The importance of vascularization in the field of bone tissue engineering has been established by previous studies. The present work proposes a novel poly(propylene fumarate) (PPF)/fibrin composite scaffold for the development of vascularized neobone tissue. The effect of prevascularization (i.e., in vitro pre-culture prior to implantation) with human mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) on in vivo vascularization of scaffolds was determined. Five conditions were studied: no pre-culture (NP), 1 week preculture (1P), 2 week pre-culture (2P), 3 week pre-culture (3P), and scaffolds without cells (control, C). Scaffolds were implanted subcutaneously in a severe combined immunodeficiency (SCID) mice model for 9 days. During in vitro studies, CD31 staining showed a significant increase in vascular network area over 3 weeks of culture. Vascular density was significantly higher in vivo when comparing NP to 3P groups. Immunohistochemical staining of human CD-31 expression indicated spreading of vascular networks with increasing pre-culture time. These vascular networks were perfused with mouse blood indicated by perfused lectin staining in human CD-31 positive vessels. Our results demonstrate that in vitro prevascularization supports in vivo vascularization in PPF/fibrin scaffolds.

Vascularization remains one of the most important challenges that must be overcome for tissue engineering to be consistently implemented for reconstruction of large volume bone defects. An extensive vascular network is needed for transport of nutrients, waste and progenitor cells required for remodelling and repair. A variety of tissue engineering strategies have been investigated in an attempt to vascularize tissues, including those applying cells, soluble factor delivery strategies, novel design and optimization of bio-active materials, vascular assembly pre-implantation and surgical techniques. However, many of these strategies face substantial barriers that must be overcome prior to their ultimate translation into clinical application. In this review recent progress in engineering vascularized bone will be presented with an emphasis on clinical feasibility.

Vascular networks provide nutrients, oxygen, and progenitor cells that are essential for bone function. It has been proposed that a preformed vascular network may enhance the performance of engineered bone. In this study vascular networks were generated from human umbilical vein endothelial cell and mesenchymal stem cell spheroids encapsulated in fibrin scaffolds, and the stability of preformed vascular networks and their effect on bone regeneration were assessed in an in vivo bone model. Under optimized culture conditions, extensive vessel-like networks formed throughout the scaffolds in vitro. After vascular network formation, the vascularized scaffolds were implanted in a critical sized calvarial defect in nude rats. Immunohistochemical staining for CD31 showed that the preformed vascular networks survived and anastomosed with host tissue within 1 week of implantation. The prevascularized scaffolds enhanced overall vascularization after 1 and 4 weeks. Early bone formation around the perimeter of the defect area was visible in X-ray images of samples after 4 weeks. Prevascularized scaffolds may be a promising strategy for engineering vascularized bone.

Poly (lactic-co-glycolic acid) (PLGA)-based materials are widely investigated for drug delivery and tissue engineering applications. Despite their popularity the genotoxic potential of PLGA has not been investigated. In this study, the comet assay, a sensitive assay for DNA damage, was used to evaluate potential genotoxicity in model cell types exposed to PLGA microspheres. Human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (MSCs) cells were exposed to PLGA microspheres (0.4-6 mg/mL) and DNA damage assessed at 24 h, 4 days, and 7 days. DNA damage was not identified after 24 h. However, after 4 and 7 days of exposure to 2 and 6 mg/mL of PLGA microspheres a significant elevation of DNA damage in both cell types was observed. The PLGA microspheres did not exhibit any cytotoxic effects on the cells under the conditions tested. Our results suggest that PLGA may have a genotoxic effect on cells. A broader investigation of the PLGA genotoxic profile in biological systems is needed. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 284-291, 2017.

Background: Cell-seeded biomaterial scaffolds have been proposed as a future option for reconstruction of bone tissue. The ability to generate larger, functional volumes of bone has been a challenge that may be addressed through the use of perfusion bioreactors. In this study, the authors investigated use of a tubular perfusion bioreactor system for the growth and differentiation of bone marrow stromal (mesenchymal stem) cells seeded onto fibrin, a highly angiogenic biomaterial. Methods: Cells were encapsulated within fibrin beads and cultured either within a tubular perfusion bioreactor system or statically for up to 14 days. Scaffolds were analyzed for osteogenic differentiation. A rodent cranial defect model (8-mm diameter) was used to assess the bone regeneration of scaffolds cultured in the bioreactor, statically, or used immediately after formation. Immunohistochemistry was used to visualize CD31+ vessel density. Micro–computed tomographic imaging was used to visualize mineral formation within the defect volume. Results: Tubular perfusion bioreactor system–cultured samples showed significantly greater osteodifferentiation, indicated by an increase in VEGF expression and mineral deposition, compared with statically cultured samples. Increased expression of OPN, RUNX2, VEGF, and CD90 was seen over time in both culture methods. After implantation, bioreactor samples exhibited greater bone formation and vessel density compared with all other groups. Analysis of micro–computed tomographic images showed full union formation through the greatest diameter of the defect in all bioreactor samples and the highest levels of mineralized volume after 8 weeks. Conclusion: Mesenchymal stem cells encapsulated in fibrin beads and cultured in the tubular perfusion bioreactor system resulted in increased vascularization and mineralized tissue formation in vivo relative to static culture.

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Innate immunity plays a vital role in detecting cytoplasmic nucleic acids resulting from viral infection or the presence of tumor cells. Cytosolic DNA is sensed by the cyclic-GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, and in the case of tumors, activation of this pathway has potential to both positively and negatively modulate cancer development. Activation of the STING pathway is a key prerequisite for type I IFN production that is needed for either endogenous or treatment-induced cancer immune responses, and reduced cGAS-STING expression is associated with poorer survival of patients with lung adenocarcinoma, invasive breast ductal carcinoma, and gastric cancer. Intrinsic activation of the cGAS-STING pathway in the tumor microenvironment (TME) promotes cancer cell-intrinsic senescence and recruitment of immune cells, resulting in antitumor effects. For cancers that are difficult to treat and characterized by immune tolerance, combination therapies of STING agonists with immune checkpoint inhibitors represents a new paradigm in disease management. To date, only two early clinical trials of the STING agonist ADU-S100 in combination with checkpoint inhibitors are underway (NCT02675439 and NCT03172936). To progress clinical trials more rapidly and with better safety outcomes for patients with advanced treatment-refractory metastatic solid tumors or lymphomas, being able to reliably screen a variety of STING agonists in different types of in vitro cancer models is of paramount importance. We have previously reported a novel functional assay that is capable of demonstrating the potency level of different STING agonists, based on the IFNβ response in THP-1 monocytic leukemia cells. We further investigated the role of newly developed STING agonists and clinical compounds in phenotypic in vitro cancer cell and TME models to inform on oncotherapeutic development. STING-expressing cancer cell lines, chosen based on microarray mRNA expression, were treated with agonists and assayed for cGAS-STING activation status using a fully automated platform for high-content imaging. In vitro analysis demonstrated high phospho-STING activation at 4 hours. In the BioMAP® Oncology Colorectal TME model, STING activation increased IL-6 release and the effect on other primary immune and tissue remodeling biomarkers will be discussed. Preclinical studies indicate that STING agonists, used as adjuvants in combination with other agents or radiation therapy, suppress tumor progression, reduce cellular toxicity, and eliminate metastases in breast and pancreatic cancer models. Phenotypic assays that provide human, translational data early in discovery are a valuable tool to accelerate progress in this area. Citation Format: Satheesh K. Sainathan, Justin H. Lipner, Jennifer I. Drake, Brogan A. Epkins, Brianna M. Roux, Alastair J. King. Measurement of cytosolic DNA sensing cGAS-STING pathway functional activity using in vitro phenotypic assay models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1877.