Engineered tissue vascularization and engraftment depends on host model

The aim of the present review has been to describe some immunobiological characteristics of the athymic nude rat and on the basis of these to describe the possibilities of establishing non-antigen-specific T-cell reactivity and inducing antigen-specific immunity. After a brief introduction, some general characteristics of the autosomal recessive rnu/rnu rat are outlined. This athymic mutant normally has a longer lifespan than athymic nude mice and is easier to breed. Inbred nude rats are now available with several rat strain backgrounds. Since the rnu/rnu rat is athymic, the morphology and function of the thymus are briefly described. The thymus has two main functions: production of T lymphocytes and production of thymic hormones. The intrathymic T-cell ontogeny is described along with the two thymocyte selection mechanisms, positive and negative selection. Different thymic hormones are mentioned and their possible function outlined. However, thymic hormones still do not play an important role in immunotherapy. There are some striking differences in the morphology of lymphatic tissues of athymic and euthymic rats. The thymic area in nude rats consists only of fat and clusters of epithelial cells. Little research has been performed on bone marrow of athymic nude rats but morphologically there seems to be no difference from findings in normal animals. The thymus-dependent areas of peripheral lymphoid organs, i.e. the paracortical area of lymph nodes, the periarteriolar sheet of the splenic white pulp, and the interfollicular areas of Peyer's patches, are severely cell-depleted in the athymic nude rat. The lower lymphocyte content of these organs is not reflected in the lymphocyte counts of peripheral blood, but the lymphocyte counts of thoracic duct lymph are much lower than those found in euthymic animals. Few T-lymphocyte marker-positive cells are found in the athymic nude rat, and in the epithelium of the intestine there are also lymphocytes bearing neither T nor B markers. These cells probably represent the tau delta receptor-bearing cells. Immunologically the athymic nude rat reflects the thymic aplasia. Normal B-lymphocyte function has been found in both in vitro and in vivo tests, whereas the T-cell function is virtually absent both in vitro and in vivo. Non-MHC-restricted cells with killer activity like NK cells are present in the nude rat, and these cells function normally both in vitro and in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Posterolateral lumbar spine fusions in athymic rats. To compare spine fusion rates of two different osteoinductive products. Many osteoinductive bone graft alternatives are available. Grafton (a demineralized bone matrix [DBM]) and Osteogenic Protein-1 (OP-1, an individual recombinant bone morphogenetic protein) are two such alternatives. The relative efficacy of products from these two classes has not been previously studied. The athymic rat spine fusion model has been validated and demonstrated useful to minimize inflammatory responses to xenogeneic or differentially expressed proteins such as those presented by DBMs of human etiology. Single-level intertransverse process fusions were performed in 60 athymic nude rats with 2 cc/kg of Grafton or OP-1 Putty. Half of each study group was killed at 3 weeks and half at 6 weeks. Fusion masses were assessed by radiography, manual palpation, and histology. At 3 weeks, manual palpation revealed a 13% fusion rate with Grafton and a 100% fusion rate with OP-1 (P = 0.0001). At 6 weeks, manual palpation revealed a 39% fusion rate of with Grafton and a 100% fusion rate with OP-1 (P = 0.0007). Similar fusion rates were found by histology at 3 and 6 weeks. Of note, one or two adjacent levels were fused in all of the OP-1 animals and none of the Grafton animals. Significant differences between the ability of Grafton and OP-1 to induce bone formation in an athymic rat posterolateral lumbar spine fusion model were found.

Comparing human “local” bone versus human cancellous bone for spinal fusion in an athymic rat model

Observation of microvasculatures in athymic nude rat transplanted tumor using synchrotron radiation microangiography system

Mucosal mast cells (MMC) and connective tissue mast cells (CTMC) of the rat contain different proteoglycans, which can be distinguished using histochemical methods. The chondroitin sulphate proteoglycan of the MMC, unlike the heparin of the CTMC, does not show fluorescent berberine binding, is susceptible to aldehyde fixatives and stains preferentially with Alcian Blue in a staining sequence with Safranin. The majority of the dermal mast cells are typical CTMC and are located in the deep part of the dermis. Subepidermal mast cells are comparatively few in normal rats but numerous in athymic rats and mice. These cells differ from other dermal mast cells in that they stain preferentially with Alcian Blue and they appear to contain little histamine. We examined some of the histochemical properties of the skin mast cells of female PVG-rnu/rnu rats and their heterozygous littermates aged from 5 to 29 weeks. The thiazine dye-binding of the subepidermal mast cells was partially blocked by formaldehyde fixation and only about half of them showed a weakly fluorescent berberine binding. The critical electrolyte concentration of the Alcian Blue staining of the subepidermal mast cells was between that of CTMC and MMC. Deaminative cleavage with nitrous acid abolished the staining of all skin mast cells, while that of the MMC was unaffected. There were no statistically significant differences in the staining patterns of the dermal mast cells between different ages or groups of rat. These results indicate that the subepidermal mast cells contain a heparin proteoglycan which is, however, different from that of the typical CTMC of other sites. They thus appear to represent a second example of a mast cell within a defined anatomical location exhibiting a distinct proteoglycan expression.

A model system is described that allows an analysis of the molecular and biochemical changes associated with expression and suppression of the oncogenic and metastatic phenotype of cloned rat embryo fibroblast (CREF) cells. Ha-ras-transformed CREF cells are morphologically transformed, anchorage-independent and both tumorigenic and metastatic in athymic nude mice and syngeneic Fischer rats. Co-expression of the Ha-ras oncogene and Krev-1 tumor suppressor gene in CREF cells results in suppression of in vitro transformation. In contrast, Ha-ras/Krev-1 transformed CREF cells retain, with greatly extended latency periods, both tumorigenic and metastatic capabilities in athymic nude mice. The present study investigates changes in the Ha-ms suppressor gene, rrg (lysyl oxidase), during expression and suppression of the oncogenic phenotype in CREF cells. Nontumorigenic CREF cells and CREF cells transformed by the Ha-ras and Krev-1 gene that express a suppression in in vitro transformation contain elevated levels of lysyl oxidase mRNA and protein. In contrast, Ha-ms and Ha-ras/Krev-1 nude mouse tumor- and nude mouse lung metastasis-derived CREF cells contain reduced levels of lysyl oxidase mRNA and protein. Nuclear run-on assays indicate that suppression of lysyl oxidase expression in transformed subclones of CREF cells correlates with a reduction in transcription of the lysyl oxidase gene. Taken together, the current studies support a transcriptional switching model in which lysyl oxidase expression correlates directly with suppression of the Ka-ms-induced transformation phenotype and escape from oncogenic suppression correlates with a transcriptional silencing of the lysyl oxidase gene and decreased lysyl oxidase mRNA and protein.

Facial osseous defects remain a major functional and esthetic challenge for the head and neck surgeon. Tissue engineering may provide advantageous alternatives to conventional therapies. The objective of the study was to evaluate the efficacy of gene therapy in the repair of osseous facial defects. Blinded, controlled, prospective animal experiment. Thirty adult athymic nude rats were divided into five groups of six animals. Three groups were used as control subjects. Two groups were treated with 3.75 x 10(8) viral particles containing recombinant type 5 adenoviral vectors. One group received viruses that coded for beta-galactosidase production, another received viruses that coded for bone morphogenetic protein (BMP-2) production. After 120 days rats were examined at necropsy with precise planimetry, histological analysis of new bone growth, and radio-densitometric analysis of bone thickness. Radio-densitometric measurements showed that BMP-2-treated nude athymic rats had significantly enhanced osseous repair compared with control subjects when evaluated by both radio-densitometry and histological examination. Gene therapy-treated, immunosuppressed rodents had an enhanced osteoinductive repair of a dorsal osseous nasal defect.

A human malignant pleural mesothelioma was xenografted serially in athymic nude Rowett rats for 27 passages during 33 months. After the two initial passages (P), the take rate during P3-9 was 100% (192/192). The tumour grew progressively during P3-9 in 99% (190/192) and regressed totally in 1% (2/192). The take rate for the tumour xenografted to athymic BALB/c mice was also 100% (17/17) and no regressions were observed. During serial passaging in nude rats, the tumour-volume doubling time (TD) decreased from 6 days in P2 to 3 days in P8-9 (P less than 0.001) and then remained around 3 days during P10-25. A TD of 11 days in P1 (man-mouse) for tumours grown in mice decreased during 10 passages in rats to 4 days (P less than 0.005) when the tumour was transplanted to mice in P11. Light microscopic morphology of the tumour was retained in rats and mice. We believe that our experimental tumour model using the nude rat as a carrier of the xenograft will be useful for studies of human mesothelioma.

This paper reports on a form of atrophy of lymph node compartments occurring in mesenteric nodes of athymic nude rats whose perinodal tissue had become unusually rich in mast cells and fibrotic. The subcapsular sinus of an affected compartment was depleted of lymphocytes, while the medullary sinuses were loaded with macrophages. A progressive expansion of medullary sinuses into the medullary cords, the extrafollicular zone, the deep cortex unit and, finally, the folliculo-nodules of the compartment was accompanied by a gradual atrophy of the usual lymphoid cell populations of these nodal components. The occurrence of such a mode of atrophy of a compartment, associated with a lack of lymphocytes in the afferent lymph, agrees with our previous proposal that lymphocytes of the afferent lymph provide the stimuli for the development and maintenance of the structures of the node. The involvement of mast cells and fibrosis in the emergence of the present form of nodal atrophy, which further weakens the immune system of an immunodeficient animal, is considered.

Severe pulmonary arterial hypertension (SPH) is a frequently lethal condition characterized by pulmonary vascular remodeling and right heart strain or failure. SPH is also often associated with autoimmune and collagen vascular disorders. To study the effects of T cells on the development of experimental SPH. Athymic nude rats lacking T cells were treated with a single subcutaneous injection of vascular endothelial growth factor (VEGF) receptor blocker SU5416 (20 mg/kg) to induce pulmonary vascular endothelial cell apoptosis. Immunohistochemical analysis and IL-4 levels of the lung tissue were performed. Cell death and proliferation were assessed by Western blot and immunohistochemistry. In contrast to SU5416-treated euthymic rats that develop SPH only in combination with chronic hypoxia, athymic nude rats developed SPH and vascular remodeling (similar to clinical SPH) at normoxic conditions as demonstrated by measurements of pulmonary artery pressure and right ventricle hypertrophy. Pulmonary arterioles became occluded with proliferating endothelial cells and were surrounded by mast cells, B cells, and macrophages. IL-4, proliferating cell nuclear antigen, and collagen type I levels were markedly increased in SU5416-treated athymic rat lungs. Antibody deposition was noted along the vascular endothelium in rats with SPH. Finally, protection from SPH was conferred by immune challenge with spleen cells from euthymic nude rats. These studies demonstrate the importance of a complete, intact immune system in protecting against pulmonary angioproliferation in this new model of SPH as well as the importance of intact VEGF receptor signaling for lung endothelial cell homeostasis.

The athymic (nude) rat (rnu/rnu) has been used for a number of years in research into various human tumours involving xenotransplantation. We now report the validation of a continuous intravenous infusion method in nude rats using a tail cuff tether, which enables the study of the efficacy of novel anti-cancer materials in this mutant strain, using intravenous infusion and with no restriction of the animals or of the tumour implantation sites by jackets. Ten animals each had a cannula surgically implanted into the vena cava via the femoral vein and exteriorized via a tail cuff. Animals were housed singly in conventional cages following surgery. Following a recovery period of 5 days all animals were continuously infused with physiological saline at an infusion rate of 0.5 ml/h for a further 37 days. Body weights and food consumption were recorded weekly. Blood samples were taken approximately 14 days post-surgery and analysed for haematology and clinical chemistry parameters. All animals were successfully cannulated, and no unexpected adverse clinical signs were noted during the recovery period and the 37 days of infusion. The results demonstrate that it is possible to surgically cannulate the femoral vein of athymic (nude) rats and infuse them in conventional cages for a period of up to 37 days with minimal adverse effects. The minimal restraint required provides benefits both to the animal and to the conduct of studies such as assessment of tumour growth in the absence of a jacket. Recent work has demonstrated that the same techniques can be successfully applied to the nude mouse.

Introduction: We have previously demonstrated the use of phthalocyanine Pc 4 for the photodynamic therapy (PDT) of ectopic human glial tumors in the athymic nude rat brain. We wish to determine whether 18F-fluorodeoxy-glucose (¹⁸F-FDG) Positron Emission Tomography (PET) imaging can detect the reduction in tumor metabolism that must occur after Pc 4-PDT-induced necrosis. Methods: 2.5 x 10⁵ U87 cells were injected into the brains of 12 athymic nude rats. After 7 days of tumor growth, all 12 animals were imaged functionally by ¹⁸F-FDG micro-PET (&mgr;PET) and structurally by micro-CT and/or micro-MR. These animals received 0.5 mg/kg b.w. Pc 4 via tail-vein injection. One day later the scalp was re-incised and the tumor illuminated with 30 J/cm² of 672-nm light from a diode laser. The next day these animals were again ¹⁸F-FDG &mgr;PET imaged. Next, the animals were euthanized and their brains were explanted for H&E histology. Results: Histology showed that tumors in the 6 Pc 4-PDT-treated animals demonstrated necrosis ranging from full to frank (severe). Preliminary analysis showed that 18F-FDG &mgr;PET activity in 3 of the 6 non-PDT group (i.e., no tumor necrosis observed) animals was seen to increase 2.28 times following tumor photoirradiation, whereas ¹⁸F-FDG &mgr;PET activity in 5 of the 6 PDT group (<i>i.e.</i>, tumor necrosis observed) animals was seen to increase 1.15 times following tumor photoirradiation. Discussion: The increased ¹⁸F-FDG &mgr;PET activity in the PDT group was unexpected. We had expected this activity to decrease and are presently investigating the cause of this observation.

Objective: To investigate the effects and mechanisms of allogeneic epidermal stem cells (ESCs) on the survival of allogeneic full-thickness skin grafts in nude mice with full-thickness skin defect wounds. Methods: Experimental research methods were applied. Primary ESCs that appeared paving stone-like after being cultured for 7 d were obtained by enzymatic digestion method from one 4-week-old male BALB/c-NU nude mouse (the same strain, age, and sex below). The cells of third passage were identified by flow cytometry to positively express ESC marker CD44 and negatively express CD45, meanwhile, the positive expression of ESC markers of p63 and integrin 6α, and negative expression of CD71 were identified by immunofluorescence method. The ESCs of third passage in the logarithmic growth phase were used for the following experiments. Twenty-six nude mice were equally divided into phosphate buffered saline (PBS) group and ESCs group according to the random number table. A full-thickness skin defect wound was made on the back of each nude mouse, and then the wounds of the two groups were sprayed with equal volumes of PBS and ESCs, respectively. The wounds were transplanted with full-thickness skin grafts cut from the backs of 4 other nude mice. Each ten nude mice from the two groups were selected, the wound healing and skin survival on post surgery day (PSD) 0 (immediately), 3, 7, 14, and 21 were observed, and the survival ratio and shrinkage rate of skin grafts on PSD 3, 7, 14, and 21 were calculated (the number of sample was the number of surviving skin grafts at each time point); the blood perfusion in the skin grafts on PSD 3, 7, and 14 was detected by the laser speckle blood flow imager, and the blood flow ratio of nude mice skin grafts in ESCs group to PBS group at each time point was calculated (the number of sample was the pair number of surviving skin grafts in group pairing at each time point). The skin graft tissue of each 3 nude mice remained in the two groups were collected on PSD 7, and the mRNA expressions and protein expressions of tumor necrosis factor α (TNF-α), interleukin 8 (IL-8), IL-10, type Ⅰ collagen, type Ⅲ collagen, and matrix metalloproteinase 9 (MMP-9) in the tissue were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction and Western blotting, respectively. Data were statistically analyzed with Log-rank test, analysis of variance for repeated measurement, one-way analysis of variance, independent sample t test, and Bonferroni correction. Results: Taking the condition on PSD 0 as a reference, the wounds of nude mice in the two groups healed gradually on PSD 3, 7, 14, and 21, and the shrinkage of skin grafts was gradually obvious. Among them, the shrinkage healing of wound of nude mice in PBS group was more significant than that in ESCs group. On PSD 3, the skin graft of 1 nude mouse failed in ESCs group, while the skin graft of 3 nude mice failed in PBS group. On PSD 7, the skin graft of another nude mouse failed in PBS group. The survival ratio of skin grafts of nude mice in the two groups was similar on PSD 3, 7, 14, and 21 (P>0.05). On PSD 3, 7, 14, and 21, the shrinkage rates of skin grafts of nude mice in ESCs group were (9.2±0.4)%, (19.7±1.2)%, (53.6±3.5)%, and (62.2±5.1)%, respectively, which was significantly lower than (11.0±0.9)%, (47.8±2.8)%, (86.1±7.1)%, and (89.7±9.0)% in PBS group (t=5.719, 26.650, 11.940, 7.617, P<0.01). On PSD 3, 7, and 14, blood perfusion signals were observed in the skin grafts of nude mice in the two groups. The average blood perfusion ratios of the skin grafts of nude mice in ESCs group to PBS group were greater than 1, and there was no statistically significant difference in the overall comparison of 3 time points (P>0.05). On PSD 7, compared with those of PBS group, the mRNA and protein expressions of TNF-α, IL-8, type Ⅰ collagen, and type Ⅲ collagen in the skin graft tissue of nude mice in ESCs group were significantly reduced, while the mRNA and protein expressions of IL-10 and MMP-9 in the skin graft tissue of nude mice in ESCs group were significantly increased (in mRNA comparison, t=2.823, 2.934, 2.845, 2.860, 3.877, 2.916, P<0.05). Conclusions: Allogeneic ESCs can reduce the shrinkage of allogeneic full-thickness skin grafts transplanted on full-thickness skin defect wounds in nude mice, promote the formation of new blood vessels between the skin graft and the wound, reduce inflammation and collagen protein expression, and promote the expression of MMP-9, thus improving the survival quality of skin grafts.

With current treatment of glioblastomas 85% of the tumors recur within the field of high dose radiation. This tumor recurrence requires formation of new blood vessels. We have developed a new therapeutic paradigm based on the dual origin of tumor blood vessels: Angiogenesis - the sprouting of endothelial cells from nearby blood vessels, and vasculogenesis - the formation of blood vessels from circulating cells. Because tumor irradiation abrogates local angiogenesis the tumor must rely on the vasculogenesis pathway for regrowth after irradiation. We have previously demonstrated that the post-irradiation recurrence of human glioblastomas implanted intracranially in the mouse brain can be delayed or prevented by inhibiting circulating blood vessel forming cells by blocking the interaction of the chemokine receptor CXCR4 with its ligand SDF-1 (CXCL12)1. However, SDF-1 has a second receptor, CXCR7, which has been implicated in endothelial cell migration, is present on tumor vasculature, and is potentially also able to activate vasculogenesis. To block both receptors we have therefore used NOX-A12, an L-enantiomeric RNA oligonucleotide (Spiegelmer®), which inhibits SDF-1 with subnanomolar affinity. We have tested the effect of this inhibitor on the response to irradiation of the U251 human GBM in nude mice and on ENU-induced brain tumors in the Sprague-Dawley rat. In the mouse model we found that inhibition of SDF-1 post-irradiation was highly effective in enhancing tumor response to a single dose of 20 Gy whole brain irradiation. In the rat model the pups from ENU-treated pregnant mothers begin to die of brain tumors from approximately 120 days of age. We performed two studies with this model: In the first we delivered a single dose of whole brain irradiation (20 Gy) on day 115 of age and began treatment with NOX-A12 immediately following irradiation and continued every 2 days with either 5 or 20 mg/kg injected subcutaneously for either 4 or 8 weeks. These doses and times were chosen as equivalent to human doses and times that based on existing data have been found to be safe and well tolerated in human volunteers and which are effective in inhibiting the action of SDF-1. We found a marked prolongation of life of the rats that was dependent both on drug dose and length of treatment. In the second study we measured the size of the tumors post-irradiation using MRI and again found a pronounced enhancement of the efficacy of irradiation that was superior to the addition of temozolomide at doses equivalent to those used with concomitant radiotherapy in treating GBM. We believe that these encouraging data justify a human trial in first line glioblastoma patients. 1. Kioi M, Vogel H, Schultz G, Hoffman RM, Harsh GR, Brown JM: Inhibition of vasculogenesis, but not angiogenesis, prevents the recurrence of glioblastoma after irradiation in mice. J Clin Invest 120:694-705, 2010 Citation Format: Shie-Chau Liu, Reem Alomran, Jason Stafford, Milton Merchant, Taichang Jang, Stefan Zollner, Anna Kruschinski, Laurence Recht, J. Martin Brown. Inhibition of recurrences of experimental brain tumors after irradiation by blocking the activity of SDF-1 (CXCL12) using the Spiegelmer® NOX-A12. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 385. doi:10.1158/1538-7445.AM2013-385

Artificial tissues constructed from therapeutic cells offer a promising approach for improving the treatment of severe peripheral nerve injuries. In this study the effectiveness of using CTX0E03, a conditionally immortalised human neural stem cell line, as a source of allogeneic cells for constructing living artificial nerve repair tissue was tested. CTX0E03 cells were differentiated then combined with collagen to form engineered neural tissue (EngNT-CTX), stable aligned sheets of cellular hydrogel. EngNT-CTX sheets were delivered within collagen tubes to repair a 12 mm sciatic nerve injury model in athymic nude rats. Autologous nerve grafts (autografts) and empty tubes were used for comparison. After 8 weeks functional repair was assessed using electrophysiology. Further, detailed histological and electron microscopic analysis of the repaired nerves was performed. Results indicated that EngNT-CTX supported growth of neurites and vasculature through the injury site and facilitated reinnervation of the target muscle. These findings indicate for the first time that a clinically validated allogeneic neural stem cell line can be used to construct EngNT. This provides a potential ‘off the shelf’ tissue engineering solution for the treatment of nerve injury, overcoming the limitations associated with nerve autografts or the reliance on autologous cells for populating repair constructs.