Acellular Extracellular Matrix Scaffolds Research Articles

Integrin-β1 aggravates paraquat-induced pulmonary fibrosis by activation of FAK/ ERK1/2 pathway depending on fibrotic ECM

BackgroundIrreversible pulmonary fibrosis induced by paraquat is the most prevalent cause of death in patients with paraquat poisoning. Pulmonary fibrosis is characterized by abnormal deposition of extracellular matrix (ECM). Currently, the role of fibrotic ECM microenvironment in paraquat-induced pulmonary fibrosis has not been established. MethodsRat pulmonary fibrosis model was induced by paraquat, ATN-161 (an integrin-β1 antagonist) was given to investigate their effect on Rat survival and pulmonary fibrosis. Lungs were decellularized to generate normal and fibrotic acellular ECM scaffolds using Triton and SDS. Fibroblasts were cocultured with ECM scaffolds to established 3D culture systems to investigate the relationship between fibrotic ECM and the differentiation of fibroblasts. Then we explored the effect of fibrotic ECM microenvironment systematically promoting on integrin-β1/FAK/ERK1/2 pathway and established 3D culture systems to investigate the relationship between fibrotic ECM and the differentiation of fibroblasts. ResultsAntagonism of integrin-β1 could alleviate paraquat-induced pulmonary fibrosis and ameliorate survival status of rats. Compared to normal ECM, fibrotic extracellular microenvironment promoted the differentiation of fibroblasts to myofibroblasts. Antagonism of integrin-β1 could also ameliorate the promotion of fibrotic extracellular microenvironment on differentiation of fibroblasts to myofibroblasts. Fibrotic ECM microenvironment promotes fibroblasts transforming into myofibroblasts through integrin-β1/FAK/ERK1/2 signaling pathway. Moreover, this phenomenon holds independent on exogenous integrin-β1. ConclusionsActivation of integrin-β1/FAK/ERK1/2 pathway aggravates paraquat-induced pulmonary fibrosis depend on fibrotic ECM and integrin-β1 may be a prospective therapeutic target for paraquat-induced pulmonary fibrosis in the future.

Which detergent is most suitable for the generation of an acellular pancreas bioscaffold?

Pancreatic bioengineering is a potential therapeutic alternative for type 1 diabetes (T1D) in which the pancreas is decellularized, generating an acellular extracellular matrix (ECM) scaffold, which may be reconstituted by recellularization with several cell types to generate a bioartificial pancreas. No consensus for an ideal pancreatic decellularization protocol exists. Therefore, we aimed to determine the best-suited detergent by comparing sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC), and Triton X-100 at different concentrations. Murine (n=12) and human pancreatic tissue from adult brain-dead donors (n=06) was harvested in accordance with Institutional Ethical Committee of the University of São Paulo Medical School (CEP-FMUSP) and decellularized under different detergent conditions. DNA content, histological analysis, and transmission and scanning electron microscopy were assessed. The most adequate condition for pancreatic decellularization was found to be 4% SDC, displaying: a) effective cell removal; b) maintenance of extracellular matrix architecture; c) proteoglycans, glycosaminoglycans (GAGs), and collagen fibers preservation. This protocol was extrapolated and successfully applied to human pancreas decellularization. The acellular ECM scaffold generated was recelullarized using human pancreatic islets primary clusters. 3D clusters were generated using 0.5×104 cells and then placed on top of acellular pancreatic slices (25 and 50 μm thickness). These clusters tended to connect to the acellular matrix, with visible cells located in the periphery of the clusters interacting with the ECM network of the bioscaffold slices and continued to produce insulin. This study provided evidence on how to improve and accelerate the pancreas decellularization process, while maintaining its architecture and extracellular structure, aiming at pancreatic bioengineering.

A Decellularized Uterine Endometrial Scaffold Enhances Regeneration of the Endometrium in Rats.

Partial or whole regeneration of the uterine endometrium using extracellular matrix (ECM)-based scaffolds is a therapeutic strategy for uterine infertility due to functional and/or structural endometrial defects. Here, we examined whether the entire endometrium can be regenerated circumferentially using an acellular ECM scaffold (decellularized endometrial scaffold, DES) prepared from rat endometrium. We placed a silicone tube alone to prevent adhesions or a DES loaded with a silicone tube into a recipient uterus in which the endometrium had been surgically removed circumferentially. Histological and immunofluorescent analyses of the uteri one month after tube placement revealed more abundant regenerated endometrial stroma in the uterine horns treated with tube-loaded DES compared to those treated with a tube alone. Luminal and glandular epithelia, however, were not fully recapitulated. These results suggest that DES can enhance the regeneration of endometrial stroma but additional intervention(s) are needed to induce epithelization. Furthermore, the prevention of adhesions alone allowed the endometrial stroma to regenerate circumferentially even without a DES, but to a lesser degree than that with a DES. The use of a DES together with the prevention of adhesions may be beneficial for efficient endometrial regeneration in the uterus that is largely deficient of endometrium.

Engineered Lung Tissues Prepared from Decellularized Lung Slices

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex vivo. Recently developed organoid models have facilitated the expansion and study of lung epithelial progenitors in vitro, but these platforms typically rely on mouse tumor-derived matrix and/or serum, and incorporate just one or two cellular lineages. Here, we describe a protocol for generating engineered lung tissues (ELTs) based on the multi-lineage recellularization of decellularized precision-cut lung slices (PCLS). ELTs contain alveolar-like structures comprising alveolar epithelium, mesenchyme, and endothelium, within an extracellular matrix (ECM) substrate closely resembling that of native lung. To generate the tissues, rat lungs are inflated with agarose, sliced into 450 µm-thick slices, cut into strips, and decellularized. The resulting acellular ECM scaffolds are then reseeded with primary endothelial cells, fibroblasts, and alveolar epithelial type 2 cells (AEC2s). AEC2s can be maintained in ELT culture for at least 7 days with a serum-free, chemically-defined growth medium. Throughout the tissue preparation and culture process, the slices are clipped into a cassette system that facilitates handling and standardized cell seeding of multiple ELTs in parallel. These ELTs represent an organotypic culture platform that should facilitate investigations of cell-cell and cell-matrix interactions within the alveolus as well as biochemical signals regulating AEC2s and their niche.

Engineered Lung Tissues Prepared from Decellularized Lung Slices.

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex vivo. Recently developed organoid models have facilitated the expansion and study of lung epithelial progenitors in vitro, but these platforms typically rely on mouse tumor-derived matrix and/or serum, and incorporate just one or two cellular lineages. Here, we describe a protocol for generating engineered lung tissues (ELTs) based on the multi-lineage recellularization of decellularized precision-cut lung slices (PCLS). ELTs contain alveolar-like structures comprising alveolar epithelium, mesenchyme, and endothelium, within an extracellular matrix (ECM) substrate closely resembling that of native lung. To generate the tissues, rat lungs are inflated with agarose, sliced into 450 µm-thick slices, cut into strips, and decellularized. The resulting acellular ECM scaffolds are then reseeded with primary endothelial cells, fibroblasts, and alveolar epithelial type 2 cells (AEC2s). AEC2s can be maintained in ELT culture for at least 7 days with a serum-free, chemically-defined growth medium. Throughout the tissue preparation and culture process, the slices are clipped into a cassette system that facilitates handling and standardized cell seeding of multiple ELTs in parallel. These ELTs represent an organotypic culture platform that should facilitate investigations of cell-cell and cell-matrix interactions within the alveolus as well as biochemical signals regulating AEC2s and their niche.

Fast Automated Approach for the Derivation of Acellular Extracellular Matrix Scaffolds from Porcine Soft Tissues

Decellularized extracellular matrix (ECM) scaffolds derived from tissues and organs are complex biomaterials used in clinical and research applications. A number of decellularization protocols have been described for ECM biomaterials derivation, each adapted to a particular tissue and use, restricting comparisons among materials. One of the major sources of variability in ECM products comes from the tissue source and animal age. Although this variability could be minimized using established tissue sources, other sources arise from the decellularization process itself. Overall, current protocols require manual work and are poorly standardized with regard to the choice of reagents, the order by which they are added, and exposure times. The combination of these factors adds variability affecting the uniformity of the final product between batches. Furthermore, each protocol needs to be optimized for each tissue and tissue source making tissue-to-tissue comparisons difficult. Automation and standardization of ECM scaffold development constitute a significant improvement to current biomanufacturing techniques but remains poorly explored. This study aimed to develop a biofabrication method for fast and automated derivation of raw material for ECM hydrogel production while preserving ECM composition and controlling lot-to-lot variability. The main result was a closed semibatch bioreactor system with automated dosing of decellularization reagents capable of deriving ECM material from pretreated soft tissues. The ECM was further processed into hydrogels to demonstrate gelation and cytocompatibility. This work presents a versatile, scalable, and automated platform for the rapid production of ECM scaffolds.

Experimental studies on the repair and restitution of cartilage by cartilage acellular extracellular matrix and adipose tissue-derived stem cells

Objective: To investigate the effects of the repair and restitution of ear-shaped cartilage by adipose tissue-derived stem cells(ADSC) and cartilage acellular extracellular matrix. Methods: ADSC were extracted by digesting with collagenase type II from the adipose tissue from 32 patients with adiposity whose fats were drawn, and were cultured and subcultured in vitro. The natural biological scaffolds were prepared by acellular method using porcine ear cartilage, and then the second generation ADSC(5.0×10(7)/ml) were inoculated on the preformed natural bio-scaffold scaffold by culturing in vitro for 3 days to form a cell scaffold complex. 32 New Zealand white rabbits were randomly divided into the experimental groups, the control group A, the control group B and the control group C. All New Zealand white rabbits were modeled by ear cartilage defects. The cell scaffolds composite was implanted into the experimental group of the ear cartilage defects of rabbits, the ADSC were implanted into the control group A, the cartilage acellular extracellular matrix scaffold was implanted into the control group B and the control group C was modeled only by ear cartilage defects. After 16 weeks, the animals were sacrificed and the repair effect was observed by gross appearance and histological examinations including HE, Toluidine blue staining, Safranin O and typeⅡ collagen staining. Its were quantitatively analyzed by positive staining results of type Ⅱ collagen. Ear cartilage tissue elasticity was detected. SPSS 17.0 software was used to analyze the data. Results: The cartilage defects in the experimental group were repaired well by general shape observation and those in the control group was filled in with granulation tissue. There were significant differences between the experimental group and the control group in the wet weight(P<0.05). HE staining showed that cartilage cavities formed in articular cartilage defects, and only the fibrous tissue was filled with the ear cartilage defect in the control groups. In the repair area, Toluidine blue staining, Safranin O and type Ⅱ collagen staining were positive in the experimental group, and negative in the control groups. There was no significant difference between the experimental group and the normal ear cartilage in the ear cartilage elastic constant detection(P>0.05). Conclusions: The mechanics and histology of rabbit ear neonatal cartilage constructed by ADSC combined with cartilage acellular matrix are close to normal ear cartilage. Cartilage acellular matrix material combined with adipose-derived stem cells has good repair and reconstruction ability for ear cartilage defects, which possesses potential clinical application value.

Decellularization of the Porcine Ear Generates a Biocompatible, Nonimmunogenic Extracellular Matrix Platform for Face Subunit Bioengineering.

The purpose of this study was to assess whether perfusion-decellularization technology could be applied to facial grafts. Facial allotransplantation remains an experimental procedure. Regenerative medicine techniques allow fabrication of transplantable organs from an individual's own cells, which are seeded into extracellular matrix (ECM) scaffolds from animal or human organs. Therefore, we hypothesized that ECM scaffolds also can be created from facial subunits. We explored the use of the porcine ear as a clinically relevant face subunit model to develop regenerative medicine-related platforms for facial bioengineering. Porcine ear grafts were decellularized and histologic, immunologic, and cell culture studies done to determine whether scaffolds retained their 3D framework and molecular content; were biocompatible in vitro and in vivo, and triggered an anti-MHC immune response from the host. The cellular compartment of the porcine ear was completely removed except for a few cartilaginous cells, leaving behind an acellular ECM scaffold; this scaffold retained its complex 3D architecture and biochemical components. The framework of the vascular tree was intact at all hierarchical levels and sustained a physiologically relevant blood pressure when implanted in vivo. Scaffolds were biocompatible in vitro and in vivo, and elicited no MHC immune response from the host. Cells from different types remained viable and could even differentiate at the scale of a whole-ear scaffold. Acellular scaffolds were produced from the porcine ear, and may be a valuable platform to treat facial deformities using regenerative medicine approaches.

The rat pancreatic body tail as a source of a novel extracellular matrix scaffold for endocrine pancreas bioengineering

BackgroundRegenerative medicine and tissue engineering are promising approaches for organ transplantation. Extracellular matrix (ECM) based scaffolds obtained through the decellularization of natural organs have become the preferred platform for organ bioengineering. In the field of pancreas bioengineering, acellular scaffolds from different animals approximate the biochemical, spatial and vascular relationships of the native extracellular matrix and have been proven to be a good platform for recellularization and in vitro culture. However, artificial endocrine pancreases based on these whole pancreatic scaffolds have a critical flaw, specifically their difficult in vivo transplantation, and connecting their vessels to the recipient is a major limitation in the development of pancreatic tissue engineering. In this study, we focus on preparing a novel acellular extracellular matrix scaffold derived from the rat pancreatic body tail (pan-body-tail ECM scaffold).ResultsSeveral analyses confirmed that our protocol effectively removes cellular material while preserving ECM proteins and the native vascular tree. DNA quantification demonstrated an obvious reduction of DNA compared with that of the natural organ (from 931.9 ± 267.8 to 11.7 ± 3.6 ng/mg, P < 0.001); the retention of the sGAG in the decellularized pancreas (0.878 ± 0.37) showed no significant difference from the natural pancreas (0.819 ± 0.1) (P > 0.05). After transplanted with the recellularized pancreas, fasting glucose levels declined to 9.08 ± 2.4 mmol/l within 2 h of the operation, and 8 h later, they had decreased to 4.7 ± 1.8 mmol/l (P < 0.05).ConclusionsThe current study describes a novel pancreatic ECM scaffold prepared from the rat pancreatic body tail via perfusion through the left gastric artery. We further showed the pioneering possibility of in vivo circulation-connected transplantation of a recellularized pancreas based on this novel scaffold. By providing such a promising pancreatic ECM scaffold, the present study might represent a key improvement and have a positive impact on endocrine pancreas bioengineering.

Preparation and characterization of acellular adipose tissue matrix using a combination of physical and chemical treatments.

Decellularized adipose extracellular matrix (ECM) has been used in the clinic to support the regeneration of adipose tissues. The methods used to produce adipose tissue ECM scaffolds exhibit distinct effects upon the structural and functional components of the resultant scaffold material. The current study presents an acellular ECM scaffold from human adipose tissues derived using successive physical and chemical treatments, including repeated freeze-thaw cycles followed by centrifugation, polar solvent extraction and enzymatic digestion. Cellular components, including nucleic acids were effectively removed without significant disruption of the morphology or structure of the ECM. The compositions of major ECM components were evaluated, including acid/pepsin soluble collagen, sulfated glycosaminoglycan and laminin. The decellularized ECM exhibited satisfactory mechanical properties. Cell seeding experiments involving human adipose-derived stem cells indicated that the decellularized ECM provided an inductive microenvironment for adipogenesis without the need for exogenous differentiation factors. Higher levels of glycerol-3-phosphate dehydrogenase activity were observed among induced cells in the ECM scaffolds when compared with induced cells in collagen type I scaffolds. In conclusion, the results suggested that the decellularized ECM, containing biological and chemical cues of native human ECM, may be an ideal scaffold material for autologous and allograft tissue engineering.

Bioactive Extracellular Matrix Scaffold Promotes Adaptive Cardiac Remodeling and Repair

Structural cardiac remodeling after ischemic injury can induce a transition to heart failure from progressive loss of cardiac function. Cellular regenerative therapies are promising but face significant translational hurdles. Tissue extracellular matrix (ECM) holds the necessary environmental cues to stimulate cell-based endogenous myocardial repair pathways and promote adaptive remodeling toward functional recovery. Heart epicardium has emerged as an important anatomic niche for endogenous repair pathways including vasculogenesis and cardiogenesis. We show that acellular ECM scaffolds surgically implanted on the epicardium following myocardial infarction (MI) can attenuate structural cardiac remodeling and improve functional recovery. We assessed the efficacy of this strategy on post-MI functional recovery by comparing intact bioactive scaffolds with biologically inactivated ECM scaffolds. We confirm that bioactive properties within the acellular ECM biomaterial are essential for the observed functional benefits. We show that interaction of human cardiac fibroblasts with bioactive ECM can induce a robust cell-mediated vasculogenic paracrine response capable of functional blood vessel assembly. Fibroblast growth factor-2 is uncovered as a critical regulator of this novel bioinductive effect. Acellular bioactive ECM scaffolds surgically implanted on the epicardium post-MI can reprogram resident fibroblasts and stimulate adaptive pro-reparative pathways enhancing functional recovery. We introduce a novel surgical strategy fortissue repair that can be performed as an adjunct to conventional surgical revascularization with minimaltranslational challenges.

Heparin Augmentation Enhances Bioactive Properties of Acellular Extracellular Matrix Scaffold.

Extracellular matrix (ECM) maintains a reservoir of bioactive growth factors and matricellular proteins that provide bioinductive effects on local cells that influence phenotype and behaviors. Bioactive acellular ECM scaffolds can be used therapeutically to stimulate adaptive tissue repair. Fibroblast growth factor-2 (FGF-2) attenuates transforming growth factor-β1 (TGF-β1)-mediated cardiac fibrosis. Heparin glycosaminoglycan can influence FGF-2 bioactivity and could be leveraged to enhance tissue engineering strategies. We explored the effects of heparin on FGF-2 enhancement of bioactive ECM scaffold biomaterials for its antifibrotic effect on attenuating human cardiac myofibroblast activation. Increasing heparin concentration at a fixed concentration of FGF-2 markedly increased the amount of FGF-2 retained and eluted by ECM scaffolds. To explore synergistic bioinductive effects of heparin and FGF-2, collagen gel contraction assay using human cardiac myofibroblasts was performed in vitro. Myofibroblast activation was induced by profibrotic cytokine, TGF-β1. FGF-2 and heparin in combination reduced human cardiac myofibroblast-mediated collagen gel contraction to a greater extent than FGF-2 alone. These observations were confirmed for both human atrial and human ventricular cardiac fibroblasts. Cell death was not different between groups. In summary, heparin is an effective adjuvant to enhance FGF-2 loading and elution of acellular ECM scaffold biomaterials. Heparin increases the bioactive effects of FGF-2 in attenuating human cardiac myofibroblast activation in response to profibrotic TGF-β1. These data may inform tissue engineering strategies for myocardial repair to prevent fibrosis.

A Porcine Urinary Bladder Matrix Does Not Recapitulate the Spatiotemporal Macrophage Response of Muscle Regeneration after Volumetric Muscle Loss Injury

Volumetric muscle loss (VML) results in irrecoverable loss of muscle tissue making its repair challenging. VML repair with acellular extracellular matrix (ECM) scaffolds devoid of exogenous cells has shown improved muscle function, but limited de novo muscle fiber regeneration. On the other hand, studies using minced autologous and free autologous muscle grafts have reported appreciable muscle regeneration. This raises the fundamental question whether an acellular ECM scaffold can orchestrate the spatiotemporal cellular events necessary for appreciable muscle fiber regeneration. This study compares the macrophage and angiogenic responses including the remodeling outcomes of a commercially available porcine urinary bladder matrix, MatriStem™, and autologous muscle grafts. The early heightened and protracted M1 response of the scaffold indicates that the scaffold does not recapitulate the spatiotemporal macrophage response of the autograft tissue. Additionally, the scaffold only supports limited de novo muscle fiber formation and regressing vessel density. Furthermore, scaffold remodeling is accompanied by increased presence of transforming growth factor and α-smooth muscle actin, which is consistent with remodeling of the scaffold into a fibrotic scar-like tissue. The limited muscle formation and scaffold-mediated fibrosis noted in this study corroborates the findings of recent studies that investigated acellular ECM scaffolds (devoid of myogenic cells) for VML repair. Taken together, acellular ECM scaffolds when used for VML repair will likely remodel into a fibrotic scar-like tissue and support limited de novo muscle fiber regeneration primarily in the proximity of the injured musculature. This is a work of the US Government and is not subject to copyright protection in the USA. Foreign copyrights may apply. Published by S. Karger AG, Basel

Targeted proteomics effectively quantifies differences between native lung and detergent-decellularized lung extracellular matrices.

The extracellular matrix (ECM) in large part defines the biochemical and mechanical properties of tissues and organs; these inherent cues make acellular ECM scaffolds potent substrates for tissue regeneration. As such, they are increasingly prevalent in the clinic and the laboratory. However, the exact composition of these scaffolds has been difficult to ascertain. This paper uses targeted proteomics to definitively quantify 71 proteins present in acellular lung ECM scaffolds. We use this technique to compare two decellularization methods and demonstrate superior retention of ECM proteins important for cell adhesion, migration, proliferation, and differentiation in scaffolds treated with low-concentration detergent solutions. In the long term, the ability to acquire quantitative biochemical data about biological substrates will facilitate the rational design of engineered tissues and organs based on precise cell-matrix interactions.

The Human Pancreas as a Source of Protolerogenic Extracellular Matrix Scaffold for a New-generation Bioartificial Endocrine Pancreas.

Our study aims at producing acellular extracellular matrix scaffolds from the human pancreas (hpaECMs) as a first critical step toward the production of a new-generation, fully human-derived bioartificial endocrine pancreas. In this bioartificial endocrine pancreas, the hardware will be represented by hpaECMs, whereas the software will consist in the cellular compartment generated from patient's own cells. Extracellular matrix (ECM)-based scaffolds obtained through the decellularization of native organs have become the favored platform in the field of complex organ bioengineering. However, the paradigm is now switching from the porcine to the human model. To achieve our goal, human pancreata were decellularized with Triton-based solution and thoroughly characterized. Primary endpoints were complete cell and DNA clearance, preservation of ECM components, growth factors and stiffness, ability to induce angiogenesis, conservation of the framework of the innate vasculature, and immunogenicity. Secondary endpoint was hpaECMs' ability to sustain growth and function of human islet and human primary pancreatic endothelial cells. Results show that hpaECMs can be successfully and consistently produced from human pancreata and maintain their innate molecular and spatial framework and stiffness, and vital growth factors. Importantly, hpaECMs inhibit human naïve CD4 T-cell expansion in response to polyclonal stimuli by inducing their apoptosis and promoting their conversion into regulatory T cells. hpaECMs are cytocompatible and supportive of representative pancreatic cell types. We, therefore, conclude that hpaECMs has the potential to become an ideal platform for investigations aiming at the manufacturing of a regenerative medicine-inspired bioartificial endocrine pancreas.

Development and Characterization of Acellular Extracellular Matrix Scaffolds from Porcine Menisci for Use in Cartilage Tissue Engineering.

Given the growing number of arthritis patients and the limitations of current treatments, there is great urgency to explore cartilage substitutes by tissue engineering. In this study, we developed a novel decellularization method for menisci to prepare acellular extracellular matrix (ECM) scaffolds with minimal adverse effects on the ECM. Among all the acid treatments, formic acid treatment removed most of the cellular contents and preserved the highest ECM contents in the decellularized porcine menisci. Compared with fresh porcine menisci, the content of DNA decreased to 4.10%±0.03%, and there was no significant damage to glycosaminoglycan (GAG) or collagen. Histological staining also confirmed the presence of ECM and the absence of cellularity. In addition, a highly hydrophilic scaffold with three-dimensional interconnected porous structure was fabricated from decellularized menisci tissue. Human chondrocytes showed enhanced cell proliferation and synthesis of chondrocyte ECM including type II collagen and GAG when cultured in this acellular scaffold. Moreover, the scaffold effectively supported chondrogenesis of human bone marrow-derived mesenchymal stem cells. Finally, in vivo implantation was conducted in rats to assess the biocompatibility of the scaffolds. No significant inflammatory response was observed. The acellular ECM scaffold provided a native environment for cells with diverse physiological functions to promote cell proliferation and new tissue formation. This study reported a novel way to prepare decellularized meniscus tissue and demonstrated the potential as scaffolds to support cartilage repair.

Glottic regeneration with a tissue-engineering technique, using acellular extracellular matrix scaffold in a canine model.

Acellular extracellular matrix scaffold derived from porcine urinary bladder (UBM) is decellularized material that has shown success for constructive remodelling of various tissues and organs. The regenerative effects of UBM were reported for the tympanic membrane, oesophagus, trachea, larynx, pleura and pericardium in animal studies, with promising results. The aim of this study was to investigate the regenerative effects of UBM on hemilarynx, using a canine model. A left partial hemilaryngectomy was performed and the surgical defects were reconstructed by insertion of UBM scaffold. Although local infection was observed in one dog in 1 week after implantation of the scaffold, all dogs showed good re-epithelialization with minimum complication in 1 month. The effect of regeneration of the larynx was evaluated 6 months after the operation. The excised larynx experiments were performed to measure phonation threshold pressure (PTP), normalized mucosal wave amplitude (NMWA) and normalized glottal gap (NGG). The results of the measurements showed that PTP was normal or near normal in two cases and NMWA was within normal range in three cases, although there were individual variations. Histological examination was completed to evaluate structural changes in the scaffold with the appearance of the new cartilaginous structure. However, the regenerated vocal fold mucosa was mostly scarred. The UBM scaffold has shown to be biocompatible, biodegradable and useful for tissue regeneration of the hemilarynx, with possible restoration of function of the vocal fold. The vocal fold mucosa was scarred, which is the next challenge to be addressed. Copyright © 2014 John Wiley & Sons, Ltd.

Acellular Porcine Urinary Bladder Matrix for the Treatment of an Avulsive Scalp Wound in Irradiated Tissue on a Chronically Immunocompromised Patient: A Case Presentation

Biological materials composed of extracellular matrix (ECM) harvested from a wide variety of tissues and organs are widely used as viable alternative options for tissue reconstruction. A new product, MatriStem by ACell, has shown promise in regards to regeneration of healthy functional tissue as opposed to scar formation. MatriStem is comprised of an acellular extracellular matrix scaffold comprised of porcine urinary bladder matrix (UBM). Its unique bimodal surface characteristics consist of an intact basement membrane and an organized connective tissue bed comprised of urinary bladder lamina propia. The basement membrane is conducive to epithelial and endothelial cell attachment, proliferation and differentiation, while the lamina propia allows for integration of the product into the wound bed and host connective tissues, and supports vascular ingrowth. In addition, the processing of the MatriStem product allows for retention of multiple growth factors including vascular endothelial growth factor (VEGF), transforming growth factor beta (TGFβ), platelet derived growth factor (PDGF), bone morphogenic protein 4 (BMP4), and basic fibroblast growth factor (BFGF) to assist in regeneration and healing. Recent studies have demonstrated antimicrobial properties of UBM-ECM to both S. aureus and E. colias well as a predominance of macrophage phenotype 2 (M2) in the early postoperative period which has been shown to correlate with less scar formation and more healthy functional tissue regeneration with increased chemotactic activity.

Maintenance of Hepatic Sinusoidal Endothelial Cell PhenotypeIn VitroUsing Organ-Specific Extracellular Matrix Scaffolds

Sinusoidal endothelial cells (SECs) are notoriously difficult to culture in vitro. SECs represent a highly specialized endothelial cell (EC) population, and traditional methods of SEC isolation from the liver initiate a process of SEC dedifferentiation. Acellular extracellular matrix (ECM) scaffolds were investigated in a physiologically relevant in vitro culture model for their ability to maintain SEC phenotype. The cell culture model used SECs only or a coculture of SECs with hepatocytes on ECM substrates derived from the liver (L-ECM), bladder (UBM-ECM), or small intestine submucosa (SIS-ECM). The effect of the ECM substrate upon SEC dedifferentiation was evaluated using scanning electron microscopy (SEM) and confocal microscopy. When SECs alone were cultured on uncoated glass slides, collagen I, UBM-ECM, or SIS-ECM, SECs showed signs of dedifferentiation after 1 day. In contrast, SECs alone cultured on L-ECM maintained their differentiated phenotype for at least 3 days, indicated by the presence of many fenestrations on SEC surface, expression of anti-rat hepatic sinusoidal endothelial cells mouse IgG MoAb (SE-1), and lack of expression of CD31. When SECs were cocultured with hepatocytes on any of the ECM scaffolds, the SECs maintained a near-normal fenestrated phenotype for at least 1 day. However, SEM revealed that the shape, size, frequency, and organization of the fenestrations varied greatly depending on ECM source. At all time points, SECs cocultured with hepatocytes on L-ECM maintained the greatest degree of differentiation. The present study demonstrated that the acellular ECM scaffold derived from the liver maintained SEC differentiation in culture longer than any of the tested substrate materials. The replacement of complex tissues and 3-dimensional organs may require specialized scaffolds to support multiple, functional cell phenotypes.

A Biodegradable, Acellular Xenogeneic Scaffold for Regeneration of the Vocal Fold Lamina Propria

A novel method for preparing an acellular xenogeneic extracellular matrix scaffold for tissue engineering was developed. Bovine vocal fold lamina propria specimens were treated with high-concentration sodium chloride, nucleic acid digestion, and ethanol dehydration for decellularization and removal of immunogenic foreign epitopes. Human vocal fold fibroblasts from primary culture were seeded onto the acellular scaffolds and cultured for 21 days. The decellularized and the recellularized scaffolds were examined by light microscopy, fluorescent microscopy, and scanning electron microscopy. Collagen synthesis and release by fibroblasts were quantified by the Sircol assay, whereas the synthesis and release of hyaluronic acid, decorin, and fibronectin were assessed by enzyme-linked immunosorbent assays. Viscoelastic shear properties of the scaffolds were quantified by a simple-shear rheometer at frequencies of up to 250 Hz. Preliminary results showed that a biodegradable, acellular extracellular matrix scaffold with an intact basement membrane and 3-dimensional structure of the matrix proteins was engineered. Vocal fold fibroblasts readily attached to and infiltrated the scaffold with high viability and active protein synthesis, demonstrating the biocompatibility. The elastic shear modulus and dynamic viscosity of the acellular scaffold and the fibroblast-repopulated scaffold were comparable to those of the human vocal fold cover. These findings support the potential of the scaffold as a xenograft for vocal fold reconstruction and regeneration.