A Tissue-Engineered Construct Based on a Decellularized Scaffold and the Islets of Langerhans: A Streptozotocin-Induced Diabetic Model

Fajarwati I., Solihin D.D., Wresdiyati T., Batubara I.

Alloxan and streptozotocin are the most popular diabetogenic agents in assessing antidiabetic activity. Self-recovery, indicated by unstable hyperglycemia conditions in animals induced by those agents, becomes a significant disturbance to accurate examination. This study aimed to evaluate and reveal the self-recovery incidence in Sprague Dawley rats induced with alloxan and streptozotocin. Each dose of alloxan (120, 150, 180 mg/kg) and streptozotocin (40, 50, 60 mg/kg) was administered through intraperitoneal injection. The results showed that each dose of alloxan induced self-recovery incidence. In rats given streptozotocin, self-recovery only occurred at a dose of 40 mg/kg. The other higher doses of streptozotocin induced stable hyperglycemia. Furthermore, this study revealed two types of self-recovery, namely temporary recovery and end recovery. Temporary recovery occurred in rats given alloxan, during end recovery in alloxan and streptozotocin. The examination of insulin levels showed a significant reduction in the temporary recovery and stable diabetic rats compared to the end recovery rats. Besides, the bodyweight of rats was also affected by different incidences of self-recovery. This study recommends paying more attention to the possibility of self-recovery in obtaining animal models of diabetes, emphasizing the determination of suitable diabetogenic agents and proper doses to reduce self-recovery incidences. The finding of temporary recovery in rats receiving alloxan indicates that alloxan induced delayed diabetes in rats.

Lim L.Y., Ding S.S., Muthukumaran P., Teoh S.H., Koh Y., Teo A.K.

Diabetes mellitus is a global disease requiring long-term treatment and monitoring. At present, pancreas or islet transplantation is the only reliable treatment for achieving stable euglycemia in Type I diabetes patients. However, the shortage of viable pancreata for transplantation limits the use of this therapy for the majority of patients. Organ decellularization and recellularization is emerging as a promising solution to overcome the shortage of viable organs for transplantation by providing a potential alternative source of donor organs. Several studies on decellularization and recellularization of rodent, porcine, and human pancreata have been performed, and show promise for generating usable decellularized pancreas scaffolds for subsequent recellularization and transplantation. In this state-of-the-art review, we provide an overview of the latest advances in pancreas decellularization, recellularization, and revascularization. We also discuss clinical considerations such as potential transplantation sites, donor source, and immune considerations. We conclude with an outlook on the remaining work that needs to be done in order to realize the goal of using this technology to create bioengineered pancreata for transplantation in diabetes patients. STATEMENT OF SIGNIFICANCE: Pancreas or islet transplantation is a means of providing insulin-independence in diabetes patients. However, due to the shortage of viable pancreata, whole-organ decellularization and recellularization is emerging as a promising solution to overcome organ shortage for transplantation. Several studies on decellularization and recellularization of rodent, porcine, and human pancreata have shown promise for generating usable decellularized pancreas scaffolds for subsequent recellularization and transplantation. In this state-of-the-art review, we highlight the latest advances in pancreas decellularization, recellularization, and revascularization. We also discuss clinical considerations such as potential transplantation sites, donor source, and immune considerations. We conclude with future work that needs to be done in order to realize clinical translation of bioengineered pancreata for transplantation in diabetes patients.

Yayla M., Binnetoglu D.

Sevastianov V.I., Ponomareva A.S., Baranova N.V., Kirsanova L.A., Basok Y.B., Nemets E.A., Kruglov D.N., Miloserdov I.A., Gautier S.V.

A significant lack of donor organs restricts the opportunity to obtain tissue-specific scaffolds for tissue-engineering technologies. One of the acceptable solutions is the development of decellularization protocols for a human donor pancreas unsuitable for transplantation. A protocol of obtaining a biocompatible tissue-specific scaffold from decellularized fragments with pronounced human pancreas lipomatosis signs with preserved basic fibrillary proteins of a pancreatic tissue extracellular matrix was developed. The scaffold supports the adhesion and proliferation of human adipose derived stem cell (hADSCs) and prolongs the viability and insulin-producing function of pancreatic islets. Experiments conducted allow for the reliance on the prospects of using the donor pancreas unsuitable for transplantation in the technologies of tissue engineering and regenerative medicine, including the development of a tissue equivalent of a pancreas.

Sevastianov V.I., Basok Y.B., Grigoriev A.M., Nemets E.A., Kirillova A.D., Kirsanova L.A., Lazhko A.E., Subbot A., Kravchik M.V., Khesuani Y.D., Koudan E.V., Gautier S.V.

AbstractOne of the approaches to restoring the structure of damaged cartilage tissue is an intra‐articular injection of tissue‐engineered medical products (TEMPs) consisting of biocompatible matrices loaded with cells. The most interesting are the absorbable matrices from decellularized tissues, provided that the cellular material is completely removed from them with the maximum possible preservation of the structure and composition of the natural extracellular matrix. The present study investigated the mechanical, biochemical, and biological properties of decellularized porcine cartilage microparticles (DCMps) obtained by techniques, differing only in physical treatments, such as freeze–thaw cycling (Protocol 1), supercritical carbon dioxide fluid (Protocol 2) and ultrasound (Protocol 3). Full tissue decellularization was achieved, as confirmed by the histological analysis and DNA quantification, though all the resultant DCMps had reduced glycosaminoglycans (GAGs) and collagen. The elastic modulus of all DCMp samples was also significantly reduced. Most notably, DCMps prepared with Protocol 3 significantly outperformed other samples in viability and the chondroinduction of the human adipose‐derived stem cells (hADSCs), with a higher GAG production per DNA content. A positive ECM staining for type II collagen was also detected only in cartilage‐like structures based on ultrasound‐treated DCMps. The biocompatibility of a xenogenic DCMps obtained with Protocol 3 has been confirmed for a 6‐month implantation in the thigh muscle tissue of mature rats (n = 18). Overall, the results showed that the porcine cartilage microparticles decellularized by a combination of detergents, ultrasound and DNase could be a promising source of scaffolds for TEMPs for cartilage reconstruction.

Eguchi N., Damyar K., Alexander M., Dafoe D., Lakey J.R., Ichii H.

Islet cell transplantation has become a favorable therapeutic approach in the treatment of Type 1 Diabetes due to the lower surgical risks and potential complications compared to conventional pancreas transplantation. Despite significant improvements in islet cell transplantation outcomes, several limitations hamper long-term graft survival due to tremendous damage and loss of islet cells during the islet cell transplantation process. Oxidative stress has been identified as an omnipresent stressor that negatively affects both the viability and function of isolated islets. Furthermore, it has been established that at baseline, pancreatic β cells exhibit reduced antioxidative capacity, rendering them even more susceptible to oxidative stress during metabolic stress. Thus, identifying antioxidants capable of conferring protection against oxidative stressors present throughout the islet transplantation process is a valuable approach to improving the overall outcomes of islet cell transplantation. In this review we discuss the potential application of antioxidative therapy during each step of islet cell transplantation.

Dubus M., Scomazzon L., Chevrier J., Ledouble C., Baldit A., Braux J., Gindraux F., Boulagnon C., Audonnet S., Colin M., Rammal H., Mauprivez C., Kerdjoudj H.

Of all biologic matrices, decellularized tissues have emerged as a promising tool in the field of regenerative medicine. Few empirical clinical studies have shown that Wharton’s jelly (WJ) of the human umbilical cord promotes wound closure and reduces wound-related infections. In this scope, we herein investigated whether decellularized (DC)-WJ could be used as an engineered biomaterial. In comparison with devitalized (DV)-WJ, our results showed an inherent effect of DC-WJ on Gram positive (S. aureus and S. epidermidis) and Gram negative (E. coli and P. aeruginosa) growth and adhesion. Although DC-WJ activated the neutrophils and monocytes in a comparable magnitude to DV-WJ, macrophages modulated their phenotypes and polarization states from the resting M0 phenotype to the hybrid M1/M2 phenotype in the presence of DC-WJ. M1 phenotype was predominant in the presence of DV-WJ. Finally, the subcutaneous implantation of DC-WJ showed total resorption after three weeks of implantation without any sign of foreign body reaction. These significant data shed light on the potential regenerative application of DC-WJ in providing a suitable biomaterial for tissue regenerative medicine and an ideal strategy to prevent wound-associated infections.

Cayabyab F., Nih L.R., Yoshihara E.

Diabetes is a complex disease that affects over 400 million people worldwide. The life-long insulin injections and continuous blood glucose monitoring required in type 1 diabetes (T1D) represent a tremendous clinical and economic burdens that urges the need for a medical solution. Pancreatic islet transplantation holds great promise in the treatment of T1D; however, the difficulty in regulating post-transplantation immune reactions to avoid both allogenic and autoimmune graft rejection represent a bottleneck in the field of islet transplantation. Cell replacement strategies have been performed in hepatic, intramuscular, omentum, and subcutaneous sites, and have been performed in both animal models and human patients. However more optimal transplantation sites and methods of improving islet graft survival are needed to successfully translate these studies to a clinical relevant therapy. In this review, we summarize the current progress in the field as well as methods and sites of islet transplantation, including stem cell-derived functional human islets. We also discuss the contribution of immune cells, vessel formation, extracellular matrix, and nutritional supply on islet graft survival. Developing new transplantation sites with emerging technologies to improve islet graft survival and simplify immune regulation will greatly benefit the future success of islet cell therapy in the treatment of diabetes.

Lepedda A.J., Nieddu G., Formato M., Baker M.B., Fernández-Pérez J., Moroni L.

Cardiovascular diseases represent the number one cause of death globally, with atherosclerosis a major contributor. Despite the clinical need for functional arterial substitutes, success has been limited to arterial replacements of large-caliber vessels (diameter > 6 mm), leaving the bulk of demand unmet. In this respect, one of the most challenging goals in tissue engineering is to design a “bioactive” resorbable scaffold, analogous to the natural extracellular matrix (ECM), able to guide the process of vascular tissue regeneration. Besides adequate mechanical properties to sustain the hemodynamic flow forces, scaffold’s properties should include biocompatibility, controlled biodegradability with non-toxic products, low inflammatory/thrombotic potential, porosity, and a specific combination of molecular signals allowing vascular cells to attach, proliferate and synthesize their own ECM. Different fabrication methods, such as phase separation, self-assembly and electrospinning are currently used to obtain nanofibrous scaffolds with a well-organized architecture and mechanical properties suitable for vascular tissue regeneration. However, several studies have shown that naked scaffolds, although fabricated with biocompatible polymers, represent a poor substrate to be populated by vascular cells. In this respect, surface functionalization with bioactive natural molecules, such as collagen, elastin, fibrinogen, silk fibroin, alginate, chitosan, dextran, glycosaminoglycans (GAGs), and growth factors has proven to be effective. GAGs are complex anionic unbranched heteropolysaccharides that represent major structural and functional ECM components of connective tissues. GAGs are very heterogeneous in terms of type of repeating disaccharide unit, relative molecular mass, charge density, degree and pattern of sulfation, degree of epimerization and physicochemical properties. These molecules participate in a number of vascular events such as the regulation of vascular permeability, lipid metabolism, hemostasis, and thrombosis, but also interact with vascular cells, growth factors, and cytokines to modulate cell adhesion, migration, and proliferation. The primary goal of this review is to perform a critical analysis of the last twenty-years of literature in which GAGs have been used as molecular cues, able to guide the processes leading to correct endothelialization and neo-artery formation, as well as to provide readers with an overall picture of their potential as functional molecules for small-diameter vascular regeneration.

Zhang Y., Zhang J., Hong M., Huang J., Wang R., Tan B., Huang P., Cao H.

Abstract Aim: This study was performed to optimize the experimental conditions in streptozotocin (STZ)-induced diabetic model by using Sprague-Dawley (SD) rats to evaluate the stability of the model.Methods: In addition to the control group, the male and female SD rats were randomly divided into the following treatment groups (with six rats per group): STZ 45 (45 mg/kg STZ); STZ 65 (65 mg/kg STZ); STZ 85 (85 mg/kg STZ); high-fat diet with STZ 45; high-fat diet with STZ 65; and high-fat diet with STZ 85. Changes in the body weight and blood glucose were observed dynamically. Results: No significant differences were found in the blood glucose or body weight between the STZ 45 and control groups in both male and female rats, whether or not the rats were on a high-fat diet. However, significant differences were found in the blood glucose between the high-dose STZ and control groups in both male and female rats, regardless of whether the rats were on a high-fat diet or not (P<0.05 or P<0.01). Compared with the control group, significant differences in the blood glucose levels (P<0.05 or P<0.01) and higher blood glucose levels were found in the male rats fed with normal diet than those of rats fed with high-fat diet.Conclusions: In this study, male rats fed with ordinary feed and injected with 65 mg/kg STZ were the most stable and ideal diabetic rats.

Uday Chandrika K., Tripathi R., Kameshwari Y., Rangaraj N., Mahesh Kumar J., Singh S.

Tissue engineering centers on creating a niche similar to the natural one, with a purpose of developing an organ construct. A natural scaffold can replace none while creating a scaffold unique to each tissue in composition, architecture and cues that regulate the character of cells. Whole pancreas from mouse was decellularized using detergent and enzymes, followed by recellularizing with MSC from human placenta. This construct was transplanted in streptozotocin induced diabetic mice. Histopathology of both decellularized and recellularized transplanted pancreas and qPCR analysis were performed to assess its recovery. Decellularization removes the cells leaving behind extracellular matrix rich natural scaffold. After reseeding with mesenchymal stem cells, these cells differentiate into pancreas specific cells. Upon transplantation in streptozotocin induced diabetic mice, this organ was capable of restoring its histomorphology and functioning. Restoration of endocrine (islets), the exocrine region (acinar) and vascular network was seen in transplanted pancreas. The process of functional recovery of endocrine system took about 20 days when the mice start showing blood glucose reduction, though none achieved gluconormalization. Natural decellularized scaffolds of soft organs can be refunctionalized using recipient’s mesenchymal stem cells to restore structure and function; and counter immune problems arising during transplantation.

Mendibil U., Ruiz-Hernandez R., Retegi-Carrion S., Garcia-Urquia N., Olalde-Graells B., Abarrategi A.

The extracellular matrix (ECM) is a complex network with multiple functions, including specific functions during tissue regeneration. Precisely, the properties of the ECM have been thoroughly used in tissue engineering and regenerative medicine research, aiming to restore the function of damaged or dysfunctional tissues. Tissue decellularization is gaining momentum as a technique to obtain potentially implantable decellularized extracellular matrix (dECM) with well-preserved key components. Interestingly, the tissue-specific dECM is becoming a feasible option to carry out regenerative medicine research, with multiple advantages compared to other approaches. This review provides an overview of the most common methods used to obtain the dECM and summarizes the strategies adopted to decellularize specific tissues, aiming to provide a helpful guide for future research development.

Norris J.M., Johnson R.K., Stene L.C.

Type 1 diabetes is a chronic, immune-mediated disease characterised by the destruction of insulin-producing cells. Standardised registry data show that type 1 diabetes incidence has increased 3-4% over the past three decades, supporting the role of environmental factors. Although several factors have been associated with type 1 diabetes, none of the associations are of a magnitude that could explain the rapid increase in incidence alone. Moreover, evidence of changing prevalence of these exposures over time is insufficient. Multiple factors could simultaneously explain the changing type 1 diabetes incidence, or the magnitude of observed associations could have been underestimated because of exposure measurement error, or the mismodelling of complex exposure-time-response relationships. The identification of environmental factors influencing the risk of type 1 diabetes and increased understanding of the cause at the individual level, regardless of the ability to explain the changing incidence at the population level, is important because of the implications for prevention.

Xia C., Mei S., Gu C., Zheng L., Fang C., Shi Y., Wu K., Lu T., Jin Y., Lin X., Chen P.

Articular cartilage lacks self-healing capacity, and there is no effective therapy facilitating cartilage repair. Osteoarthritis (OA) due to cartilage defects represents large and increasing healthcare burdens worldwide. Nowadays, the generation of scaffolds to preserve bioactive factors and the biophysical environment has received increasing attention. Furthermore, improved decellularization technology has provided novel insights into OA treatment. This review provides a comparative account of different cartilage defect therapies. Furthermore, some recent effective decellularization protocols have been discussed. In particular, this review focuses on the decellularization ratio of each protocol. Moreover, these protocols were compared particularly on the basis of immunogenicity and mechanical functionality. Further, various recellularization methods have been enlisted and the reparative capacity of decellularized cartilage scaffolds is evaluated herein. The advantages and limitations of different recellularization processes have been described herein. This provides a basis for the generation of decellularized cartilage scaffolds, thereby potentially promoting the possibility of decellularization as a clinical therapeutic target.

Gazia C., Gaffley M., Asthana A., Chaimov D., Orlando G.

Emerging technologies in regenerative medicine are crucial to overcome the organ donor shortage and the limitations in conventional diabetes treatments. A bioengineered pancreas, obtained through the development of a three-dimensional scaffold from both animal and human extracellular matrix, represents the endeavor to provide alternative therapies to pancreatic transplantation. This alternative hopes to provide the needed components, supply a temporary substrate that enhances cellular function, and aid organized development before implantation. This chapter will present an overview of the different techniques used to engineer scaffolds from natural and synthetic materials. We will discuss their main properties and applications in the regenerative medicine pancreatic research field.