Functional characterization of a bioengineered liver after heterotopic implantation in pigs.

Organ bioengineering offers a promising solution to the persistent shortage of donor organs. However, the progression of this technology toward clinical use has been hindered by the challenges of reconstituting a functional vascular network, directing the engraftment of specific functional cell types, and defining appropriate culture conditions to concurrently support the health and phenotypic stability of diverse cell lineages. We previously demonstrated the ability to functionally reendothelialize the vasculature of a clinically scaled decellularized liver scaffold with human umbilical vein endothelial cells (HUVECs) and to sustain continuous perfusion in a large animal recovery model. We now report a method for seeding and engrafting primary porcine hepatocytes into a bioengineered liver (BEL) scaffold previously reendothelialized with HUVECs. The resulting BELs were competent for albumin production, ammonia detoxification and urea synthesis, indicating the presence of a functional hepatocyte compartment. BELs additionally slowed ammonia accumulation during in vivo perfusion in a porcine model of surgically induced acute liver failure. Following explant of the graft, BEL parenchyma showed maintenance of canonical endothelial and hepatocyte markers. Taken together, these results support the feasibility of engineering a clinically scaled functional BEL and establish a platform for optimizing the seeding and engraftment of additional liver specific cells.

Urinary Bladder Matrix Scaffolds Promote Pericardium Repair in a Porcine Model.

Pericardium closure after cardiac surgery is recommended to prevent postoperative adhesions to the sternum. Synthetic materials have been used as substitutes, with limited results because of impaired remodeling and fibrotic tissue formation. Urinary bladder matrix (UBM) scaffolds promote constructive remodeling that more closely resemble the native tissue. The aim of the study is to evaluate the host response to UBM scaffolds in a porcine model of partial pericardial resection. Twelve Landrace pigs were subjected to a median sternotomy. A 5 × 7 cm pericardial defect was created and then closed with a 5 × 7 cm multilayer UBM patch (UBM group) or left as an open defect (control group). Animals were survived for 8 wk. End points included gross morphology, biomechanical testing, histology with semiquantitative score, and cardiac function. The UBM group showed mild adhesions, whereas the control group showed fibrosis at the repair site, with robust adhesions and injury to the coronary bed. Load at failure (gr) and stiffness (gr/mm) were lower in the UBM group compared with the native pericardium (199.9 ± 59.2 versus 405.3 ± 99.89 g, P = 0.0536 and 44.23 ± 15.01 versus 146.5 ± 24.38 g/mm, P = 0.0025, respectively). In the UBM group, the histology resembled native pericardial tissue, with neovascularization, neofibroblasts, and little inflammatory signs. In contrast, control group showed fibrotic tissue with mononuclear infiltrates and a lack of organized collagen fibers validated with a histologic score. Both groups had normal ultrasonography results without cardiac motility disorders. In this setting, UBM scaffolds showed appropriate features for pericardial repair, restoring tissue properties that could help reduce postsurgical adhesions and prevent its associated complications.

Biomechanical Features of Reinforced Esophageal Hiatus Repair in a Porcine Model.

Recurrence rates in the laparoscopic repair of the hiatal hernia range from 12% to 59%. Limitation of reinforcement has been principally the risk of adverse events caused by synthetic materials. Biologic and resorbable synthetic materials are valid alternatives. This study compares the host response to all these materials after hiatal hernia repair. A total of 20 Landrace pigs, underwent laparoscopic primary hiatal hernia repair and reinforced with a polypropylene mesh (PROLENE: polypropylene [PP]), an absorbable synthetic scaffold (GOREBIO-A: polyglycolic acid [PGA]), a urinary bladder matrix scaffold, (Gentrix: urinary bladder matrix [UBM]), or without reinforcement, control group (C). Animals were survived for 3 months. Endpoints included gross morphology, biomechanical testing, and histology. Pigs in PP and PGA groups showed fibrosis at the repair site, with robust adhesions. In UBM and C groups, only mild adhesions were found. Load at failure (gr) and stiffness (gr/mm) of PP were higher than C group (PP:2103 ± 548.3 versus C:951.1 ± 372.7, P = 0.02; PP:643.3 ± 301 versus C:152.6 ± 142.7, P = 0.01). PGA and UBM values for both parameters were in between PP and C samples. However, stiffness in UBM was tended to be lower than PP group, and approached a significant difference (643.3 ± 301 versus 243 ± 122.1, P = 0.0536). In UBM group, the histology resembled native tissue. By contrast, PP and PGA groups showed mononuclear infiltrates, fibroencapsulation, necrosis, remnants of mesh, and disorganized tissue that was validated with a histologic score. In this setting, UBM scaffolds showed the most appropriate features for hiatal hernia repair, recovering the tissue properties that can help reduce the possibility of early failure and prevent complications associated with the implanted material.

Evaluation of magnesium alloys for use as an intraluminal tracheal for pediatric applications in a rat tracheal bypass model.

Tracheal stenting currently using non-degradable stents is commonplace for treatment of trauma, prolonged intubation related adult airway obstructions, and pediatric patients-associated tracheal stenosis conditions. Degradable tracheal stent placement will avoid complications of stent removal and restenosis. Widespread reports exist on degradable magnesium alloys success for orthopedic and cardiovascular applications but none to date for intra tracheal use. This research explores the use of pure Mg, AZ31, and Mg-3Y alloys for degradable tracheal stent assessment. In vitro evaluation of magnesium, prototype stents in a bioreactor simulate the airway environment and corrosion. Micro-CT imaging and biocompatibility evaluation helped assess the 24-week degradation of intraluminal alloy stents following implantation in a rat tracheal in vivo bypass model. Histological analysis indicate tissue response of the harvested stented trachea segments after each time point. Corrosion studies for each alloy indicate significant differences between the simulated and control in vitro conditions. AZ31 exhibited the lowest volume loss of 6.8% in saline, while pure Mg displayed the lowest volume loss of 4.6% in simulated airway fluid (SAF), both at 1-week time points. Significant differences in percentage of total volume lost after 6 months were determined between the alloys over time. MgY alloy displayed the slowest corrosion losing only 15.1% volume after 24 weeks of immersion. Additionally, in vitro magnesium alloy corrosion was not significantly different from the percentage of total volume lost in vivo at 1-week time point. The study demonstrates promise of magnesium alloys for intraluminal tracheal stent application albeit viability of a clinically translatable model warrants further studies. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1844-1853, 2019.

Urinary Bladder Matrix Protects Host in a Murine Model of Bacterial-Induced Lung Infection.

IMPACT STATEMENT: Lung infection is a leading cause of human life lost to morbidity and/or mortality. This problem is exacerbated by the alarming emergence of increasingly antibiotic-resistant (AR) microorganisms worldwide and the lack of effective antimicrobials to overcome the AR bacterial infection. Urinary bladder matrix (UBM) is a biologically derived scaffold material that has been used to promote site-appropriate tissue regeneration and remodeling in a variety of body systems. Our novel findings demonstrate that the preformulated UBM effectively protects the host from methicillin-resistant Staphylococcus aureus (MRSA)- and Pseudomonas aeruginosa-induced murine pneumonia and may provide a viable alternative/supplement for protection against respiratory AR bacterial infection.

Comparison of in vivo remodeling of urinary bladder matrix and acellular dermal matrix in an ovine model.

AIM: Biologically derived surgical graft materials come from a variety of sources with varying mechanical properties. This study aimed to evaluate the host response and mechanical performance of two extracellular matrix devices in a large animal preclinical model. MATERIALS & METHODS: Bilateral defects were created in the fascia lata of sheep and repaired with either an acellular dermal matrix (ADM) or urinary bladder matrix (UBM). After 1 or 3 months, the repair site was explanted for histological and mechanical analysis. RESULTS & CONCLUSION: Despite pre-implantation mechanical differences, both UBM and ADM demonstrated similar mechanical performance at 3 months. However, UBM was completely remodeled into site-appropriate tissue by 3 months, while ADM showed limited tissue incorporation.

Retrorectus repair of incisional ventral hernia with urinary bladder matrix reinforcement in a long-term porcine model.

AIM: Not all biologically derived materials elicit the same host response when used for reinforcement of ventral hernia repairs. This study aimed to evaluate the remodeling characteristics of the abdominal wall following reinforcement with urinary bladder matrix (UBM) in a large animal preclinical model of ventral hernia repair. MATERIALS & METHODS: Midline defects in 36 Yucatan minipigs were reinforced with UBM-derived surgical devices using a classic Rives-Stoppa-Wantz approach, and compared with primary repair controls. After 3 or 8 months, the abdominal wall was explanted for histological and mechanical analysis. RESULTS & CONCLUSION: All UBM-derived surgical devices were completely resorbed within 8 months and facilitated deposition of vascularized, biomechanically functional connective tissue in the retrorectus plane, with no evidence of hernia formation.

Urinary bladder matrix scaffolds strengthen esophageal hiatus repair.

BACKGROUND: Laparoscopic repair of the hiatal hernia is associated with a recurrence rate between 12% and 42% depending on the defect size. Although the impact of hiatal reinforcement on long-term recurrence remains controversial, the main limitation of this approach has been the risk of adverse events related with the use of synthetic materials in the vicinity of the esophagus. METHODS: A total of 14 female domestic pigs underwent laparoscopic primary hiatal hernia repair of a simulated defect in the esophageal hiatus. Seven of the hiatal repairs were reinforced with an extracellular matrix (ECM) scaffold, whereas the remaining seven served as primary repair controls. Animals were survived for 8 wk. At necropsy, after gross morphologic evaluation, samples were sent for mechanical testing and histology. RESULTS: The repaired defect site reinforced with ECM scaffolds showed a robust closure of the crura in all cases with a smooth peritoneal-like structure covering the entire repair. Average load at failure of the treated group was found to be significantly stronger than that of the controls (185.8 ± 149.7 g versus 57.5 ± 57.5 g, P < 0.05). Similarly, the stiffness was significantly higher in the treated animals (57.5 ± 26.9 g/mm versus 19.1 ± 17.5 g/mm; P < 0.01). Interestingly, there was no difference in elongation at failure (7.62 ± 2.02 mm versus 7.87 ± 3.28 mm; P = 0.44). CONCLUSIONS: In our animal survival model, we have provided evidence that the addition of an ECM to augment a primary hiatal repair leads to tissue characteristics that may decrease the possibility of early failure of the repair. This may translate to decreased recurrence rates. Further study is necessary.

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.

Reprint of: Extracellular matrix as a biological scaffold material: Structure and function.

Biological scaffold materials derived from the extracellular matrix (ECM) of intact mammalian tissues have been successfully used in a variety of tissue engineering/regenerative medicine applications both in preclinical studies and in clinical applications. Although it is recognized that the materials have constructive remodeling properties, the mechanisms by which functional tissue restoration is achieved are not well understood. There is evidence to support essential roles for both the structural and functional characteristics of the biological scaffold materials. This paper provides an overview of the composition and structure of selected ECM scaffold materials, the effects of manufacturing methods upon the structural properties and resulting mechanical behavior of the scaffold materials, and the in vivo degradation and remodeling of ECM scaffolds with an emphasis on tissue function.

Decellularized tracheal extracellular matrix supports epithelial migration, differentiation, and function.

Tracheal loss is a source of significant morbidity for affected patients with no acceptable solution. Interest in engineering tracheal transplants has created a demand for small animal models of orthotopic tracheal transplantation. Here, we examine the use of a decellularized graft in a murine model of tracheal replacement. Fresh or decellularized tracheas harvested from age-matched female donor C57BL/6 mice were transplanted into syngeneic recipients. Tracheas were decellularized using repeated washes of water, 3% Triton X-100, and 3 M NaCl under cyclic pressure changes, followed by disinfection with 0.1% peracetic acid/4% ethanol, and terminal sterilization by gamma irradiation. Tracheas were explanted for immunolabeling at 1, 4, and 8 weeks following surgery. Video microscopy and computed tomography were performed to assess function and structure. Decellularized grafts supported complete reepithelialization by 8 weeks and motile cilia were observed. Cartilaginous portions of the trachea were maintained in mice receiving fresh transplants, but repopulation of the cartilage was not seen in mice receiving decellularized transplants. We observed superior postsurgical survival, weight gain, and ciliary function in mice receiving fresh transplants compared with those receiving decellularized transplants. The murine orthotopic tracheal transplant provides an appropriate model to assess the repopulation and functional regeneration of decellularized tracheal grafts.

In vivo assessment of a biological occluder for NOTES gastrotomy closure.

BACKGROUND: The objective of this study is to evaluate the closure of a transgastric natural orifice transluminal endoscopic surgery (NOTES) access using a multilayer extracellular matrix (ECM) occluder in a canine model. MATERIALS AND METHODS: Mongrel dogs (n = 4) underwent a transgastric NOTES peritoneoscopy and the gastrotomy was closed by deploying a 2-sided ECM occluder. Animals were killed at 7 days (n = 2) and 8 weeks (n = 2) for macroscopic and microscopic assessment. RESULTS: All procedures were completed without any complications. No air leaks were detected immediately after the procedure and at 48 hours postoperatively. At 7 days, ECM appeared to be resolved and mild mucosal inflammation was found at the site of gastrotomy. At 8 weeks, the gastrotomy site was macroscopically and microscopically covered with a normal-appearing gastric mucosa. There was an absence of inflammatory cells and no evidence of the ECM. CONCLUSION: The ECM occluder is safe and effective in this "proof-of-concept" preclinical model.

Evaluation of magnesium-yttrium alloy as an extraluminal tracheal stent.

Tracheomalacia is a relatively rare problem, but can be challenging to treat, particularly in pediatric patients. Due to the presence of mechanically deficient cartilage, the trachea is unable to resist collapse under physiologic pressures of respiration, which can lead to acute death if left untreated. However, if treated, the outcome for patients with congenital tracheomalacia is quite good because the cartilage tends to spontaneously mature over a period of 12 to 18 months. The present study investigated the potential for the use of degradable magnesium-3% yttrium alloy (W3) to serve as an extraluminal tracheal stent in a canine model. The host response to the scaffold included the formation of a thin, vascularized capsule consisting of collagenous tissue and primarily mononuclear cells. The adjacent cartilage structure was not adversely affected as observed by bronchoscopic, gross, histologic, and mechanical analysis. The W3 stents showed reproducible spatial and temporal fracture patterns, but otherwise tended to corrode quite slowly, with a mix of Ca and P rich corrosion product formed on the surface and observed focal regions of pitting. The study showed that the approach to use degradable magnesium alloys as an extraluminal tracheal stent is promising, although further development of the alloys is required to improve the resistance to stress corrosion cracking and improve the ductility.

Effect of Mucin and Bicarbonate Ion on Corrosion Behavior of AZ31 Magnesium Alloy for Airway Stents.

The biodegradable ability of magnesium alloys is an attractive feature for tracheal stents since they can be absorbed by the body through gradual degradation after healing of the airway structure, which can reduce the risk of inflammation caused by long-term implantation and prevent the repetitive surgery for removal of existing stent. In this study, the effects of bicarbonate ion (HCO3-) and mucin in Gamble's solution on the corrosion behavior of AZ31 magnesium alloy were investigated, using immersion and electrochemical tests to systematically identify the biodegradation kinetics of magnesium alloy under in vitro environment, mimicking the epithelial mucus surfaces in a trachea for development of biodegradable airway stents. Analysis of corrosion products after immersion test was performed using scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD). Electrochemical impedance spectroscopy (EIS) was used to identify the effects of bicarbonate ions and mucin on the corrosion behavior of AZ31 magnesium alloys with the temporal change of corrosion resistance. The results show that the increase of the bicarbonate ions in Gamble's solution accelerates the dissolution of AZ31 magnesium alloy, while the addition of mucin retards the corrosion. The experimental data in this work is intended to be used as foundational knowledge to predict the corrosion behavior of AZ31 magnesium alloy in the airway environment while providing degradation information for future in vivo studies.

Urinary bladder matrix promotes site appropriate tissue formation following right ventricle outflow tract repair.

The current prevalence and severity of heart defects requiring functional replacement of cardiac tissue pose a serious clinical challenge. Biologic scaffolds are an attractive tissue engineering approach to cardiac repair because they avoid sensitization associated with homograft materials and theoretically possess the potential for growth in similar patterns as surrounding native tissue. Both urinary bladder matrix (UBM) and cardiac ECM (C-ECM) have been previously investigated as scaffolds for cardiac repair with modest success, but have not been compared directly. In other tissue locations, bone marrow derived cells have been shown to play a role in the remodeling process, but this has not been investigated for UBM in the cardiac location, and has never been studied for C-ECM. The objectives of the present study were to compare the effectiveness of an organ-specific C-ECM patch with a commonly used ECM scaffold for myocardial tissue repair of the right ventricle outflow tract (RVOT), and to examine the role of bone marrow derived cells in the remodeling response. A chimeric rat model in which all bone marrow cells express green fluorescent protein (GFP) was generated and used to show the ability of ECM scaffolds derived from the heart and bladder to support cardiac function and cellular growth in the RVOT. The results from this study suggest that urinary bladder matrix may provide a more appropriate substrate for myocardial repair than cardiac derived matrices, as shown by differences in the remodeling responses following implantation, as well as the presence of site appropriate cells and the formation of immature, myocardial tissue.

Preservation of micro-architecture and angiogenic potential in a pulmonary acellular matrix obtained using intermittent intra-tracheal flow of detergent enzymatic treatment.

Tissue engineering of autologous lung tissue aims to become a therapeutic alternative to transplantation. Efforts published so far in creating scaffolds have used harsh decellularization techniques that damage the extracellular matrix (ECM), deplete its components and take up to 5 weeks to perform. The aim of this study was to create a lung natural acellular scaffold using a method that will reduce the time of production and better preserve scaffold architecture and ECM components. Decellularization of rat lungs via the intratracheal route removed most of the nuclear material when compared to the other entry points. An intermittent inflation approach that mimics lung respiration yielded an acellular scaffold in a shorter time with an improved preservation of pulmonary micro-architecture. Electron microscopy demonstrated the maintenance of an intact alveolar network, with no evidence of collapse or tearing. Pulsatile dye injection via the vasculature indicated an intact capillary network in the scaffold. Morphometry analysis demonstrated a significant increase in alveolar fractional volume, with alveolar size analysis confirming that alveolar dimensions were maintained. Biomechanical testing of the scaffolds indicated an increase in resistance and elastance when compared to fresh lungs. Staining and quantification for ECM components showed a presence of collagen, elastin, GAG and laminin. The intratracheal intermittent decellularization methodology could be translated to sheep lungs, demonstrating a preservation of ECM components, alveolar and vascular architecture. Decellularization treatment and methodology preserves lung architecture and ECM whilst reducing the production time to 3 h. Cell seeding and in vivo experiments are necessary to proceed towards clinical translation.

Human-scale whole-organ bioengineering for liver transplantation: a regenerative medicine approach.

At this time, the only definitive treatment of hepatic failure is liver transplantation. However, transplantation has been limited by the severely limited supply of human donor livers. Alternatively, a regenerative medicine approach has been recently proposed in rodents that describe the production of three-dimensional whole-organ scaffolds for assembly of engineered complete organs. In the present study, we describe the decellularization of porcine livers to generate liver constructs at a scale that can be clinically relevant. Adult ischemic porcine livers were successfully decellularized using a customized perfusion protocol, the decellularization process preserved the ultrastructural extracellular matrix components, functional characteristics of the native microvascular and the bile drainage network of the liver, and growth factors necessary for angiogenesis and liver regeneration. Furthermore, isolated hepatocytes engrafted and reorganized in the porcine decellularized livers using a human-sized organ culture system. These results provide proof-of-principle for the generation of a human-sized, three-dimensional organ scaffold as a potential structure for human liver grafts reconstruction for transplantation to treat liver disease.

Prevention of seroma formation with TissuGlu® surgical adhesive in a canine abdominoplasty model: long term clinical and histologic studies.

BACKGROUND: Seroma formation is a common postoperative complication following many surgical procedures including abdominoplasty. Recently, a lysine-derived urethane (LDU) surgical adhesive was shown to prevent seroma formation in short term studies in a canine model of abdominoplasty. This current study evaluates efficacy of the adhesive (TissuGlu®, Cohera Medical, Inc.) in the same model at longer time points, and examines the histological tissue response to extended exposure to the adhesive. MATERIALS AND METHODS: Bilateral subcutaneous pockets were created in the ventrolateral abdominal wall and additional tissue damage was inflicted using electrocautery. On one side, the tissue layers were treated with the adhesive prior to closure, whereas the control side received no treatment prior to standard closure of the incision. Seroma fluid accumulation was measured and histologic analysis was performed at 3 and 12 weeks. RESULTS: Seroma formation (mean±SD, 690±870 ml; median volume of 348.5 ml) was observed on the control side, whereas the treated side had adherence between the tissue layers, and minimal if any fluid accumulation (mean±SD, 44±53 ml; median volume of 15 ml) (p<0.01) (n=8) at 3 week necropsy. In animals survived to 12 weeks, two of the four control sides required aspiration of serous fluid, and dead space persisted for the entirety of the study in one animal. For the adhesive treated sites, none of the four animals showed signs of seroma at euthanasia, although serial aspiration was performed in one treatment site within the first month and resulted in resolution of the process. The adhesive was detected in the surgical site at 3 and 12 weeks, and independent histological analysis found it to be a non-irritant compared to control (no treatment). CONCLUSIONS: Long term evaluation of TissuGlu® Surgical Adhesive showed that it is capable of preventing the formation of seroma in this canine abdominoplasty model, indicating that it may be of clinical benefit in the prevention of seroma formation in patients undergoing abdominoplasty.