Alejandro Nieponice

An Extracellular Matrix Scaffold for Esophageal Stricture Prevention After Circumferential EMR

BACKGROUND EMR is an accepted treatment for early esophageal cancer and high-grade dysplasia. One of the limitations of this technique is that extensive mucosal resection and endoscopic submucosal dissection may be required to obtain complete removal of the neoplasm, which may result in significant stricture formation. OBJECTIVE The objective of the current study was to evaluate the efficacy of an endoscopically deployed extracellular matrix (ECM) scaffold material for prevention of esophageal stenosis after circumferential EMR. DESIGN Ten mongrel dogs were subjected to surgical plane anesthesia and circumferential esophageal EMR by the cap technique. In 5 animals, an ECM scaffold material was endoscopically placed at the resection site; the remaining 5 animals were subjected to circumferential esophageal EMR without ECM placement. Follow-up endoscopy was performed at 4 and 8 weeks; necropsy with histologic assessment was performed at 8 weeks. SETTING Animal laboratory. INTERVENTIONS Circumferential esophageal EMR by the cap technique, followed by endoscopic placement of an ECM scaffold material. MAIN OUTCOME MEASUREMENTS Degree of esophageal stricture and histologic assessment of remodeled esophageal tissue. RESULTS All 5 control dogs had endoscopic evidence of esophageal stenosis. Three required early euthanasia because of inability to tolerate oral intake. Incomplete epithelialization and inflammation persisted at the EMR site in control animals. Endoscopic placement of an ECM scaffold material prevented clinically significant esophageal stenosis in all animals. Histologic assessment showed near-normal esophageal tissue with a lack of inflammation or scar tissue at 8 weeks. CONCLUSIONS Endoscopic placement of an ECM scaffold material prevented esophageal stricture formation after circumferential EMR in this canine model during short-term observation.

A small diameter, fibrous vascular conduit generated from a poly(ester urethane)urea and phospholipid polymer blend

The thrombotic and hyperplastic limitations associated with synthetic small diameter vascular grafts have generated sustained interest in finding a tissue engineering solution for autologous vascular segment generation in situ. One approach is to place a biodegradable scaffold at the site that would provide acute mechanical support while vascular tissue develops. To generate a scaffold that possessed both non-thrombogenic character and mechanical properties appropriate for vascular tissue, a biodegradable poly(ester urethane)urea (PEUU) and non-thrombogenic bioinspired phospholipid polymer, poly(2-methacryloyloxyethyl phosphorylcholine-co-methacryloyloxyethyl butylurethane) (PMBU) were blended at PMBU weight fractions of 0-15% and electrospun to create fibrous scaffolds. The composite scaffolds were flexible with breaking strains exceeding 300%, tensile strengths of 7-10MPa and compliances of 2.9-4.4 x 10(-4) mmHg(-1). In vitro platelet deposition on the scaffold surfaces significantly decreased with increasing PMBU content. Rat smooth muscle cell proliferation was also inhibited on PEUU/PMBU blended scaffolds with greater inhibition at higher PMBU content. Fibrous vascular conduits (1.3mm inner diameter) implanted in the rat abdominal aorta for 8 weeks showed greater patency for grafts with 15% PMBU blending versus PEUU without PMBU (67% versus 40%). A thin neo-intimal layer with endothelial coverage and good anastomotic tissue integration was seen for the PEUU/PMBU vascular grafts. These results are encouraging for further evaluation of this technique in larger diameter applications for longer implant periods.

Pericyte-based human tissue engineered vascular grafts.

The success of small-diameter tissue engineered vascular grafts (TEVGs) greatly relies on an appropriate cell source and an efficient cellular delivery and carrier system. Pericytes have recently been shown to express mesenchymal stem cell features. Their relative availability and multipotentiality make them a promising candidate for TEVG applications. The objective of this study was to incorporate pericytes into a biodegradable scaffold rapidly, densely and efficiently, and to assess the efficacy of the pericyte-seeded scaffold in vivo. Bi-layered elastomeric poly(ester-urethane)urea scaffolds (length = 10 mm; inner diameter = 1.3 mm) were bulk seeded with 3 x 10(6) pericytes using a customized rotational vacuum seeding device in less than 2 min (seeding efficiency > 90%). The seeded scaffolds were cultured in spinner flasks for 2 days and then implanted into Lewis rats as aortic interposition grafts for 8 weeks. Results showed pericytes populated the porous layer of the scaffolds evenly and maintained their original phenotype after the dynamic culture. After implantation, pericyte-seeded TEVGs showed a significant higher patency rate than the unseeded control: 100% versus 38% (p < 0.05). Patent pericyte-seeded TEVGs revealed extensive tissue remodeling with collagen and elastin present. The remodeled tissue consisted of multiple layers of alpha-smooth muscle actin- and calponin-positive cells, and a von Willebrand factor-positive monolayer in the lumen. These results demonstrate the feasibility of a pericyte-based TEVG and suggest that the pericytes play a role in maintaining patency of the TEVG as an arterial conduit.

Constructive remodeling of biologic scaffolds is dependent on early exposure to physiologic bladder filling in a canine partial cystectomy model.

Biologic scaffolds composed of extracellular matrix (ECM) have been used to facilitate the constructive remodeling of several tissue types. Previous studies suggest that the ECM scaffold remodeling process is dependent on microenvironmental factors, including tissue-specific biomechanical loading. The objective of the present study was to evaluate the effects of long-term catheterization (LTC), with its associated inhibition of bladder filling and physiologic biomechanical loading, on ECM scaffold remodeling following partial cystectomy in a canine model. Reconstruction of the partial cystectomy site was performed using ECM scaffolds prepared from porcine small intestinal submucosa (SIS) or porcine urinary bladder matrix (UBM). Animals were randomly assigned to either a long-term catheterization (LTC) group (n=5, catheterized 28 d) or a short-term catheterization group (STC, n=5, catheterized 24 h), and scaffold remodeling was assessed by histologic methods at 4 and 12 wk postoperatively. By 4 wk, animals in the STC group showed a well-developed and highly differentiated urothelium, a robust vascularization network, abundant smooth muscle actin (SMA), and smooth muscle myosin heavy chain (smMHC) expressing spindle-shaped cells, and many neuronal processes associated with newly formed arterioles. In contrast, at 4 wk the scaffolds in LTC animals were not epithelialized, and did not express neuronal markers. The scaffolds in the LTC group developed a dense granulation tissue containing SMA+, smMHC-, spindle-shaped cells that were morphologically and phenotypically consistent with myofibroblasts, but not smooth muscle cells. By 12 wk postoperatively, the ECM scaffolds in the STC animals showed a constructive remodeling response, with a differentiated urothelium and islands of smooth muscle cells within the remodeled scaffold. In contrast, at 12 wk the scaffolds in LTC animals had a remodeling response more consistent with fibrosis even though catheters had been removed 8 wk earlier. These findings show that early exposure of site-appropriate mechanical loading (i.e., bladder filling) mediates a constructive remodeling response after ECM repair in a canine partial cystectomy model.

In vivo performance of a phospholipid-coated bioerodable elastomeric graft for small-diameter vascular applications

There remains a great need for vascular substitutes for small-diameter applications. The use of an elastomeric biodegradable material, enabling acute antithrombogenicity and long-term in vivo remodeling, could be beneficial for this purpose. Conduits (1.3 mm internal diameter) were obtained by electrospinning biodegradable poly(ester urethane)urea (PEUU), and by luminally immobilizing a non-thrombogenic, 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymer. Platelet adhesion was characterized in vitro after contact with ovine blood. The conduits were implanted as aortic interposition grafts in the rat for 4, 8, 12, and 24 weeks. Surface treatment resulted in a 10-fold decrease in platelet adhesion compared to untreated material. Patency at 8 weeks was 92% for the coated grafts compared to 40% for the non-coated grafts. Histology at 8 and 12 weeks demonstrated formation of cellularized neotissue consisting of aligned collagen and elastin. The lumen of the grafts was confluent with cells qualitatively aligned in the direction of blood flow. Immunohistochemistry suggested the presence of smooth muscle cells in the medial layer of the neotissue and endothelial cells lining the lumen. Mechanically, the grafts were less compliant than rat aortas prior to implantation (4.5 ± 2.0 × 10(-4) mmHg(-1) vs. 14.2 ± 1.1 × 10(-4) mmHg(-1) , respectively), then after 4 weeks in vivo they approximated native values, but subsequently became stiffer again at later time points. The novel coated grafts exhibited promising antithrombogenic and mechanical properties for small-diameter arterial revascularization. Further evaluation in vivo will be required to demonstrate complete remodeling of the graft into a native-like artery.

Polypropylene Surgical Mesh Coated with Extracellular Matrix Mitigates the Host Foreign Body Response

Surgical mesh devices composed of synthetic materials are commonly used for ventral hernia repair. These materials provide robust mechanical strength and are quickly incorporated into host tissue; factors that contribute to reduced hernia recurrence rates. However, such mesh devices cause a foreign body response with the associated complications of fibrosis and patient discomfort. In contrast, surgical mesh devices composed of naturally occurring extracellular matrix (ECM) are associated with constructive tissue remodeling, but lack the mechanical strength of synthetic materials. A method for applying a porcine dermal ECM hydrogel coating to a polypropylene mesh is described herein with the associated effects upon the host tissue response and biaxial mechanical behavior. Uncoated and ECM coated heavy-weight BARD™ Mesh were compared to the light-weight ULTRAPRO™ and BARD™ Soft Mesh devices in a rat partial thickness abdominal defect overlay model. The ECM coated mesh attenuated the pro-inflammatory response compared to all other devices, with a reduced cell accumulation and fewer foreign body giant cells. The ECM coating degraded by 35 days, and was replaced with loose connective tissue compared to the dense collagenous tissue associated with the uncoated polypropylene mesh device. Biaxial mechanical characterization showed that all of the mesh devices were of similar isotropic stiffness. Upon explanation, the light-weight mesh devices were more compliant than the coated or uncoated heavy-weight devices. This study shows that an ECM coating alters the default host response to a polypropylene mesh, but not the mechanical properties in an acute in vivo abdominal repair model.

Patch Esophagoplasty: Esophageal Reconstruction Using Biologic Scaffolds

BACKGROUND Standard techniques for surgical reconstruction of the esophagus remain suboptimal. Primary closure of diseased or injured esophagus has been associated with high morbidity, primarily due to leak and stricture, and synthetic materials are contraindicated due to the high risk of erosion and infection. Degradable bioscaffolds composed of extracellular matrix (ECM) have recently shown promising results in both pre-clinical and clinical settings to prevent stricture after extended endoscopic mucosal resection. We propose a novel surgical technique that utilizes an ECM scaffold as a reconstructive patch to augment the esophageal diameter during primary repair. METHODS Four patients requiring esophageal reconstruction underwent a patch esophagoplasty using an ECM scaffold composed of porcine urinary bladder ECM. The full thickness wall of the esophagus was replaced with an ECM patch that was sutured to the edges of the remaining esophagus, similar to the patch angioplasty performed in vascular procedures. RESULTS All patients had a favorable clinical outcome with immediate recovery from the procedure and reinstated oral intake after 7 days. One patient had a micro leak at day 5 that closed spontaneously 2 days after drainage. Follow-up studies including barium swallow and esophagogastroduodenoscopy (EGD) showed adequate esophageal emptying through the surgical segment in all patients. The EGD showed complete mucosal remodeling at 2 months, with approximately 20% area contraction at the patch level. The area of the defect was indistinguishable from surrounding healthy tissue. Biopsy of the patch area showed normal squamous epithelium. One of the patients had a separate intrathoracic stricture that required further surgery. Clinical outcomes were otherwise favorable in all cases. CONCLUSIONS An alternative for the treatment of esophageal stenosis is presented which uses a biological scaffold and an innovative surgical procedure. Additional work, including prospective studies and long-term follow-up, is required to fully evaluate the potential of this bioscaffold-based regenerative medicine approach for esophageal reconstruction.

Molecular aspects of vascular tissue engineering.

Cardiovascular disease remains the number one cause of death in the United States. Most current surgical procedures to alleviate this disease rely on the availability of suitable small diameter vascular grafts, which are constrained by several limitations. Tissue engineering brings new hope to this field, but still faces many challenges. This review focuses on the molecular aspects of the different components of vascular tissue engineering. The topics addressed include the cell type, extracellular matrix, and physical and biochemical stimulation with respect to their role in the development of a tissue engineered vascular graft.

Bone marrow–derived cells participate in the long-term remodeling in a mouse model of esophageal reconstruction

BACKGROUND The default response of the esophagus to injury includes inflammation and scar tissue formation often leading to stricture. Biologic scaffolds composed of extracellular matrix (ECM) have been associated with the reconstitution of functional esophageal tissue in preclinical studies and clinical case reports of esophageal mucosal resection, anastomotic reinforcement, and full circumferential replacement. However, the mechanisms responsible for this change in the default response to esophageal injury are not fully understood. METHODS The objective of the present study was to determine whether bone marrow-derived cells (BMCs) participate in the long-term remodeling of ECM scaffolds in the esophageal location in a mouse model. RESULTS BMCs were present in low numbers in remodeling ECM scaffolds. Compared with the untreated control mice, the ECM-implanted animals showed better remodeling of the epithelial layer. CONCLUSIONS BMCs are involved in ECM remodeling process during tissue repair after esophageal injury, but the low numbers argue against any significant involvement in the constructive remodeling process.

A New Experimental System for the Extended Application of Cyclic Hydrostatic Pressure to Cell Culture

Mechanical forces have been shown to be important stimuli for the determination and maintenance of cellular phenotype and function. Many cells are constantly exposed in vivo to cyclic pressure, shear stress, and/or strain. Therefore, the ability to study the effects of these stimuli in vitro is important for understanding how they contribute to both normal and pathologic states. While there exist commercial as well as custom-built devices for the extended application of cyclic strain and shear stress, very few cyclic pressure systems have been reported to apply stimulation longer than 48 h. However, pertinent responses of cells to mechanical stimulation may occur later than this. To address this limitation, we have designed a new cyclic hydrostatic pressure system based upon the following design variables: minimal size, stability of pressure and humidity, maximal accessibility, and versatility. Computational fluid dynamics (CFD) was utilized to predict the pressure and potential shear stress within the chamber during the first half of a 1.0 Hz duty cycle. To biologically validate our system, we tested the response of bone marrow progenitor cells (BMPCs) from Sprague Dawley rats to a cyclic pressure stimulation of 120/80 mm Hg, 1.0 Hz for 7 days. Cellular morphology was measured using Scion Image, and cellular proliferation was measured by counting nuclei in ten fields of view. CFD results showed a constant pressure across the length of the chamber and no shear stress developed at the base of the chamber where the cells are cultured. BMPCs from Sprague Dawley rats demonstrated a significant change in morphology versus controls by reducing their size and adopting a more rounded morphology. Furthermore, these cells increased their proliferation under cyclic hydrostatic pressure. We have demonstrated that our system imparts a single mechanical stimulus of cyclic hydrostatic pressure and is capable of at least 7 days of continuous operation without affecting cellular viability. Furthermore, we have shown for the first time that BMPCs respond to cyclic hydrostatic pressure by alterations in morphology and increased proliferation.