Recellularized Scaffolds Research Articles

Kidney Mesenchymal Stem Cell Differentiation: Effect of Scaffold and Basic Fibroblast Growth Factor.

Background: Chronic kidney disease (CKD) poses a global health challenge, and it needs alternative therapeutic approaches for patients with end-stage renal disease (ESRD). Although organ transplantation is effective, it faces challenges such as declining quality of life, immunological responses, transplant rejection, and donor shortages. Tissue engineering, by using suitable scaffolds, cells, and growth factors, emerges as a promising treatment option for kidney regeneration. Experiment: We precisely decellularized scaffold, derived from rat kidneys while maintaining its native three-dimensional (3D) architecture. The efficiency of decellularization was evaluated through histological examinations, including hematoxylin and eosin, periodic acid-Schiff, and DAPI staining, as well as scanning electron microscopy. The scaffolds were then recellularized with kidney mesenchymal stem cells (kMSCs), and their adhesion, proliferation, and differentiation were assessed over 1, 2, and 3 weeks. The expression of specific renal markers, including Wt-1, ZO-1, AQP-1, and ANG-1, was examined through quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in monolayer and 3D cultures. Results: The infiltration rate of cells into the scaffold increased in a time-dependent manner, and the expression of specific renal markers significantly increased, demonstrating successful differentiation of kMSCs within the scaffold. The application of basic fibroblast growth factor (bFGF) could intensify the expression of kidney-specific genes. Conclusions: The study highlighted the importance of preserving the 3D architecture of the scaffold during decellularization to achieve optimal cellular responses. Moreover, the capacity of mesenchymal stem cells in recellularized scaffolds facilitated tissue regeneration.

Strategies for decellularization, re-cellularIzation and crosslinking in liver bioengineering.

Liver transplantation is the only definitive treatment for end-stage liver disease and its availability is restricted by organ donor shortages. The development of liver bioengineering provides the probability to create a functional alternative to reduce the gap in organ demand and supply. Decellularized liver scaffolds have been widely applied in bioengineering because they can mimic the native liver microenvironment and retain extracellular matrix (ECM) components. Multiple approaches including chemical, physical and biological methods have been developed for liver decellularization in current studies, but a full set of unified criteria has not yet been established. Each method has its advantages and drawbacks that influence the microstructure and ligand landscape of decellularized liver scaffolds. Optimizing a decellularization method to eliminate cell material while retaining as much of the ECM intact as possible is therefore important for biological scaffold applications. Furthermore, crosslinking strategies can improve the biological performance of scaffolds, including reinforcing biomechanics, delaying degradation in vivo and reducing immune rejection, which can better promote the integration of re-cellularized scaffolds with host tissue and influence the reconstruction process. In this review, we aim to present the different liver decellularization techniques, the crosslinking methods to improve scaffold characteristics with crosslinking and the preparation of soluble ECM.

Evaluation of Vascular Endothelial Growth Factor Gene Expression in Recellularized Liver Tissue by Mouse Embryo Fibroblast.

The aim of the present study was to evaluate alterations in the vegf gene expression as an angiogenic factor in mouse embryo fibroblasts seeded on the decellularized liver fragments. Liver tissue samples (n = 10) collected from adult male mice were randomly divided into decellularized and native control groups. Tissues were decellularized by treating with 1% Triton X-100 and 0.1% SDS for 24 hours and assessed by H&E staining and SEM. Then DNA content analysis and toxicity tests were performed. By centrifugation, DiI-labeled mouse embryo fibroblasts were seeded on each scaffold and cultured for one week. The recellularized scaffolds were studied by H&E staining, SEM, and LSCM. After RNA extraction and cDNA synthesis, the expression of the vegf gene in these samples was investigated using real-time RT-PCR. Our observations showed that the decellularized tissues had morphology and porous structure similar to the control group, and their DNA content significantly reduced (p < 0.05) and reached to 4.12% of the control group. The MTT test indicated no significant cellular toxicity for the decellularized scaffolds. Light microscopy, SEM, and LSCM observations confirmed the attachment and penetration of embryonic fibroblast cells on the surface and into different depths of the scaffolds. There was no statistically significant difference in terms of vegf gene expression in the cultured cells in the presence and absence of a scaffold. The reconstructed scaffold had no effect on vegf gene expression. Decellularized mouse liver tissue recellularized by embryonic fibroblasts could have an application in regenerative medicine.

Fabrication of bioartificial pancreas using decellularized rat testicular tissue

AimsDiabetes is a chronic disease that is associated with a decrease or disfunction of β-cell. In the present study, fabrication of bioartificial pancreas using MIN-6 β-cell line seeded in decellularized rat testicles was investigated. Main methodsIn this experimental study, the whole body of testes were decellularized and after characterization, were seeded by MIN-6 cell line. The expression of insulin-related genes and proteins including PDX-1, Glut2, Insulin, and Neurogenin-3 were evaluated. Insulin secretion was assessed under different concentrations of glucose. Seeded scaffolds with or without MIN-6 cells were transplanted to the rat's mesentery and their blood sugar and body weight were evaluated every three days for 28 days and analyzed with H&E staining. ResultsHistological assessments indicated the cells were completely removed after decellularization. The scaffold had no toxic impacts on the MIN-6 cells (P˂ 0.02).Insulin release in response to different concentrations of glucose in 3D culture (testis-ECM) was significantly more than the traditional 2D monolayer culture (P < 0.001). Moreover, the relative genes and proteins expression were significantly higher in the 3D culture, compared to the 2D control group. In vivo transplantation of the (testis- Extra Cellular Matrix) testis-ECM scaffolds showed appropriate positions for transplantation with angiogenesis and low infiltration of inflammatory cells. The recellularized scaffolds could drop blood sugar levels and increase the body-weight of STZ-diabetic rats (P < 0.01). SignificanceOur study clearly confirmed that ECM valuable organ scaffolds prepared by decellularization of the testicular tissue is suitable for the fabrication of bioartificial pancreas for transplantation.

Re-Cellularised Kidney Scaffold for Chikungunya Virus Propagation: A Novel Approach.

Background:Re-emerging viral attacks are catastrophic for health and economy. It is crucial to grasp the viral life cycle, replication and mutation policies and attack strategies. It is also absolute to fathom the cost-efficient antiviral remedies earliest possible.Methods:We propose to use a lab-grown organ (re-cellularized scaffold of sheep kidney) for viral culture and understand its interaction with extra-cellular matrices of the host tissue.Results:Our findings showed that the chikungunya virus (CHIKV) could be better replicated in tissue-engineered bio models than cell culture. A decrease in ds-DNA levels emphasized that CHIKV propagates within the re-cellularized and cell culture models. There was an increase in the viral titres (pfu/ml) in re-cellularized scaffolds and control groups. The lipid peroxidation levels were increased as the infection was progressed in cell culture as well as re-cellularized and control groups. The onset and progress of the CHIKV attacks (cellular infection) lead to transmembrane domain fatty acid peroxidation and DNA breakdown, landing in cellular apoptosis. Simultaneously cell viability was inversely proportional to non-viability, and it decreased as the infection progressed in all infected groups. Histological findings and extracellular matrix evaluation showed the impairment in medullary, cortex regions due to propagation of CHIKV and plaques generations.Conclusion:This method will be a breakthrough for future virus culture, drug interaction and to study its effect on extracellular matrix alterations. This study will also allow us to investigate the correct role of any vaccine or antiviral drugs and their effects on re-engineered organ matrices before moving towards the animal models.Graphical abstract

In vivo biocompatibility analysis of the recellularized canine tracheal scaffolds with canine epithelial and endothelial progenitor cells

ABSTRACT Decellularized extracellular matrix (ECM) has frequently been applied as a biomaterial for tissue engineering purposes. When implanted, their role can be essential for partial trachea replacement in patients that require a viable transplant solution. Acellular canine tracheal scaffolds with preserved ECM structure, flexibility, and proteins were obtained by high pressure vacuum decellularization. Here, we aimed to evaluate the cell adhesion and proliferation of canine tracheal epithelial cells (EpC) and canine yolk sac endothelial progenitor cells (YS) cultivated on canine decellularized tracheal scaffolds and test the in vivo biocompatibility of these recellularized scaffolds implanted in BALB-c nude mice. In order to evaluate the recellularization efficiency, scaffolds were evaluated by scanning electron microscopy (SEM), immunofluorescence, DNA quantification, mycoplasma test, and in vivo biocompatibility. The scaffolds sterility was confirmed, and EpC and YS cells were cultured by 7 and 14 days. We demonstrated by SEM, immunofluorescence, and genomic DNA analyzes cell adhesion to tracheal ECM. Then, recellularized scaffolds were in vivo subcutaneously implanted in mice and after 45 days, the fragments were collected and analyzed by Hematoxylin-Eosin and Gömori Trichrome staining and PCNA, CD4, CD8, and CD68 immunohistochemistry. In vivo results confirmed that the implanted tissue remains preserved and proliferative, and no fibrotic tissue process was observed in animals. Finally, our results showed the recellularization success due the preserved ECM proteins, and that these may be suitable to future preclinical studies applications for partial trachea replacement in tissue engineering.

Decellularized Splenic Matrix as a Scaffold for Spleen Bioengineering.

The spleen is considered a non-essential organ. However, its importance is increasingly clear, given the serious disorders caused by its absence or dysfunction, e.g., greater susceptibility to infections, thromboembolism and cancer. Surgical techniques to preserve the spleen and maintain splenic function have become increasingly common. However, the morbidity and mortality associated with its absence and dysfunction are still high. We used the decellularization technique to obtain a viable splenic scaffold for recellularization in vitro and propose the idea of bioengineered spleen transplantation to the host. We observed the maintenance of important structural components such as white pulp, marginal zone and red pulp, in addition to the network of vascular ducts. The decellularized scaffold presents minimal residual DNA and SDS, which are essential to prevent immunogenic responses and transplantation failure. Also, the main components of the splenic matrix were preserved after decellularization, with retention of approximately 72% in the matrisomal protein content. The scaffold we developed was partially recellularized with stromal cells from the spleen of neonatal rats, demonstrating adhesion, proliferation and viability of cells. Therefore, the splenic scaffold is very promising for use in studies on spleen reconstruction and transplantation, with the aim of complete recovery of splenic function.

Sequential adaptation of perfusion and transport conditions significantly improves vascular construct recellularization and biomechanics.

Recellularization of ex vivo-derived scaffolds remains a significant hurdle primarily due to the scaffolds subcellular pore size that restricts initial cell seeding to the scaffolds periphery and inhibits migration over time. With the aim to improve cell migration, repopulation, and graft mechanics, the effects of a four-step culture approach were assessed. Using an ex vivo-derived vein as a model scaffold, human smooth muscle cells were first seeded onto its ablumen (Step 1: 3 hr) and an aggressive 0-100% nutrient gradient (lumenal flow under hypotensive pressure) was created to initiate cell migration across the scaffold (Step 2: Day 0 to 19). The effects of a prolonged aggressive nutrient gradient created by this single lumenal flow was then compared with a dual flow (lumenal and ablumenal) in Step 3 (Day 20 to 30). Analyses showed that a single lumenal flow maintained for 30 days resulted in a higher proportion of cells migrating across the scaffold toward the vessel lumen (nutrient source), with improved distribution. In Step 4 (Day 31 to 45), the transition from hypotensive pressure (12/8 mmHg) to normotensive (arterial-like) pressure (120/80 mmHg) was assessed. It demonstrated that recellularized scaffolds exposed to arterial pressures have increased glycosaminoglycan deposition, physiological modulus, and Young's modulus. By using this stepwise conditioning, the challenging recellularization of a vein-based scaffold and its positive remodeling toward arterial biomechanics were obtained.

Induced cardiac progenitor cells repopulate decellularized mouse heart scaffolds and differentiate to generate cardiac tissue

Native myocardium has limited regenerative potential post injury. Advances in lineage reprogramming have provided promising cellular sources for regenerative medicine in addition to research applications. Recently we have shown that adult mouse fibroblasts can be reprogrammed to expandable, multipotent, induced cardiac progenitor cells (iCPCs) by employing forced expression of five cardiac factors along with activation of canonical Wnt and JAK/STAT signaling. Here we aim to further characterize iCPCs by highlighting their safety, ease of attainability, and functionality within a three-dimensional cardiac extracellular matrix scaffold. Specifically, iCPCs did not form teratomas in contrast to embryonic stem cells when injected into immunodeficient mice. iCPC reprogramming was achieved in wild type mouse fibroblasts without requiring a cardiac-specific reporter, solely utilizing morphological changes to identify, clonally isolate, and expand iCPCs, thus increasing the versatility of this technology. iCPCs also show the ability to repopulate decellularized native heart scaffolds and differentiated into organized structures containing cardiomyocytes, smooth muscle, and endothelial cells. Optical mapping of recellularized scaffolds shows field-stimulated calcium transients that propagate across islands of reconstituted tissue and bipolar local stimulation demonstrates cell-cell coupling within scaffolds. Overall, iCPCs provide a readily attainable, scalable, safe, and functional cell source for a variety of application including drug discovery, disease modeling, and regenerative therapy.

Rat lung decellularization using chemical detergents for lung tissue engineering

ABSTRACTAlthough pulmonary diseases account for a large number of deaths in the world, most have no treatment other than transplantation. New therapeutic methods for lung treatment include lung tissue engineering and regenerative medicine. Lung decellularization has been used to produce an appropriate scaffold for recellularization and implantation. We investigated 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) with sodium dodecyl sulfate (SDS) and Triton X-100 detergents for effecting rat lung decellularization. We evaluated using conventional histology, immunofluorescence staining and SEM methods for removing nuclear material while leaving intact extracellular matrix proteins and three-dimensional architecture. We investigated different concentrations of CHAPS, SDS and Triton X-100 for different periods. We found that 2 mM CHAPS + 0/1% SDS for 48 h was the best among the treatments investigated. Our method can be used to produce an appropriate scaffold for recellularization by stem cells and for investigations ex vivo and in vivo.

Lung bioengineering: advances and challenges in lung decellularization and recellularization.

Bioengineering the lung based on its natural extracellular matrix (ECM) offers novel opportunities to overcome the shortage of donors, to reduce chronic allograft rejections, and to improve the median survival rate of transplanted patients. During the last decade, lung tissue engineering has advanced rapidly to combine scaffolds, cells, and biologically active molecules into functional tissues to restore or improve the lung's main function, gas exchange. This review will inspect the current progress in lung bioengineering using decellularized and recellularized lung scaffolds and highlight future challenges in the field. Lung decellularization and recellularization protocols have provided researchers with tools to progress toward functional lung tissue engineering. However, there is continuous evolution and refinement particularly for optimization of lung recellularization. These further the possibility of developing a transplantable bioartificial lung. Bioengineering the lung using recellularized scaffolds could offer a curative option for patients with end-stage organ failure but its accomplishment remains unclear in the short-term. However, the state-of-the-art of techniques described in this review will increase our knowledge of the lung ECM and of chemical and mechanical cues which drive cell repopulation to improve the advances in lung regeneration and lung tissue engineering.

Evaluation of the host immune response to decellularized lung scaffolds derived from α-Gal knockout pigs in a non-human primate model

Whole organ tissue engineering is a promising approach to address organ shortages in many applications, including lung transplantation for patients with chronic pulmonary disease. Engineered lungs may be derived from animal sources after removing cellular content, exposing the extracellular matrix to serve as a scaffold for recellularization with human cells. However, the use of xenogeneic tissue sources in human transplantation raises concerns due to the presence of the antigenic Gal epitope. In the present study, lungs from wild type or α-Gal knockout pigs were harvested, decellularized, and implanted subcutaneously in a non-human primate model to evaluate the host immune response. The decellularized porcine implants were compared to a sham surgery control, as well as native porcine and decellularized macaque lung implants. The results demonstrated differential profiles of circulating and infiltrating immune cell subsets and histological outcomes depending on the implanted tissue source. Upon implantation, the decellularized α-Gal knockout lung constructs performed similarly to the decellularized wild type lung constructs. However, upon re-implantation into a chronic exposure model, the decellularized wild type lung constructs resulted in a greater proportion of infiltrating CD45+ cells, including CD3+ and CD8+ cytotoxic T-cells, likely mediated by an increase in production of Gal-specific antibodies. The results suggest that removal of the Gal epitope can potentially reduce adverse inflammatory reactions associated with chronic exposure to engineered organs containing xenogeneic components.

Recellularized Native Kidney Scaffolds as a Novel Tool in Nephrotoxicity Screening.

Drug-induced kidney injury in medicinal compound development accounts for over 20% of clinical trial failures and involves damage to different nephron segments, mostly the proximal tubule. Yet, currently applied cell models fail to reliably predict nephrotoxicity; neither are such models easy to establish. Here, we developed a novel three-dimensional (3D) nephrotoxicity platform on the basis of decellularized rat kidney scaffolds (DS) recellularized with conditionally immortalized human renal proximal tubule epithelial cells overexpressing the organic anion transporter 1 (ciPTEC-OAT1). A 5-day SDS-based decellularization protocol was used to generate DS, of which 100-μm slices were cut and used for cell seeding. After 8 days of culturing, recellularized scaffolds (RS) demonstrated 3D-tubule formation along with tubular epithelial characteristics, including drug transporter function. Exposure of RS to cisplatin (CDDP), tenofovir (TFV), or cyclosporin A (CsA) as prototypical nephrotoxic drugs revealed concentration-dependent reduction in cell viability, as assessed by PrestoBlue and Live/Dead staining assays. This was most probably attributable to specific uptake of CDDP by the organic cation transporter 2 (OCT2), TFV through organic anion transporter 1 (OAT1), and CsA competing for P-glycoprotein-mediated efflux. Compared with 2D cultures, RS showed an increased sensitivity to cisplatin and tenofovir toxicity after 24-hour exposure (9 and 2.2 fold, respectively). In conclusion, we developed a physiologically relevant 3D nephrotoxicity screening platform that could be a novel tool in drug development.

Development of a novel human recellularized endometrium that responds to a 28-day hormone treatment†

Three-dimensional (3D) in vitro models have been established to study the physiology and pathophysiology of the endometrium. With emerging evidence that the native extracellular matrix (ECM) provides appropriate cues and growth factors essential for tissue homeostasis, we describe, a novel 3D endometrium in vitro model developed from decellularized human endometrial tissue repopulated with primary endometrial cells. Analysis of the decellularized endometrium using mass spectrometry revealed an enrichment of cell adhesion molecules, cytoskeletal proteins, and ECM proteins such as collagen IV and laminin. Primary endometrial cells within the recellularized scaffolds proliferated and remained viable for an extended period of time in vitro. In order to evaluate the hormonal response of cells within the scaffolds, the recellularized scaffolds were treated with a modified 28-day hormone regimen to mimic the human menstrual cycle. At the end of 28 days, the cells within the endometrial scaffold expressed both estrogen and progesterone receptors. In addition, decidualization markers, IGFBP-1 and prolactin, were secreted upon addition of dibutyryl cyclic AMP indicative of a decidualization response. This 3D model of the endometrium provides a new experimental tool to study endometrial biology and drug testing.

The effective bioengineering method of implantation decellularized renal extracellular matrix scaffolds.

End stage renal disease (ESRD) is a progressive loss of kidney function with a high rate of morbidity and mortality. Transplantable organs are hard to come by and hold a high risk of recipient immune rejection. We intended to establish a more effective and faster method to decellularize and recellularize the kidney scaffold for transplant and regeneration. We successfully produced renal scaffolds by decellularizing rat kidneys with 0.5% sodium dodecyl sulfate (SDS), while still preserving the extracellular matrix (ECM) 3D architecture, an intact vascular tree and biochemical components. We recellularized the kidney scaffolds with mouse embryonic stem (ES) cells that then populated and proliferated within the glomerular, vascular, and tubular structures. After in vivo implantation, these recellularized scaffolds were easily reperfused, tolerated blood pressure and produced urine with no blood leakage. Our methods can successfully decellularize and recellularize rat kidneys to produce functional renal ECM scaffolds. These scaffolds maintain their basic components, retain intact vasculature and show promise for kidney regeneration.

Cell Stratification, Spheroid Formation and Bioscaffolds Used to Grow Cells in Three Dimensional Cultures.

The cell culture became an invaluable tool for studying cell behaviour, development, function, gene expression, toxicity of compounds and efficacy of novel drugs. Although most results were obtained from cell cultivation in two-dimensional (2D) systems, in which cells are grown in a monolayer, three-dimensional (3D) cultures are more promising as they correspond closely to the native arrangement of cells in living tissues. In our study, we focused on three types of 3D in vitro systems used for cultivation of one cell type. Cell morphology, their spatial distribution inside of resulting multicellular structures and changes in time were analysed with histological examination of samples harvested at different time periods. In multilayered cultures of WRL 68 hepatocytes grown on semipermeable membranes and non-passaged neurospheres generated by proliferation of neural progenitor cells, the cells were tightly apposed, showed features of cell differentiation but also cell death that was observable in short-term cultures. Biogenic scaffolds composed of extracellular matrix of the murine tibial anterior muscle were colonized with C2C12 myoblasts in vitro. The recellularized scaffolds did not reach high cell densities comparable with the former systems but supported well cell anchorage and migration without any signs of cell regression.

Decellularization protocols of porcine heart valves differ importantly in efficiency of cell removal and susceptibility of the matrix to recellularization with human vascular cells

Objective We compared 3 different decellularization protocols in porcine heart valves for efficiency of complete cell removal and potential for recellularization. Methods Porcine aortic and pulmonary roots were treated with trypsin, sodium-dodecyl-sulphate, or a new method using 0.25% tert-octylphenyl-polyoxyethylen in combination with sodium-deoxycholate. After a subsequent ribonuclease digestion, specimens were seeded with in vitro expanded human saphenous vein endothelial cells and myofibroblasts. Results After treatment with trypsin and subsequent ribonuclease digestion, endothelial attachment took place; however, xenogenic cells were still visible within the matrix. Unexpectedly, when human cells were seeded onto specimens that had been decellularized with sodium-dodecyl-sulphate, the matrices were surrounded by nonviable endothelial cell fragments, indicating a toxic influence of the ionic detergent; 0.25% tert-octylphenyl-polyoxyethylen together with sodium-deoxycholate completely removed porcine cells and enabled host recellularization. Conclusion Compared with trypsin and sodium-dodecyl-sulphate involving decellularization procedures, reported to be effective in cell removal and susceptible to recellularization with human cells, only the porcine matrix treated with a new detergent-based decellularization method using 0.25% tert-octylphenyl-polyoxyethylen/sodium-deoxycholate followed by nuclease digestion presented an excellent scaffold for recellularization with human cells.