Valuation of angiogenesis in bovine xenografts implanted in intracorporal sites of rabbits as models of in-vivo bioreactors / Avaliação da angiogênese em xenoenxertos bovinos implantados em sítios intracorpóreos de coelhos como modelos de biorreatores in vivo

The aim of this study was to evaluate neovascularization of bovine xenografts implanted in intracorporeal sites of rabbits (bioreactors). 30 rabbits were used, divided into 6 groups, according to the evaluation time (7, 15, 30, 45, and 60 days); each animal received xenogenic implants in 3 different intracorporeal sites (A1 - omentum bag; A2 - intermuscular space of quadriceps femoris; A3 - subperiosteal of ilium bone). Histological assessments graded the presence of angiogenesis, the number of inflammatory cells, newly formed bone tissue, and the presence of giant cells. Histological analyses showed intense angiogenesis in all implanted xenografts. Presence of inflammatory infiltrate and giant cells at the A1 implant site and presence of bone neoformation at the A3 implant site were noted. Degeneration of implants and formation of a fibrous capsule were noted. When comparing the interaction of the site with the days of evaluation, statistical analysis showed a significant difference (p≤0.05) in any time of neovascularization analysis. The vascular endothelial growth factor (VEGF) and inflammatory cells of the omentum in its structure, may have contributed to the greater presence of neovessels and inflammatory cells, a fact that may indicate functionality as a possible bone substitute.(AU)

O objetivo deste estudo foi avaliar a neovascularização de xenoenxertos bovinos implantados em sítios intracorpóreos de coelhos (biorreatores). Foram utilizados 30 coelhos, os quais foram divididos em seis grupos, de acordo com o tempo de avaliação (sete, 15, 30, 45 e 60 dias); cada animal recebeu implantes xenogênicos em três diferentes sítios intracorpóreos (A1 - bolsa de omento; A2 - espaço intermuscular do quadríceps femoral; A3 - subperiosteal do osso ílio). Avaliações histológicas classificaram a presença de angiogênese, o número de células inflamatórias, de tecido ósseo neoformado e a presença de células gigantes. As análises histológicas mostraram intensa angiogênese em todos os xenoenxertos implantados. Observou-se presença de infiltrado inflamatório e células gigantes no local do implante A1 e presença de neoformação óssea no local do implante A3. Ao mesmo tempo, a degeneração dos implantes e a formação de uma cápsula fibrosa foram observadas. Ao comparar a interação do local com os dias de avaliação, a análise estatística mostrou diferença significativa (P≤0,05) em qualquer momento da análise de neovascularização. O fator de crescimento endotelial vascular (VEGF) e as células inflamatórias do omento em sua estrutura podem ter contribuído para a maior presença de neovasos e células inflamatórias, fato que pode indicar funcionalidade como possível substituto ósseo.(AU)

Insulin-producing organoids engineered from islet and amniotic epithelial cells to treat diabetes.

Maintaining long-term euglycemia after intraportal islet transplantation is hampered by the considerable islet loss in the peri-transplant period attributed to inflammation, ischemia and poor angiogenesis. Here, we show that viable and functional islet organoids can be successfully generated from dissociated islet cells (ICs) and human amniotic epithelial cells (hAECs). Incorporation of hAECs into islet organoids markedly enhances engraftment, viability and graft function in a mouse type 1 diabetes model. Our results demonstrate that the integration of hAECs into islet cell organoids has great potential in the development of cell-based therapies for type 1 diabetes. Engineering of functional mini-organs using this strategy will allow the exploration of more favorable implantation sites, and can be expanded to unlimited (stem-cell-derived or xenogeneic) sources of insulin-producing cells.

Fat Graft Safety after Oncologic Surgery: Addressing the Contradiction between In Vitro and Clinical Studies.

BACKGROUND: The authors investigate the in vitro and in vivo interaction of human breast cancer cells and human adipose-derived stem cells to address the controversy on the safety of postmastectomy fat grafting. METHODS: The authors co-cultured human adipose-derived stem cells and MDA-MB-231 breast cancer cells in an in vitro cell migration assay to examine the migration of breast cancer cells. In the in vivo arm, the authors injected breast cancer cells (group I), human breast cancer cells plus human adipose-derived stem cells (group II), human breast cancer cells plus human fat graft (group III), and human breast cancer cells plus human fat graft plus human adipose-derived stem cells (group IV) to the mammary fat pads of female nude mice (n = 20). The authors examined the tumors, livers, and lungs histologically after 2 weeks. RESULTS: Migration of breast cancer cells increased significantly when co-cultured with adipose-derived stem cells (p < 0.05). The tumor growth rate in group IV was significantly higher than in groups I and II (p < 0.05). The tumor growth rate in group III was also higher than in groups I and II, but this difference was not statistically significant (p > 0.05). Histologically, there was no liver/lung metastasis at the end of 2 weeks. The vascular density in the tumors from group IV was significantly higher than in other groups (p < 0.01). CONCLUSION: The injection of breast cancer cells, fat graft, and adipose-derived stem cells together increases breast cancer xenograft growth rates significantly.

Equine chorionic gonadotrophin improved vascularization of feline ovarian tissue xenografted into immunosuppressed mice.

The objective of this study was to evaluate effects of eCG on vascularization and development of feline ovarian tissue xenografted to immunosuppressed mice. Feline ovarian fragments (∼1 mm3) were transplanted under the renal capsule of 20 adult, ovariectomized, C57BL/6 SCID female mice. At 45 d after transplantation, 10 mice (controls) were euthanized and the remainder given 10 IU of eCG (and sacrificed 48 h later). Transplants were recovered immediately after death, fixed, sectioned, and stained with periodic acid-Schiff (PAS). Fragment volume (Cavallieri principle) and vascularization were assessed. Mean xenotransplant volume for control and treatment groups was 0.17 ±â€¯0.03 and 0.37 ±â€¯0.13 mm3, respectively (P = 0.0952); vascular volume density, 30.3 ±â€¯11.3 and 49.1 ±â€¯8.9% (P = 0.0281); surface density, 4.1 ±â€¯2.4 and 6.2 ±â€¯1.7 µm-1 (P = 0.2222); and vessel total surface, 0.63 ±â€¯0.24 µm2 and 2.28 ±â€¯1.05 µm2 (P = 0.0079). In conclusion, eCG significantly increased vascular volume density of xenotransplanted ovarian tissue and improved its development.

Efficacy of a Nanofabricated Electrospun Wound Matrix in Treating Full-thickness Cutaneous Wounds in a Porcine Model.

OBJECTIVE: This study aims to evaluate the comparative performance of a resorbable nanofiber wound matrix (Restrata Wound Matrix; Acera Surgical Inc, St Louis, MO) and a bilayered collagen xenograft (Integra Bilayer Matrix Wound Dressing; Integra, Plainsboro, NJ) in healing critical full-thickness cutaneous wounds in a preclinical porcine model. MATERIALS AND METHODS: Full-thickness cutaneous wounds were created in Yucatan miniature swine and treated with either the nanofiber wound matrix or xenograft. Wound area was measured and inflammation and healing were assessed until euthanasia at day 15 or 30, at which time tissue samples were harvested for histopathology. RESULTS: Wounds treated with the nanofiber wound matrix demonstrated significantly faster wound areal reduction, less inflammation, greater neovascularization, more collagen maturation, and superior quality of healing compared with wounds treated with the xenograft. CONCLUSIONS: The nanofiber wound matrix is an effective wound healing material that may offer a unique alternative in the treatment of challenging refractory wounds.

Positive Effect of Incubated Adipose-Derived Mesenchymal Stem Cells on Microfat Graft Survival.

Autologous fat grafting is commonly used for soft tissue augmentation, but its unpredictably high resorption rate remains a major limitation. Although adipose-derived mesenchymal stem cells (ASCs) are an attractive candidate for enhancing graft retention, their poor posttransplantation viability limits their application. The authors aimed to evaluate the effect of incubated ASCs on microfat graft survival in an immunocompromised mouse model. Lipoaspirates for microfat injection were collected from the wasted lower abdominal adipose tissues of 5 patients who had undergone breast reconstructive surgery with an abdominal flap. Adipose-derived mesenchymal stem cells were also isolated and proliferated from these fat tissues. Sixty athymic mice were randomly allocated to a control group (microfat grafting alone; n = 30) or ASCs group (microfat grafting plus simultaneous human ASCs injection; n = 30). The volume and weight of survived fat were measured at 8 and 16 weeks, and histopathological and immunologic staining was performed at 16 weeks. The survived fat volume of the ASCs group was significantly greater than that of the control group at 8 and 16 weeks, whereas the weight of survived fat tissues did not significantly differ. Histologic evaluation of the harvested fat indicated significantly higher levels of adipocytes, and fewer cysts and fibrosis in the tissues in the ASCs group than in the control group. The ASCs group also exhibited a significantly higher number of capillary vessels than the control group on CD31 and alpha-smooth muscle actin staining. In conclusion, transplanted fat survival is markedly higher when simultaneous microfat graft and ASCs injection were performed, as compared with that in the classical microfat graft alone method in mice; this improvement was primarily attributed to the increased ability to produce blood vessels.

Biomechanical Analysis of Bilateral Facet Joint Stabilization Using Bioderived Tendon for Posterior Cervical Spine Motion Reservation in Goats.

OBJECTIVES: To investigate the biomechanical properties of a novel stabilization method for posterior cervical motion preservation using bioderived freeze-dried tendon. METHODS: Experiments were conducted both in vitro and in vivo. For the in vitro group, 15 fresh-frozen goat spines (C1-C7) were randomly divided into 3 subgroups: intact (INT-vitro, n = 5), injury model (IM-vitro, n = 5), and bilateral facet joint stabilization (BFJS-vitro, n = 5) subgroups. For the in vivo group, 15 adult goats were randomly divided into 3 experimental subgroups: INT-vivo subgroup (n = 5), IM-vivo subgroup (n = 5), and BFJS-vivo subgroup (n = 5). Goats in the in vivo group were euthanized 12 weeks after surgery. Biomechanical tests were performed to evaluate range of motion. Histologic analysis was conducted to evaluate survival and reactions associated with the bioderived tendon. RESULTS: Compared with the INT-vitro and INT-vivo subgroups, the flexion of IM-vitro and IM-vivo subgroups increased significantly, respectively (P < 0.05). The flexion of the BFJS-vitro and BFJS-vivo subgroups was significantly smaller than in the IM-vitro and IM-vivo subgroups, respectively (P < 0.05). Significant differences between the BFJS-vitro and BFJS-vivo subgroups were observed in flexion, lateral bending, and rotation (P < 0.05). Histologic evaluation demonstrated that fibers arranged regularly and stained homogeneously. New vessels in growth indicated that the bioderived tendon was survival and processed good regeneration. CONCLUSIONS: Bilateral facet joint stabilization can significantly limit excessive flexion motion and maintain adequate stability. Furthermore, the preservation of extension motions without limiting lateral bending and rotation ideally simulates the features of the posterior ligamentous complex. This preserves the dynamic stability of the lower cervical spine.

Dynamic contrast-enhanced MRI of the microenvironment of pancreatic adenocarcinoma xenografts.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with poor outcome. Resistance to treatment is associated with impaired vascularity, extensive hypoxia, and interstitial hypertension. In this study, the potential of dynamic contrast-enhanced (DCE)-MRI as a method for assessing the microvascular density (MVD), the fraction of hypoxic tissue, and the interstitial fluid pressure (IFP) of PDACs was investigated. MATERIAL AND METHODS: Intramuscular BxPC-3, Capan-2, MIAPaCa-2, and Panc-1 PDAC xenografts were used as preclinical models of human PDACs. DCE-MRI with Gd-DOTA as contrast agent was conducted with a 7.05-T scanner, and the DCE-MRI series were analyzed voxelwise by using the Tofts pharmacokinetic model. Tumor MVD and hypoxia were measured in histological preparations by using pimonidazole as a hypoxia marker and CD31 as a marker of endothelial cells. IFP was measured with a Millar catheter. RESULTS: Ktrans (the volume transfer constant of Gd-DOTA) increased with increasing MVD and decreased with increasing hypoxic fraction, but was not associated with IFP. Any association between ve (the fractional distribution volume of Gd-DOTA) and MVD, hypoxic fraction, or IFP could not be detected. CONCLUSIONS: This study shows that DCE-MRI is a useful modality for assessing important features of the microenvironment of PDAC xenografts and thus provides the basis for future preclinical and clinical DCE-MRI investigations of PDAC.

Hypoxia-controlled EphA3 marks a human endometrium-derived multipotent mesenchymal stromal cell that supports vascular growth.

Eph and ephrin proteins are essential cell guidance cues that orchestrate cell navigation and control cell-cell interactions during developmental tissue patterning, organogenesis and vasculogenesis. They have been extensively studied in animal models of embryogenesis and adult tissue regeneration, but less is known about their expression and function during human tissue and organ regeneration. We discovered the hypoxia inducible factor (HIF)-1α-controlled expression of EphA3, an Eph family member with critical functions during human tumour progression, in the vascularised tissue of regenerating human endometrium and on isolated human endometrial multipotent mesenchymal stromal cells (eMSCs), but not in other highly vascularised human organs. EphA3 affinity-isolation from human biopsy tissue yielded multipotent CD29+/CD73+/CD90+/CD146+ eMSCs that can be clonally propagated and respond to EphA3 agonists with EphA3 phosphorylation, cell contraction, cell-cell segregation and directed cell migration. EphA3 silencing significantly inhibited the ability of transplanted eMSCs to support neovascularisation in immunocompromised mice. In accord with established roles of Eph receptors in mediating interactions between endothelial and perivascular stromal cells during mouse development, our findings suggest that HIF-1α-controlled expression of EphA3 on human MSCs functions during the hypoxia-initiated early stages of adult blood vessel formation.

Generation, expansion and functional analysis of endothelial cells and pericytes derived from human pluripotent stem cells.

Human endothelial cells (ECs) and pericytes are of great interest for research on vascular development and disease, as well as for future therapy. This protocol describes the efficient generation of ECs and pericytes from human pluripotent stem cells (hPSCs) under defined conditions. Essential steps for hPSC culture, differentiation, isolation and functional characterization of ECs and pericytes are described. Substantial numbers of both cell types can be derived in only 2-3 weeks: this involves differentiation (10 d), isolation (1 d) and 4 or 10 d of expansion of ECs and pericytes, respectively. We also describe two assays for functional evaluation of hPSC-derived ECs: (i) primary vascular plexus formation upon coculture with hPSC-derived pericytes and (ii) incorporation in the vasculature of zebrafish xenografts in vivo. These assays can be used to test the quality and drug sensitivity of hPSC-derived ECs and model vascular diseases with patient-derived hPSCs.

Effect of oxygen tensions on the proliferation and angiogenesis of endometriosis heterograft in severe combined immunodeficiency mice.

OBJECTIVE: To investigate the effects of oxygen on the proliferation and angiogenesis of endometriosis in vivo. DESIGN: Animal studies. SETTING: Animal research facility. ANIMAL(S): Thirty-six female severe combined immunodeficiency (SCID) mice, implanted with eutopic endometrium from seven endometriosis patients. INTERVENTION(S): Human eutopic endometrial tissues were randomized to normoxia, hyperoxia, or hypoxia pretreatment and were subcutaneously implanted into estrogen-treated ovariectomized SCID mice. MAIN OUTCOME MEASURE(S): The growth and quality of the implants were measured, and the expression of proliferation- and angiogenesis-associated markers (i.e., Ki67, CD31, vascular endothelial growth factor, and hypoxia-inducible factor-1α) were assessed using immunohistochemistry and Western blot analyses. RESULT(S): The growth curves of the implants were distinct with different oxygen pretreatments. The growth of the implants of the hypoxia group was significantly increased compared with the normoxia group, but the growth of the implants of the hyperoxia group was significantly decreased compared with the normoxia group. Microscopic examination indicated that lesions with hyperplastic cylindrical glandular epithelium were surrounded by the endometrial stroma in the hypoxia group, but the glandular epithelium was partially depauperate in the hyperoxia group. The expression of Ki67, CD31, vascular endothelial growth factor, and hypoxia-inducible factor-1α in the hypoxia-pretreated implants was significantly higher compared with the hyperoxia or normoxia groups. CONCLUSION(S): Oxygen can alter the growth patterns of endometriosis implants in a SCID mouse model. Hypoxia pretreatment promoted the proliferation and angiogenesis of endometriosis, whereas hyperoxia pretreatment exhibited the opposite effect.

Histological advantages of the tumor graft: a murine model involving transplantation of human pancreatic cancer tissue fragments.

OBJECTIVES: Experimental data based on cell line-derived xenograft models (cell xenograft) seldom reproduce the clinical situation, and therefore we demonstrated here the superiority of a murine model involving transplantation of human pancreatic cancer tissue fragments (tumor graft), focusing on the histological features and drug delivery characteristics. METHODS: Tumor pieces from 10 pancreatic cancer patients were transplanted into SCID (severe combined immunodeficient) mice. Histological characteristics of tumor grafts, including morphology, desmoplastic reaction, and vascularization, were compared with those of cell xenografts. Drug delivery was evaluated by quantifying the concentrations of injected drug, and the results were compared with its histological features. RESULTS: Eight of the 10 transplanted tumors successfully engrafted. Histological comparisons between tumor grafts and cell xenografts revealed the following: the amount of stroma was more (22.9% ± 11.8% vs 10.8% ± 5.4%; P < 0.05), vessel-cancer cell distance was longer (35.3 ± 39.0 vs 3.9 ± 3.1 µm; P < 0.001), and microvessel density was lower (6.8 ± 1.9 vs 10.8 ± 2.1 vessels/0.4 mm(2); P < 0.05) in tumor grafts. Drug concentrations in tumor grafts were lower than those in cell xenografts (3.3 ± 1.2 vs 6.0±0.2 µg/mL; P = 0.003), and the differences were correlated with the histological differences. CONCLUSIONS: Pancreatic tumor grafts better reproduce the histological nature of clinical cancer and thus provide a more realistic model that is applicable for pharmacokinetic studies.

Xenograft tumors vascularized with murine blood vessels may overestimate the effect of anti-tumor drugs: a pilot study.

Recent evidence demonstrated that endothelial cells initiate signaling events that enhance tumor cell survival, proliferation, invasion, and tumor recurrence. Under this new paradigm for cellular crosstalk within the tumor microenvironment, the origin of endothelial cells and tumor cells may have a direct impact on the pathobiology of cancer. The purpose of this pilot study was to evaluate the effect of endothelial cell species (i.e. murine or human) on xenograft tumor growth and response to therapy. Tumor xenografts vascularized either with human or with murine microvascular endothelial cells were engineered, side-by-side, subcutaneously in the dorsum of immunodefficient mice. When tumors reached 200 mm(3), mice were treated for 30 days with either 4 mg/kg cisplatin (i.p.) every 5 days or with 40 mg/kg sunitinib (p.o.) daily. Xenograft human tumors vascularized with human endothelial cells grow faster than xenograft tumors vascularized with mouse endothelial cells (P<0.05). Notably, human tumors vascularized with human endothelial cells exhibited nuclear translocation of p65 (indicative of high NF-kB activity), and were more resistant to treatment with cisplatin or sunitinib than the contralateral tumors vascularized with murine endothelial cells (P<0.05). Collectively, these studies suggest that the species of endothelial cells has a direct impact on xenograft tumor growth and response to treatment with the chemotherapeutic drug cisplatin or with the anti-angiogenic drug sunitinib.