Identification of a humanized mouse model for functional testing of immune-mediated biomaterial foreign body response.

Biomedical devices comprise a major component of modern medicine, however immune-mediated fibrosis and rejection can limit their function over time. Here, we describe a humanized mouse model that recapitulates fibrosis following biomaterial implantation. Cellular and cytokine responses to multiple biomaterials were evaluated across different implant sites. Human innate immune macrophages were verified as essential to biomaterial rejection in this model and were capable of cross-talk with mouse fibroblasts for collagen matrix deposition. Cytokine and cytokine receptor array analysis confirmed core signaling in the fibrotic cascade. Foreign body giant cell formation, often unobserved in mice, was also prominent. Last, high-resolution microscopy coupled with multiplexed antibody capture digital profiling analysis supplied spatial resolution of rejection responses. This model enables the study of human immune cell-mediated fibrosis and interactions with implanted biomaterials and devices

Neutrophil Responses to Sterile Implant Materials

In vivo implantation of sterile materials and devices results in a foreign body immune response leading to fibrosis of implanted material. Neutrophils, one of the first immune cells to be recruited to implantation sites, have been suggested to contribute to the establishment of the inflammatory microenvironment that initiates the fibrotic response. However, the precise numbers and roles of neutrophils in response to implanted devices remains unclear. Using a mouse model of peritoneal microcapsule implantation, we show 30–500 fold increased neutrophil presence in the peritoneal exudates in response to implants. We demonstrate that these neutrophils secrete increased amounts of a variety of inflammatory cytokines and chemokines. Further, we observe that they participate in the foreign body response through the formation of neutrophil extracellular traps (NETs) on implant surfaces. Our results provide new insight into neutrophil function during a foreign body response to peritoneal implants which has implications for the development of biologically compatible medical devices

Long term Glycemic Control Using Polymer Encapsulated, Human Stem-Cell Derived β-cells in Immune Competent mice

The transplantation of glucose-responsive, insulin-producing cells offers the potential for restoring glycemic control in diabetic patients1. Pancreas transplantation and the infusion of cadaveric islets are currently implemented clinically2, but are limited by the adverse effects of lifetime immunosuppression and the limited supply of donor tissue3. The latter concern may be addressed by recently described glucose responsive mature β-cells derived from human embryonic stem cells; called SC-β, these cells may represent an unlimited human cell source for pancreas replacement therapy4. Strategies to address the immunosuppression concern include immunoisolation of insulin-producing cells with porous biomaterials that function as an immune barrier5,6. However, clinical implementation has been challenging due to host immune responses to implant materials7. Here, we report the first long term glycemic correction of a diabetic, immune-competent animal model with human SC-β cells. SC-β cells were encapsulated with alginate-derivatives capable of mitigating foreign body responses in vivo, and implanted into the intraperitoneal (IP) space of streptozotocin-treated (STZ) C57BL/6J mice. These implants induced glycemic correction until removal at 174 days without any immunosuppression. Human C-peptide concentrations and in vivo glucose responsiveness demonstrate therapeutically relevant glycemic control. Implants retrieved after 174 days contained viable insulin-producing cells

Combinatorial hydrogel library enables identification of materials that mitigate the foreign body response in primates

The foreign body response is an immune-mediated reaction that can lead to the failure of implanted medical devices and discomfort for the recipient. There is a critical need for biomaterials that overcome this key challenge in the development of medical devices. Here we use a combinatorial approach for covalent chemical modification to generate a large library of variants of one of the most widely used hydrogel biomaterials, alginate. We evaluated the materials in vivo and identified three triazole-containing analogs that substantially reduce foreign body reactions in both rodents and, for at least 6 months, in non-human primates. The distribution of the triazole modification creates a unique hydrogel surface that inhibits recognition by macrophages and fibrous deposition. In addition to the utility of the compounds reported here, our approach may enable the discovery of other materials that mitigate the foreign body response.Leona M. and Harry B. Helmsley Charitable Trust (3-SRA-2014-285-M-R)United States. National Institutes of Health (EB000244)United States. National Institutes of Health (EB000351)United States. National Institutes of Health (DE013023)United States. National Institutes of Health (CA151884)United States. National Institutes of Health (P41EB015871-27)National Cancer Institute (U.S.) (P30-CA14051

Structure-function study on pepsin and comparative digestion of albumin and gliadins

Thesis (M.A.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at [email protected]. Thank you.Background: Although pepsin's crystal structure has been analyzed extensively, enzymatic components and sequential characteristics that make pepsin so functional and resilient in the acidity of the stomach, remain to be determined. Upon comprehending this phenomenon, researchers can begin to utilize and exploit such properties from other enzymes in an attempt to engineer enzymes that are gastric-active, with potential application in gastrointestinal (GI) disorders. Objective: The aim of this study was to investigate pepsin protease activities under ionic conditions representative of the stomach and representative of the duodenum. Two proteins were selected as protease substrates: albumin (test protein) and gliadin, which is an abundant dietary protein contained in cereals. The latter was chosen since it is a protein that is implicated in celiac disease. Celiac disease is a gastrointestinal (GI) disorder that renders patients intolerant to gluten and its molecular components, gliadins and gluten ins. Once dietary gluten reaches the duodenum of a celiac patient, T cells trigger an inflammatory response. Besides pepsin, we also investigated the susceptibility of both substrates to trypsin and chymotrypsin. All enzymes were standardized to the same unit concentration and digestion was carried out in solutions mimicking those of the stomach (pH 2.0 for pepsin) and the duodenum (pH 8.0, for trypsin and chymotrypsin). Enzymatic efficacy was assessed to determine which enzyme, at the standardized concentration, is most effective at digesting albumin and gliadins. Methods: Stimulated gastric fluid (SGF) was prepared, consisting of NaCl (35 mM) and HCl (84 mM). Albumin was dissolved to 2 mg/ml in water and gliadins in 60% ethanol stock solution to 2 mg/ml. Pepsin (3 ,900 U/mg solid) was dissolved in SGF to 1 mg/ml (3,900 U/ml). Chymotrypsin (66 U/mg solid) and trypsin (9 ,700 U/solid) were each dissolved to 1 mg/ml in duodenal buffer (DB). Gliadin or albumin were mixed with aliquots of pepsin, trypsin, or chymotrypsin yielding final albumin and gliadin concentrations of 100 µg/ml and final enzyme concentrations of 3.9 U/ml. The samples were incubated at 37 degrees in a waterbath. After various time points (t=O, 2, 5, 10, 20, and 30 min.), sample aliquots were removed and boiled to abolish pepsin enzymatic activity. Samples were then dried, re-suspended, and analyzed by SDS PAGE. After staining the gels with coomassie brilliant blue, proteolysis of albumin by pepsin was assessed visually and by densitometric analysis. Results: The SDS-PAGE gels revealed that the added amount of albumin was completely digested by pepsin after approximately 10 minutes of incubation. Mixed gliadins, however, remained mostly undigested throughout the entire incubatory period. Trypsin and chymotrypsin, however, at the tested unit concentration of 3.9 U/ml, were not effective at digesting albumin or gliadins throughout the entire incubatory period. Conclusion: Our results confirmed that gliadin is incompletely digested by pepsin in SGF solution. It is feasible that upon prolonged incubation of gliadin with pepsin, some degradation might occur, which will be further investigated. Furthermore, Trypsin and chymotrypsin are both ineffective when diluted to the same unit concentration as pepsin. Prolonging the incubatory time frame (t=0-30 minutes) may have some impact on digestion, however, it seems the most logical means of determining efficacy would be to determine the effective U/ml concentrations for trypsin and chymotrypsin. Only then can the efficacy of both duodenal enzymes towards albumin and gliadins be quantitatively compared to that of pepsin

Frontline Science: Splenic progenitors aid in maintaining high neutrophil numbers at sites of sterile chronic inflammation

Neutrophils are constantly generated from hematopoietic stem and progenitor cells in the bone marrow to maintain high numbers in circulation. A considerable number of neutrophils and their progenitors have been shown to be present in the spleen too; however, their exact role in this organ remains unclear. Herein, we sought to study the function of splenic neutrophils and their progenitors using a mouse model for sterile, peritoneal inflammation. In this microcapsule device implantation model, we show chronic neutrophil presence at implant sites, with recruitment from circulation as the primary mechanism for their prevalence in the peritoneal exudate. Furthermore, we demonstrate that progenitor populations in the spleen play a key role in maintaining elevated neutrophil numbers. Our results provide new insight into the role for splenic neutrophils and their progenitors and establish a model to study neutrophil function during sterile inflammation

Colony Stimulating Factor-1 Receptor is a central component of the foreign body response to biomaterial implants in rodents and non-human primates

Host recognition and immune-mediated foreign body response (FBR) to biomaterials can compromise the performance of implanted medical devices. To identify key cell and cytokine targets, here we perform in-depth systems analysis of innate and adaptive immune system responses to implanted biomaterials in rodents and non-human primates. While macrophages are indispensable to the fibrotic cascade, surprisingly neutrophils and complement are not. Macrophages, via CXCL13, lead to downstream B cell recruitment, which further potentiated fibrosis, as confirmed by B cell knock out and CXCL13 neutralization. Interestingly, Colony Stimulating Factor-1 Receptor (CSF1R) is significantly increased following implantation of multiple biomaterial classes: ceramic, polymer, and hydrogel. Its inhibition, like macrophage depletion, leads to complete loss of fibrosis, but spares other macrophage functions such as wound healing, ROS production, and phagocytosis. Our results indicate targeting CSF1R may allow for a more selective method of fibrosis inhibition, and improve biomaterial biocompatibility without the need for broad immunosuppression

Long-term glycemic control using polymer-encapsulated human stem cell–derived beta cells in immune-competent mice

The transplantation of glucose-responsive, insulin-producing cells offers the potential for restoring glycemic control in individuals with diabetes. Pancreas transplantation and the infusion of cadaveric islets are currently implemented clinically, but these approaches are limited by the adverse effects of immunosuppressive therapy over the lifetime of the recipient and the limited supply of donor tissue. The latter concern may be addressed by recently described glucose-responsive mature beta cells that are derived from human embryonic stem cells (referred to as SC-β cells), which may represent an unlimited source of human cells for pancreas replacement therapy. Strategies to address the immunosuppression concerns include immunoisolation of insulin-producing cells with porous biomaterials that function as an immune barrier. However, clinical implementation has been challenging because of host immune responses to the implant materials. Here we report the first long-term glycemic correction of a diabetic, immunocompetent animal model using human SC-β cells. SC-β cells were encapsulated with alginate derivatives capable of mitigating foreign-body responses in vivo and implanted into the intraperitoneal space of C57BL/6J mice treated with streptozotocin, which is an animal model for chemically induced type 1 diabetes. These implants induced glycemic correction without any immunosuppression until their removal at 174 d after implantation. Human C-peptide concentrations and in vivo glucose responsiveness demonstrated therapeutically relevant glycemic control. Implants retrieved after 174 d contained viable insulin-producing cells.Leona M. and Harry B. Helmsley Charitable Trust (Grant 3-SRA-2014-285-M-R)National Institutes of Health (U.S.) (Grants EB000244, EB000351, DE013023, and CA151884)Tayebati Family FoundationUnited States. Dept. of Defense. Congressionally Directed Medical Research Programs (Grant W81XWH-13-1-0215)Juvenile Diabetes Research Foundation International (Grant 3-2013-178

Size- and shape-dependent foreign body immune response to materials implanted in rodents and non-human primates

The efficacy of implanted biomedical devices is often compromised by host recognition and subsequent foreign body responses. Here, we demonstrate the role of the geometry of implanted materials on their biocompatibility in vivo. In rodent and non-human primate animal models, implanted spheres 1.5 mm and above in diameter across a broad spectrum of materials, including hydrogels, ceramics, metals, and plastics, significantly abrogated foreign body reactions and fibrosis when compared to smaller spheres. We also show that for encapsulated rat pancreatic islet cells transplanted into streptozotocin-treated diabetic C57BL/6 mice, islets prepared in 1.5 mm alginate capsules were able to restore blood-glucose control for up to 180 days, a period more than 5-fold longer than for transplanted grafts encapsulated within conventionally sized 0.5-mm alginate capsules. Our findings suggest that the in vivo biocompatibility of biomedical devices can be significantly improved by simply tuning their spherical dimensions