on demand in the rat spinal cord. We genetically modified hNPCs to stably overexpress human ferritin. Ferritin-overexpressing (FT) hNPCs labeled with 0.2 mM manganese provided significant T1-induced bright contrast on in vitro MRI, with no adverse effect on cell viability, morphology, proliferation, and differentiation. In vivo, 2 M cells were injected into the cervical spinal cord of Rowett nude rats. MRI employed T1-weighted acquisitions and T1 mapping on a 3 T scanner. Conventional short-term cell tracking was performed using exogenous Mn labeling prior to cell transplantation, which displayed transient bright contrast on MRI 1 day after cell transplantation and disappeared after 1 week. In contrast, long-term cell tracking using bright-ferritin allowed on-demand signal recall upon Mn supplementation and precise visualization of the surviving hNPC graft. In fact, this new cell tracking technology identified 7 weeks post-transplantation as the timepoint by which substantial hNPC integration occurred. Spatial distribution of hNPCs on MRI matched that on histology. In summary, bright-ferritin provides the first demonstration of long-term, on-demand, high-resolution, and specific tracking of hNPCs in the rat spinal cord.

Comparative Study of Immunodeficient Rat Strains in Engraftment of Human-Induced Pluripotent Stem Cell-Derived Airway Epithelia.

The airway epithelia (AE) play a role in the clearance of foreign substances through ciliary motility and mucus secreted. We developed an artificial trachea that is made of collagen sponges and polypropylene mesh for the regeneration of the tracheal defect, and it was used for a clinical study. Then, a model in which the luminal surface of an artificial trachea was covered with a human-induced pluripotent stem cell-derived AE (hiPSC-AE) was transplanted into the tracheal defect of nude rats to promote epithelialization. In the future, this model was expected to be applied to research on infectious diseases and drug discovery as a trachea-humanized rat model. However, at present, sufficient engraftment has not been achieved to evaluate functional recovery in transplanted cells. Therefore, this study focused on immunosuppression in recipient rats. Nude rats lack T cell function and are widely used for transplantation experiments; however, more severe immunosuppressed recipients are preferred for xenotransplantation. Several strains of immunodeficient rats were created as rats that exhibit more severe immunodeficiency until now. In this study, to establish a trachea-humanized rat model in which human AE function can be analyzed to improve engraftment efficiency, engraftment efficiency in nude rats and X-linked severe combined immunodeficiency (X-SCID) rats following hiPSC-AE transplantation was compared. In the analysis of the proportion of engrafted cells in total cells at the graft site, the engraftment efficiency of epithelial cells tended to be high in X-SCID rats, although no statistical difference was found between the two groups, whereas the engraftment efficiency of mesenchymal cells was higher in X-SCID rats. Furthermore, the number of immune cells that accumulated in the grafts showed that a pan T cell marker, that is, CD3-positive cells, did not differ between the two strains; however, CD45-positive cells and major histocompatibility complex (MHC) class II-positive cells significantly decreased in X-SCID rats. These results indicate that X-SCID rats are more useful for the transplantation of hiPSC-AE into the tracheae to generate trachea-humanized rat models.

A nude mutant rat derived from Sprague Dawley-National Institute of Nutrition rat colony with normal thymus: A potential model for noncommunicable diseases.

BACKGROUND: A spontaneous mutant rat with a hairless phenotype and an intact thymus was discovered in a long-standing Sprague Dawley-National Institute of Nutrition (SD/NIN) rat colony at a national animal resource facility. OBJECTIVE: We conducted extensive phenotypic and biochemical analyses on this mutant strain to determine its suitability as a preclinical model for immunocompetent testing in noncommunicable disease research. MATERIALS AND METHODS: We subjected the mutant rats to strict and frequent phenotypic and genetic surveillance to accomplish this objective. The animals were assessed for food intake, body weight, blood cell profile, clinical chemistry, adipose tissue deposition, and bone mineral density (BMD) using total electrical body conductance (TOBEC) and dual-energy X-ray absorptiometry (DXA) analysis. RESULTS: Initially, only two hairless mutant rats, a male and a female, were born from a single dam in the SD/NIN rat strain. However, the results indicate that the mutant colony propagated from these unique pups displayed distinct phenotypic features and exhibited differences in feeding behavior, weight gain, and clinical biochemistry. The food conversion rate was significantly higher in nude females (2.8-fold) while 26% lower in nude males. Both sexes of nude rats had significantly higher triglycerides and lower glucose levels in females. However, glucose levels did not change in male nude rats. Furthermore, nude female and male rats had significantly lower fat (TOBEC) and bone mineral content (DXA). Nonetheless, BMD was only slightly lower (7%-8%) compared to the heterozygous groups. CONCLUSIONS: These findings indicate that the spontaneous mutant rat has the potential to serve as an immunopotent and modulatory testing system in pharmacokinetics/pharmacodynamics and toxicology, which can be further explored for therapeutic drug discovery.

Calvaria defect regeneration via human periodontal ligament stem cells and prevascularized scaffolds in athymic rats.

BACKGROUND: Vascularization plays an important role in dental and craniofacial regenerations. Human periodontal ligament stem cells (hPDLSCs) are a promising cell source and, when co-cultured with human umbilical vein endothelial cells (hUVECs), could promote vascularization. The objectives of this study were to develop a novel prevascularized hPDLSC-hUVEC-calcium phosphate construct, and investigate the osteogenic and angiogenic efficacy of this construct with human platelet lysate (hPL) in cranial defects in rats for the first time. METHODS: hPDLSCs and hUVECs were co-cultured on calcium phosphate cement (CPC) scaffolds with hPL. Cell proliferation, angiogenic gene expression, angiogenesis, alkaline phosphatase activity, and cell-synthesized minerals were determined. Bone and vascular regenerations were investigated in rat critical-sized cranial defects in vivo. RESULTS: hPDLSC-hUVEC-CPC-hPL group had 2-fold greater angiogenic expressions and cell-synthesized mineral synthesis than hPDLSC-hUVEC-CPC group (p < 0.05). Microcapillary-like structures were formed on scaffolds in vitro. hPDLSC-hUVEC-CPC-hPL group had more vessels than hPDLSC-hUVEC-CPC group (p < 0.05). In cranial defects in rats, hPDLSC-hUVEC-CPC-hPL group regenerated new bone amount that was 2.1 folds and 4.0 folds, respectively, that of hPDLSC-hUVEC-CPC group and CPC control (p < 0.05). New blood vessel density of hPDLSC-hUVEC-CPC-hPL group was 2 folds and 7.9 folds, respectively, that of hPDLSC-hUVEC-CPC group and CPC control (p < 0.05). CONCLUSION: The hPL pre-culture method is promising to enhance bone regeneration via prevascularized CPC. Novel hPDLSC-hUVEC-CPC-hPL prevascularized construct increased new bone formation and blood vessel density by 4-8 folds over CPC control. CLINICAL SIGNIFICANCE: Novel hPDLSC-hUVEC-hPL-CPC prevascularized construct greatly increased bone and vascular regeneration in vivo and hence is promising for a wide range of craniofacial applications.

Tumour status prediction by means of carbon-ion beam irradiation: comparison of washout rates between in-beam PET and DCE-MRI in rats.

Objective.Tumour response to radiation therapy appears as changes in tumour vascular condition. There are several methods for analysing tumour blood circulatory changes one of which is dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), but there is no method that can observe the tumour vascular condition and physiological changes at the site of radiation therapy. Positron emission tomography (PET) has been applied for treatment verification in charged particle therapy, which is based on the detection of positron emitters produced through nuclear fragmentation reactions in a patient's body. However, the produced positron emitters are washed out biologically depending on the tumour vascular condition. This means that measuring the biological washout rate may allow evaluation of the tumour radiation response, in a similar manner to DCE-MRI. Therefore, this study compared the washout rates in rats between in-beam PET during12C ion beam irradiation and DCE-MRI.Approach.Different vascular conditions of the tumour model were prepared for six nude rats. The tumour of each nude rat was irradiated by a12C ion beam with simultaneous in-beam PET measurement. In 10-12 h, the DCE-MRI experiment was performed for the same six nude rats. The biological washout rate of the produced positron emitters (k2,1st) and the MRI contrast agent (k2a) were derived using the single tissue compartment model.Main results.A linear correlation was observed betweenk2,1standk2a, and they were inversely related to fractional necrotic volume.Significance.This is the first animal study which confirmed the biological washout rate of in-beam PET correlates closely with tumour vascular condition measured with the MRI contrast agent administrated intravenously.

In Vivo Ultrasound Imaging of Macrophages Using Acoustic Vaporization of Internalized Superheated Nanodroplets.

Activating patients' immune cells, either by reengineering them or treating them with bioactive molecules, has been a breakthrough in the field of immunotherapy and has revolutionized treatment, especially against cancer. As immune cells naturally home to tumors or injured tissues, labeling such cells holds promise for non-invasive tracking and biologic manipulation. Our study demonstrates that macrophages loaded with extremely low boiling point perfluorocarbon nanodroplets not only survive ultrasound-induced phase change but also maintain their phagocytic function. Unlike observations made when using higher boiling point perfluorocarbon nanodroplets, our results show that phase change occurs intracellularly at a low mechanical index using a clinical scanner operating within the energy limit set by the Food and Drug Administration (FDA). After nanodroplet-loaded macrophages were given intravenously to nude rats, they were invisible in the liver when imaged at a very low mechanical index using a clinical ultrasound scanner. They became visible when power was increased but still within the FDA limits up to 8 h after administration. The acoustic labeling and in vivo detection of macrophages using a clinical ultrasound scanner represent a paradigm shift in the field of cell tracking and pave the way for potential therapeutic strategies in the clinical setting.

Laryngeal Cartilage Regeneration of Nude Rats by Transplantation of Mesenchymal Stem Cells Derived from Human-Induced Pluripotent Stem Cells.

Previous studies transplanted human-induced pluripotent stem cells (hiPSCs)-derived mesenchymal stem cells (iMSCs) into thyroid cartilage defect of X-liked severe combined immunodeficiency (X-SCID) rats and confirmed transplanted cell survival and cartilage regeneration. Thus, this study aimed to investigate the contribution of iMSC transplantation to thyroid cartilage regeneration of nude rats. iMSCs were induced from hiPSCs via a neural crest cell lineage. Then, clumps formed from an iMSC/extracellular matrix complex were transplanted into thyroid cartilage defects in nude rats. The larynx was removed and histological and immunohistochemical analyses were performed 4 or 8 weeks after the transplantation. Human nuclear antigen (HNA)-positive cells were observed in 11 of 12 (91.7%) rats, which indicated that transplanted iMSCs survived in thyroid cartilage defects in nude rats. HNA-positive cells co-expressed SOX9, and type II collagen was identified around HNA-positive cells in 8 of 12 rats (66.7%), which indicated cartilage-like regeneration. Cartilage-like regeneration in nude rats in this study was comparable to the previous report on X-SCID rats (HNA-positive cells were observed in all 14 rats and cartilage-like regeneration was observed in 10 of 14 rats). This result suggests that nude rats could be an alternative to X-SCID rats in thyroid cartilage regeneration experiments using iMSCs, and this nude rat cartilage transplantation model may develop cartilage regeneration research concerning fewer problems such as infection due to immunosuppression.

Comparison of demineralized bone matrix with different cycling crushing times in posterolateral fusion model of athymic rats.

Decalcified bone matrix (DBM) is a widely used alternative material for bone transplantation. In the DBM production process, an effective particle size and the highest utilization rate of raw materials can be achieved only through multiple high-speed circulating comminution. The rat posterolateral lumbar fusion model (PLF) is the most mature small animal model for the initial evaluation of the efficacy of graft materials for bone regeneration and spinal fusion. To evaluate the differences in the in vivo osteogenic effects of DBM pulverization through 1, 5, 9, and 14 high-speed cycles, sixty athymic rats were divided into six groups: single cycling crushing (CC1), 5 cycles of crushing (CC5), 9 cycles of crushing (CC9), 13 cycles of crushing (CC13), autogenous bone graft (ABG) and negative control (NC). Posterolateral lumbar fusion was performed. Six weeks after surgery, the bilateral lumbar fusion of athymic rats was evaluated through manual palpation, X-ray, micro-CT and histological sections. Rank data were tested by the rank-sum test, and nonparametric data were tested by the Kruskal‒Wallis H test. The manual palpation and X-ray results showed that the fusion rate did not significantly differ between the CC1, CC5, CC9, CC13 and ABG groups. However, cavities appeared in CC9 and CC13 on the micro-CT image. The bone mass (BV/TV) of CC1, CC5, CC9 and CC13 was better than that of the ABG group, while almost no osteogenesis was observed in the NC group. Histologically, there was no obvious difference between the four groups except that the CC9 group and CC13 group had more fibrous tissues in the new bone. In conclusion, DMB with different cycling crushing times has no obvious difference in fusion rate of PLF, but it is slightly better than the ABG group.

Comparison of Omentum and Subcutis as Implant Sites for Device-Encapsulated Human iPSC-Derived Pancreatic Endoderm in Nude Rats.

Subcutaneous implants of device-encapsulated stem cell-derived pancreatic endoderm (PE) can establish a functional beta cell mass (FBM) with metabolic control in immune-compromised mice. In a study with human-induced pluripotent stem cell-PE, this outcome was favored by a preformed pouch which allowed lesion-free insertion of devices in a pre-vascularized site. This was not reproduced in nude rats, known to exhibit a higher innate reactivity than mice and therefore relevant as preclinical model: a dense fibrotic capsule formed around subcutis (SC) implants with virtually no FBM formation. Placement in omentum (OM) of nude rats provided a less fibrous, better vascularized environment than SC. It resulted in less donor cell loss (56% recovery at post-transplant-PT week 3 versus 16% in SC) allowing FBM-formation. At PT week 30, 6/13 OM-recipients exhibited glucose-induced plasma hu-C-peptide to 0.1-0.4 ng/ml, versus 0/8 in SC-recipients. These levels are more than 10-fold lower than in a state of metabolic control. This shortcoming is not caused by inadequate glucose responsiveness of the beta cells but by their insufficient number. The size of the formed beta cell mass (0.4 ± 0.2 µl) was lower than that reported in mice receiving the same cell product subcutaneously; the difference is attributed to a lower expansion of pancreatic progenitor cells and to their lower degree of differentiation to beta cells. This study in the nude rat model demonstrates that OM provides a better environment for formation of beta cells in device-encapsulated PE-implants than SC. It also identified targets for increasing their dose-efficacy.

Engineered tissue vascularization and engraftment depends on host model.

Developing vascular networks that integrate with the host circulation and support cells engrafted within engineered tissues remains a key challenge in tissue engineering. Most previous work in this field has focused on developing new methods to build human vascular networks within engineered tissues prior to their implant in vivo, with substantively less attention paid to the role of the host in tissue vascularization and engraftment. Here, we assessed the role that different host animal models and anatomic implant locations play in vascularization and cardiomyocyte survival within engineered tissues. We found major differences in the formation of graft-derived blood vessels and survival of cardiomyocytes after implantation of identical tissues in immunodeficient athymic nude mice versus rats. Athymic mice supported robust guided vascularization of human microvessels carrying host blood but relatively sparse cardiac grafts within engineered tissues, regardless of implant site. Conversely, athymic rats produced substantive inflammatory changes that degraded grafts (abdomen) or disrupted vascular patterning (heart). Despite disrupted vascular patterning, athymic rats supported > 3-fold larger human cardiomyocyte grafts compared to athymic mice. This work demonstrates the critical importance of the host for vascularization and engraftment of engineered tissues, which has broad translational implications across regenerative medicine.

Behavioral and axon histomorphometric analyses after intraneural transplantation of human skin- and nerve-derived Schwann cells in nude rats.

INTRODUCTION/AIMS: We have recently isolated and expanded skin-derived Schwann cells (Sk-SCs) from human skin and showed that they are largely similar to nerve-derived Schwann cells (N-SCs). Here, we extend our investigation into functional assessments of the nude rats that received human Sk-SCs and N-SCs after intraneural delivery into crushed and decellularized tibial nerve in adult nude rats. METHODS: Sk-SCs, N-SCs, dermal fibroblasts, or control culture medium was injected into the crushed and decellularized tibial nerve using in situ repeated freeze-thaw cycles. Animals were then subjected to a ladder rung walking test, nociceptive von Frey testing, and walking gait analysis weekly. Animals were euthanized 6 weeks after surgery, gastrocnemius and soleus muscles were weighed, distal nerves were harvested, and whole semithin cross-sections were analyzed using segmentation software. RESULTS: N-SC-injected and dermal fibroblast-injected animals improved significantly at 4 to 6 weeks postinjury in nociceptive assessment compared with medium-injected controls. Sk-SCs recovered more rapidly in tibial functional index at 2 weeks postinjury compared with medium-injected controls. No significant difference was observed for the ladder rung walking test or muscle weight ratio. Histologically, the number of myelinated axons was significantly higher in all cell injection groups compared with medium-injected controls. No significant difference was observed in g ratio, axon diameter, or myelin thickness. DISCUSSION: Cell injection significantly improved axon regeneration across an in situ decellularized nerve segment. However, a more human cell-permissive animal model is required to delineate functional differences between cell types for preclinical transplantation studies.

Human primary muscle stem cells regenerate injured urethral sphincter in athymic rats.

BACKGROUND: The aim of the study was to demonstrate the efficacy of human muscle stem cells (MuSCs) isolated using innovative technology in restoring internal urinary sphincter function in a preclinical animal model. METHODS: Colonies of pure human MuSCs were obtained from muscle biopsy specimens. Athymic rats were subjected to internal urethral sphincter damage by electrocauterization. Five days after injury, 2 × 105 muscle stem cells or medium as control were injected into the area of sphincter damage (n = 5 in each group). Peak bladder pressure and rise in pressure were chosen as outcome measures. To repeatedly obtain the necessary pressure values, telemetry sensors had been implanted into the rat bladders 10 days prior to injury. RESULTS: There was a highly significant improvement in the ability to build up peak pressure as well as a pressure rise in animals that had received muscle stem cells as compared to control (p = 0.007) 3 weeks after the cells had been injected. Only minimal histologic evidence of scarring was observed in treated rats. CONCLUSION: Primary human muscle stem cells obtained using innovative technology functionally restore internal urethral sphincter function after injury. Translation into use in clinical settings is foreseeable.

A Method for Perfusion of Tissue-Isolated Human Tumor Xenografts in Nude Rats to Investigate the Oncostatic Role of the Physiological Nocturnal Melatonin Signal.

The tissue-isolated human tumor perfusion methodology enables the elucidation of physiological melatonin's oncostatic impact on cancer metabolism and physiology. Here we describe an apparatus and surgical technique for perfusing tissue-isolated human tumor xenografts in nude rats in situ that ensures continuous blood flow to and from the tissue. This system and methodology have proven quite successful in examining the receptor-mediated oncostatic effects of the physiological nocturnal melatonin signal on metabolism and physiology in a variety of epithelial and mesenchymal human tumors.

A Method for Growing Tissue-Isolated Human Tumor Xenografts in Nude Rats for Melatonin/Cancer Studies.

The tissue-isolated tumor model permits the investigation of melatonin's influence on human tumor growth and metabolism in laboratory rats in vivo. Here we describe a unique surgical technique for implanting and growing human tumor xenografts on a vascular stalk composed of the nude rat epigastric artery and vein that provides a continuous blood supply from a single source to the tissue-isolated tumor while insuring the absence of extraneous vascular connections. A variety of human tumor types may be implanted and grown utilizing this unique model that may provide a plethora of scientific data from a single tumor examined.

Local transplantation of syngeneic adipose stromal vascular fraction ameliorates damaged anal sphincter function in a rat model of vaginal distension.

BACKGROUND: The adipose stromal vascular fraction contains abundant mesenchymal stem cells and is utilized for cell therapy of male stress urinary incontinence. The purpose of this paper was to explore the effect of local transplantation of the stromal vascular fraction on improvement of damaged anal sphincter function. METHODS: A rat model of vaginal distension was used as a model of damaged anal sphincter function. The adipose stromal vascular fraction was separated from the inguinal fat of syngeneic green fluorescent protein transgenic rats and delivered into the internal anal sphincter of vaginal distension rats. The maximum resting pressure was evaluated during insertion and withdrawal of the catheter at 4 or 10 days after vaginal distension treatment to estimate anal sphincter function. Green fluorescent protein-transfected human-adipose-derived mesenchymal stem cells were transplanted into the internal anal sphincter of nude rats. Hematoxylin-eosin and Masson trichrome staining were performed to evaluate tissue damage and collagen synthesis. Transplanted cells were identified using a green fluorescent protein antibody and a human-specific antibody. Activation of the transplanted human-ADSC was evaluated by quantitative RT-PCR RESULTS: The mean maximum resting pressure (during catheter withdrawal) of vaginal distension rats was significantly lower than that of control rats, and stromal vascular fraction injection normalized it 4 days after treatment (control: 5.66 ± 0.98, vaginal distension: 4.04 ± 1.28, vaginal distension + stromal vascular fraction: 5.92 ± 1.28 [mmHg, control versus vaginal distension: P = .039; vaginal distension versus vaginal distension + stromal vascular fraction: P = .007]). Histological examination showed that vaginal distension disrupted the internal anal sphincter, and the transplanted syngeneic stromal vascular fraction survived for 10 days. Transplanted xenogeneic human-adipose-derived mesenchymal stem cells survived in the internal anal sphincter of nude rats for 4 and 10 days. Genes related to extracellular remodeling were up-regulated in the transplanted human-adipose-derived mesenchymal stem cells CONCLUSION: Syngeneic and heterotopic transplanted adipose-derived mesenchymal stem cells engrafted in the internal anal sphincter and ameliorated damaged anal sphincter function in a rat model of vaginal distension.

Do Liquid Nitrogen-treated Tumor-bearing Nerve Grafts Have the Capacity to Regenerate, and Do They Pose a Risk of Local Recurrence? A Study in Rats.

BACKGROUND: Under most circumstances, the resection of soft tissue sarcomas of the extremities can be limb-sparing, function-preserving oncologic resections with adequate margins. However, en bloc resection may require resection of the major peripheral nerves, causing poor function in the extremities. Although liquid nitrogen treatment has been used to sterilize malignant bone tumors, its use in the preparation of nerve grafts has, to our knowledge, not been reported. Hence, this study aimed to investigate the tumor recurrence and function after peripheral nerve reconstruction using liquid nitrogen-treated tumor-bearing nerves in a rat model. QUESTIONS/PURPOSES: (1) Do liquid nitrogen-treated frozen autografts have regeneration capabilities? (2) Do liquid nitrogen-treated tumor-bearing nerves cause any local recurrences in vivo in a rat model? METHODS: Experiment 1: Twelve-week-old female Wistar rats, each weighing 250 g to 300 g, were used. A 10-mm-long section of the right sciatic nerve was excised; the prepared nerve grafts were bridge-grafted through end-to-end suturing. The rats were grouped as follows: an autograft group, which underwent placement of a resected sciatic nerve after it was sutured in the reverse orientation, and a frozen autograft group, which underwent bridging of the nerve gap using a frozen autograft. The autograft was frozen in liquid nitrogen, thawed at room temperature, and then thawed in distilled water before application. The third group was a resection group in which the nerve gap was not reconstructed. Twenty-four rats were included in each group, and six rats per group were evaluated at 4, 12, 24, and 48 weeks postoperatively. To assess nerve regeneration after reconstruction using the frozen nerve graft in the nontumor rat model, we evaluated the sciatic functional index, tibialis anterior muscle wet weight ratio, electrophysiologic parameters (amplitude and latency), muscle fiber size (determined with Masson trichrome staining), lower limb muscle volume, and immunohistochemical findings (though neurofilament staining and S100 protein produced solely and uniformly by Schwann cells associated with axons). Lower limb muscle volume was calculated via CT before surgery (0 weeks) and at 4, 8, 12, 16, 20, 24, 32, 40, and 48 weeks after surgery. Experiment 2: Ten-week-old female nude rats (F344/NJcl-rnu/rnu rats), each weighing 100 g to 150 g, were injected with HT1080 (human fibrosarcoma) cells near the bilateral sciatic nerves. Two weeks after injection, the tumor grew to a 10-mm-diameter mass involving the sciatic nerves. Subsequently, the tumor was resected with the sciatic nerves, and tumor-bearing sciatic nerves were obtained. After liquid nitrogen treatment, the frozen tumor-bearing nerve graft was trimmed to a 5-mm-long tissue and implanted into another F344/NJcl-rnu/rnu rat, in which a 5-mm-long section of the sciatic nerve was resected to create a nerve gap. Experiment 2 was performed with 12 rats; six rats were evaluated at 24 and 48 weeks postoperatively. To assess nerve regeneration and tumor recurrence after nerve reconstruction using frozen tumor-bearing nerve grafts obtained from the nude rat with human fibrosarcoma involving the sciatic nerve, the sciatic nerve's function and histologic findings were evaluated in the same way as in Experiment 1. RESULTS: Experiment 1: The lower limb muscle volume decreased once at 4 weeks in the autograft and frozen autograft groups and gradually increased thereafter. The tibialis anterior muscle wet weight ratio, sciatic functional index, muscle fiber size, and electrophysiologic evaluation showed higher nerve regeneration potential in the autograft and frozen autograft groups than in the resection group. The median S100-positive areas (interquartile range [IQR]) in the autograft group were larger than those in the frozen autograft group at 12 weeks (0.83 [IQR 0.78 to 0.88] versus 0.57 [IQR 0.53 to 0.61], difference of medians 0.26; p = 0.04) and at 48 weeks (0.86 [IQR 0.83 to 0.99] versus 0.74 [IQR 0.69 to 0.81], difference of median 0.12; p = 0.03). Experiment 2: Lower limb muscle volume decreased at 4 weeks and gradually increased thereafter. The median muscle fiber size increased from 0.89 (IQR 0.75 to 0.90) at 24 weeks to 1.20 (IQR 1.08 to 1.34) at 48 weeks (difference of median 0.31; p< 0.01). The median amplitude increased from 0.60 (IQR 0.56 to 0.67) at 24 weeks to 0.81 (IQR 0.76 to 0.90) at 48 weeks (difference of median 0.21; p < 0.01). Despite tumor involvement and freezing treatment, tumor-bearing frozen grafts demonstrated nerve regeneration activity, with no local recurrence observed at 48 weeks postoperatively in nude rats. CONCLUSION: Tumor-bearing frozen nerve grafts demonstrated nerve regeneration activity, and there was no tumor recurrence in rats in vivo. CLINICAL RELEVANCE: A frozen nerve autograft has a similar regenerative potential to that of a nerve autograft. Although the findings in a rat model do not guarantee efficacy in humans, if they are substantiated by large-animal models, clinical trials will be needed to evaluate the efficacy of tumor-bearing frozen nerve grafts in humans.

Establishment and Image based evaluation of a New Preclinical Rat Model of Osteoblastic Bone Metastases.

Bone remodeling is disrupted in the presence of metastases and can present as osteolytic, osteoblastic or a mixture of the two. Established rat models of osteolytic and mixed metastases have been identified changes in structural and tissue-level properties of bone. The aim of this work was to establish a preclinical rat model of osteoblastic metastases and characterize bone quality changes through image-based evaluation. Female athymic rats (n = 22) were inoculated with human breast cancer cells ZR-75-1 and tumor development tracked over 3-4 months with bioluminescence and in-vivo µCT imaging. Bone tissue-level stereological features were quantified on ex-vivo µCT imaging. Histopathology verified the presence of osteoblastic bone. Bone mineral density distribution was assessed via backscattered electron microscopy. Newly formed osteoblastic bone was associated with reduced mineral content and increased heterogeneity leading to an overall degraded bone quality. Characterizing changes in osteoblastic bone properties is relevant to pre-clinical therapeutic testing and treatment planning.

Assessing fetal human neural stem cells tumorigenicity potential in athymic rats with penetrating traumatic brain injury (pTBI).

Traumatic brain injuries (TBI) often produce disability in survivors due to unresolved inflammation and progressive neurodegeneration. The central nervous system in mammals is incapable of self-repair. Two decades of preclinical studies and clinical trials have provided insights into TBI pathophysiology that could be utilized to develop clinically relevant therapy. Our laboratory recently reported efficacy of clinical trial grade fetal human neural stem cells (hNSCs) in immunosuppressed rats with penetrating traumatic brain injury (pTBI). Next, in compliance with the United States Food and Drug Administration (USFDA) guidance, this study explores safety by assessing the tumorigenicity potential of intracranial hNSC transplants in athymic rats with pTBI. First, the maximum tolerated dose (MTD) was determined. Then, forty athymic pTBI rats were randomized to either: Group A. pTBI + vehicle or Group B. pTBI + hNSCs at MTD one week after injury with 6-months survival, sufficient time to uncover transplant associated tumorigenicity. A board-certified Pathologist examined hematoxylin-eosin (H&E), Ki67 immunostained brain and spinal cord, serial sections along with several abnormal peripheral masses for evidence of lesion, transplant, and oncogenesis. There was no evidence of transplant derived tumors or oncogenic tissue necrosis. Consistent with athymic literature, the lesion remained unchanged even after robust hNSC engraftment. This safety study supports the conclusion that hNSCs are safe for transplantation in pTBI. The differences in lesion expansion between immunosuppressed and athymic rats in the presence of hNSCs suggests an unexpected role for thymus derived cells in resolution of trauma induced inflammation.