Monitoring the macrophage response towards biomaterial implants using label-free imaging.

Understanding the immune system's foreign body response (FBR) is essential when developing and validating a biomaterial. Macrophage activation and proliferation are critical events in FBR that can determine the material's biocompatibility and fate in vivo. In this study, two different macro-encapsulation pouches intended for pancreatic islet transplantation were implanted into streptozotocin-induced diabetes rat models for 15 days. Post-explantation, the fibrotic capsules were analyzed by standard immunohistochemistry as well as non-invasive Raman microspectroscopy to determine the degree of FBR induced by both materials. The potential of Raman microspectroscopy to discern different processes of FBR was investigated and it was shown that Raman microspectroscopy is capable of targeting ECM components of the fibrotic capsule as well as pro and anti-inflammatory macrophage activation states, in a molecular-sensitive and marker-independent manner. In combination with multivariate analysis, spectral shifts reflecting conformational differences in Col I were identified and allowed to discriminate fibrotic and native interstitial connective tissue fibers. Moreover, spectral signatures retrieved from nuclei demonstrated changes in methylation states of nucleic acids in M1 and M2 phenotypes, relevant as indicator for fibrosis progression. This study could successfully implement Raman microspectroscopy as complementary tool to study in vivo immune-compatibility providing insightful information of FBR of biomaterials and medical devices, post-implantation.

Raman microspectroscopy identifies fibrotic tissues in collagen-related disorders via deconvoluted collagen type I spectra.

Fibrosis is a consequence of the pathological remodeling of extracellular matrix (ECM) structures in the connective tissue of an organ. It is often caused by chronic inflammation, which over time, progressively leads to an excess deposition of collagen type I (COL I) that replaces healthy tissue structures, in many cases leaving a stiff scar. Increasing fibrosis can lead to organ failure and death; therefore, developing methods that potentially allow real-time monitoring of early onset or progression of fibrosis are highly valuable. In this study, the ECM structures of diseased and healthy human tissue from multiple organs were investigated for the presence of fibrosis using routine histology and marker-independent Raman microspectroscopy and Raman imaging. Spectral deconvolution of COL I Raman spectra allowed the discrimination of fibrotic and non-fibrotic COL I fibers. Statistically significant differences were identified in the amide I region of the spectral subpeak at 1608 cm-1, which was deemed to be representative for structural changes in COL I fibers in all examined fibrotic tissues. Raman spectroscopy-based methods in combination with this newly discovered spectroscopic biomarker potentially offer a diagnostic approach to non-invasively track and monitor the progression of fibrosis. STATEMENT OF SIGNIFICANCE: Current diagnosis of fibrosis still relies on histopathological examination with invasive biopsy procedures. Although, several non-invasive imaging techniques such as positron emission tomography, single-photon emission computed tomography and second harmonic generation are gradually employed in preclinical or clinical studies, these techniques are limited in spatial resolution and the morphological interpretation highly relies on individual experience and knowledge. In this study, we propose a non-destructive technique, Raman microspectroscopy, to discriminate fibrotic changes of collagen type I based on a molecular biomarker. The changes of the secondary structure of collagen type I can be identified by spectral deconvolution, which potentially can provide an automatic diagnosis for fibrotic tissues in the clinical applicaion.

Decorin improves human pancreatic ß-cell function and regulates ECM expression in vitro.

Transplantation of islets of Langerhans is a promising alternative treatment strategy in severe cases of type 1 diabetes mellitus; however, the success rate is limited by the survival rate of the cells post-transplantation. Restoration of the native pancreatic niche during transplantation potentially can help to improve cell viability and function. Here, we assessed for the first time the regulatory role of the small leucine-rich proteoglycan decorin (DCN) in insulin secretion in human ß-cells, and its impact on pancreatic extracellular matrix (ECM) protein expression in vitro. In depth analyses utilizing next-generation sequencing as well as Raman microspectroscopy and Raman imaging identified pathways related to glucose metabolism to be upregulated in DCN-treated cells, including oxidative phosphorylation within the mitochondria as well as proteins and lipids of the endoplasmic reticulum. We further showed the effectiveness of DCN in a transplantation setting by treating collagen type 1-encapsulated ß-cell-containing pseudo-islets with DCN. Taken together, in this study, we demonstrate the potential of DCN to improve the function of insulin-secreting ß-cells while reducing the expression of ECM proteins affiliated with fibrotic capsule formation, making DCN a highly promising therapeutic agent for islet transplantation.

Type 1 diabetes and engineering enhanced islet transplantation.

The development of new therapeutic approaches to treat type 1 diabetes mellitus (T1D) relies on the precise understanding and deciphering of insulin-secreting ß-cell biology, as well as the mechanisms responsible for their autoimmune destruction. ß-cell or islet transplantation is viewed as a potential long-term therapy for the millions of patients with diabetes. To advance the field of insulin-secreting cell transplantation, two main research areas are currently investigated by the scientific community: (1) the identification of the developmental pathways that drive the differentiation of stem cells into insulin-producing cells, providing an inexhaustible source of cells; and (2) transplantation strategies and engineered transplants to provide protection and enhance the functionality of transplanted cells. In this review, we discuss the biology of pancreatic ß-cells, pathology of T1D and current state of ß-cell differentiation. We give a comprehensive view and discuss the different possibilities to engineer enhanced insulin-secreting cell/islet transplantation from a translational perspective.

The Foreign Body Response to an Implantable Therapeutic Reservoir in a Diabetic Rodent Model.

Advancements in type 1 diabetes mellitus treatments have vastly improved in recent years. The move toward a bioartificial pancreas and other fully implantable systems could help restore patient's glycemic control. However, the long-term success of implantable medical devices is often hindered by the foreign body response. Fibrous encapsulation "walls off" the implant to the surrounding tissue, impairing its functionality. In this study we aim to examine how streptozotocin-induced diabetes affects fibrous capsule formation and composition surrounding implantable drug delivery devices following subcutaneous implantation in a rodent model. After 2 weeks of implantation, the fibrous capsule surrounding the devices were examined by means of Raman spectroscopy, micro-computed tomography (µCT), and histological analysis. Results revealed no change in mean fibrotic capsule thickness between diabetic and healthy animals as measured by µCT. Macrophage numbers (CCR7 and CD163 positive) remained similar across all groups. True component analysis also showed no quantitative difference in the alpha-smooth muscle actin and extracellular matrix proteins. Although principal component analysis revealed significant secondary structural difference in collagen I in the diabetic group, no evidence indicates an influence on fibrous capsule composition surrounding the device. This study confirms that diabetes did not have an effect on the fibrous capsule thickness or composition surrounding our implantable drug delivery device. Impact Statement Understanding the impact diabetes has on the foreign body response (FBR) to our implanted material is essential for developing an effective drug delivery device. We used several approaches (Raman spectroscopy and micro-computed tomography imaging) to demonstrate a well-rounded understanding of the diabetic impact on the FBR to our devices, which is imperative for its clinical translation.

Fluorescence lifetime metabolic mapping of hypoxia-induced damage in pancreatic pseudo-islets.

Pancreatic islet isolation from donor pancreases is an essential step for the transplantation of insulin-secreting ß-cells as a therapy to treat type 1 diabetes mellitus. This process however damages islet basement membranes, which can lead to islet dysfunction or death. Posttransplantation, islets are further stressed by a hypoxic environment and immune reactions that cause poor engraftment and graft failure. The current standards to assess islet quality before transplantation are destructive procedures, performed on a small islet population that does not reflect the heterogeneity of large isolated islet batches. In this study, we incorporated fluorescence lifetime imaging microscopy (FLIM) into a pancreas-on-chip system to establish a protocol to noninvasively assess the viability and functionality of pancreatic ß-cells in a three-dimensional in vitro model (= pseudo-islets). We demonstrate how (pre-) hypoxic ß-cell-composed pseudo-islets can be discriminated from healthy functional pseudo-islets according to their FLIM-based metabolic profiles. The use of FLIM during the pretransplantation pancreatic islet selection process has the potential to improve the outcome of ß-cell islet transplantation.