Porous honeycomb film membranes enhance endothelial barrier integrity in human vascular wall bilayer model compared to standard track-etched membranes.

In vitro vascular wall bilayer models for drug testing and disease modeling must emulate the physical and biological properties of healthy vascular tissue and its endothelial barrier function. Both endothelial cell (EC)-vascular smooth muscle cell (SMC) interaction across the internal elastic lamina (IEL) and blood vessel stiffness impact endothelial barrier integrity. Polymeric porous track-etched membranes (TEM) typically represent the IEL in laboratory vascular bilayer models. However, TEM stiffness exceeds that of diseased blood vessels, and the membrane pore architecture limits EC-SMC interaction. The mechanical properties of compliant honeycomb film (HCF) membranes better simulate the Young's modulus of healthy blood vessels, and HCFs are thinner (4 vs. 10 µm) and more porous (57 vs. 6.5%) than TEMs. We compared endothelial barrier integrity in vascular wall bilayer models with human ECs and SMCs statically cultured on opposite sides of HCFs and TEMs (5 µm pores) for up to 12 days. Highly segregated localization of tight junction (ZO-1) and adherens junction (VE-cadherin) proteins and quiescent F-actin cytoskeletons demonstrated superior and earlier maturation of interendothelial junctions. Quantifying barrier integrity based on transendothelial electrical resistance (TEER), membranes showed only minor but significant TEER differences despite enhanced junctional protein localization on HCF. Elongated ECs on HCF likely experienced greater paracellular diffusion than blocky ECs on TEM. Also, larger populations of plaques of connexin 43 subunit-containing gap junctions suggested enhanced EC-SMC communication across the more porous, thinner HCF. Compared with standard TEMs, engineered vascular wall bilayers cultured on HCFs better replicate physiologic endothelial barrier integrity.

Correction to: Bone Marrow Derived Pluripotent Cells are Pericytes which Contribute to Vascularization.

Please note the following errors in the original version.

Fluorescent Reporter Mice for Nerve Guidance Conduit Assessment: A High-Throughput in vivo Model.

OBJECTIVE: Use of cell culture and conventional in vivo mammalian models to assess nerve regeneration across guidance conduits is resource-intensive. Herein we describe a high-throughput platform utilizing transgenic mice for stain-free axon visualization paired with rapid cryosection techniques for low-cost screening of novel bioengineered nerve guidance conduit performance. METHODS: Interposition repair of sciatic nerve transection in mice expressing yellow fluorescent protein in peripheral neurons (Thy1.2 YFP-16) was performed with various bioengineered neural conduit compositions using a rapid sutureless entubulation technique under isoflurane anesthesia. Axonal ingrowth was assessed at 3 and 6 weeks using epifluorescent microscopy following cryosectioning. RESULTS: Mean procedure time (incision-to-closure) was less than 2½ minutes. Direct operational costs of a 3-week experiment was calculated at $21.47 per animal. Tissue processing steps were minimized to aldehyde fixation, cryoprotection and sectioning, and rapid fluorescent dye staining for conduit visualization. Fluorescent microscopy readily resolved robust axonal sprouting at 3 weeks, with clear elucidation of ingrowth-permissive, semipermissive, or restrictive nerve guidance conduit environments. CONCLUSION: A rapid and cost-efficient in vivo platform for screening of nerve guidance conduit performance has been described. LEVEL OF EVIDENCE: NA. Laryngoscope, E392-E392, 2018.

Freeze-cast Porous Chitosan Conduit for Peripheral Nerve Repair.

A novel freeze-cast porous chitosan conduit for peripheral nerve repair with highly-aligned, double layered porosity, which provides the ideal mechanical and chemical properties was designed, manufactured, and assessed in vivo. Efficacies of the conduit and the control inverted nerve autograft were evaluated in bridging 10-mm Lewis rat sciatic nerve gap at 12 weeks post-implantation. Biocompatibility and regenerative efficacy of the porous chitosan conduit were evaluated through the histomorphometric analysis of longitudinal and transverse sections. The porous chitosan conduit was found to have promising regenerative characteristics, promoting the desired neovascularization, and axonal ingrowth and alignment through a combination of structural, mechanical and chemical cues.

In vivo response to decellularized mesothelium scaffolds.

Biological surgical scaffolds are used in plastic and reconstructive surgery to support structural reinforcement and regeneration of soft tissue defects. Macrophage and fibroblast cell populations heavily regulate scaffold integration into host tissue following implantation. In the present study, the biological host response to a commercially available surgical scaffold (Meso BioMatrix Surgical Mesh (MBM)) was investigated for up to 9 weeks after subcutaneous implantation; this scaffold promoted superior cell migration and infiltration previously in in vitro studies relative to other commercially available scaffolds. Infiltrating macrophages and fibroblasts phenotypes were assessed for evidence of inflammation and remodeling. At week 1, macrophages were the dominant cell population, but fibroblasts were most abundant at subsequent time points. At week 4, the scaffold supported inflammation modulation as indicated by M1 to M2 macrophage polarization; the foreign body giant cell response resolved by week 9. Unexpectedly, a fibroblast subpopulation expressed macrophage phenotypic markers, following a similar trend in transitioning from a proinflammatory to anti-inflammatory phenotype. Also, α-smooth muscle actin-expressing myofibroblasts were abundant at weeks 4 and 9, mirroring collagen expression and remodeling activity. MBM supported physiologic responses observed during normal wound healing, including cellular infiltration, host tissue ingrowth, remodeling of matrix proteins, and immune modulation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 716-725, 2018.

Chondrogenesis by bone marrow-derived mesenchymal stem cells grown in chondrocyte-conditioned medium for auricular reconstruction.

Bone marrow-derived mesenchymal stem cells (BMSCs) can be obtained by minimally invasive means and would be a favourable source for cell-based cartilage regeneration. However, controlling the differentiation of the BMSCs towards the desired chondrogenic pathway has been a challenge hampering their application. The major aim of the present study was to determine if conditioned medium collected from cultured auricular chondrocytes could promote chondrogenic differentiation of BMSCs. Auricular chondrocytes were isolated and grown in BMSC standard culture medium (SM) that was collected and used as chondrocyte-conditioned medium (CCM). The BMSCs were expanded in either CCM or SM for three passages. Cells were seeded onto fibrous collagen scaffolds and precultured for 2 weeks with or without transforming growth factor-beta 3 (TGF-ß3). After preculture, constructs were implanted subcutaneously in nude mice for 6 and 12 weeks and evaluated with real-time polymerase chain reaction, histology, immunohistochemistry and biochemistry. Real-time polymerase chain reaction results showed upregulation of COL2A1 in the constructs cultured in CCM compared with those in SM. After 12 weeks in vivo, abundant neocartilage formation was observed in the implants that had been cultured in CCM, with or without TGF-ß3. In contrast, very little cartilage matrix formation was observed within the SM groups, regardless of the presence of TGF-ß3. Osteogenesis was only observed in the SM group with TGF-ß3. In conclusion, CCM even had a stronger influence on chondrogenesis than the supplementation of the standard culture medium with TGF-ß3, without signs of endochondral ossification. Efficient chondrogenic differentiation of BMSCs could provide a promising alternative cell population for auricular regeneration. Copyright © 2016 John Wiley & Sons, Ltd.

In vitro evaluation of decellularized ECM-derived surgical scaffold biomaterials.

Decellularized extracellular matrix (ECM) biomaterials are increasingly used in regenerative medicine for abdominal tissue repair. Emerging ECM biomaterials with greater compliance target surgical procedures like breast and craniofacial reconstruction to enhance aesthetic outcome. Clinical studies report improved outcomes with newly designed ECM scaffolds, but their comparative biological characteristics have received less attention. In this study, we investigated scaffolds derived from dermis (AlloDerm Regenerative Tissue Matrix), small intestinal submucosa (Surgisis 4-layer Tissue Graft and OASIS Wound Matrix), and mesothelium (Meso BioMatrix Surgical Mesh and Veritas Collagen Matrix) and evaluated biological properties that modulate cellular responses and recruitment. An assay panel was utilized to assess the ECM scaffold effects upon cells. Results of the material-conditioned media study demonstrated Meso BioMatrix and OASIS best supported cell proliferation. Meso BioMatrix promoted the greatest migration and chemotaxis signaling, followed by Veritas and OASIS; OASIS had superior suppression of cell apoptosis. The direct adhesion assay indicated that AlloDerm, Meso BioMatrix, Surgisis, and Veritas had sidedness that affected cell-material interactions. In the chick chorioallantoic membrane assay, Meso BioMatrix and OASIS best supported cell infiltration. Among tested materials, Meso BioMatrix and OASIS demonstrated characteristics that facilitate scaffold incorporation, making them promising choices for many clinical applications. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 585-593, 2017.

Ear-Shaped Stable Auricular Cartilage Engineered from Extensively Expanded Chondrocytes in an Immunocompetent Experimental Animal Model.

Advancement of engineered ear in clinical practice is limited by several challenges. The complex, largely unsupported, three-dimensional auricular neocartilage structure is difficult to maintain. Neocartilage formation is challenging in an immunocompetent host due to active inflammatory and immunological responses. The large number of autologous chondrogenic cells required for engineering an adult human-sized ear presents an additional challenge because primary chondrocytes rapidly dedifferentiate during in vitro culture. The objective of this study was to engineer a stable, human ear-shaped cartilage in an immunocompetent animal model using expanded chondrocytes. The impact of basic fibroblast growth factor (bFGF) supplementation on achieving clinically relevant expansion of primary sheep chondrocytes by in vitro culture was determined. Chondrocytes expanded in standard medium were either combined with cryopreserved, primary passage 0 chondrocytes at the time of scaffold seeding or used alone as control. Disk and human ear-shaped scaffolds were made from porous collagen; ear scaffolds had an embedded, supporting titanium wire framework. Autologous chondrocyte-seeded scaffolds were implanted subcutaneously in sheep after 2 weeks of in vitro incubation. The quality of the resulting neocartilage and its stability and retention of the original ear size and shape were evaluated at 6, 12, and 20 weeks postimplantation. Neocartilage produced from chondrocytes that were expanded in the presence of bFGF was superior, and its quality improved with increased implantation time. In addition to characteristic morphological cartilage features, its glycosaminoglycan content was high and marked elastin fiber formation was present. The overall shape of engineered ears was preserved at 20 weeks postimplantation, and the dimensional changes did not exceed 10%. The wire frame within the engineered ear was able to withstand mechanical forces during wound healing and neocartilage maturation and prevented shrinkage and distortion. This is the first demonstration of a stable, ear-shaped elastic cartilage engineered from auricular chondrocytes that underwent clinical-scale expansion in an immunocompetent animal over an extended period of time.

Conditions for seeding and promoting neo-auricular cartilage formation in a fibrous collagen scaffold.

BACKGROUND: Carved autologous costal cartilage and porous polyethylene implants (Medpor) are the most common approaches for total ear reconstruction, but these approaches may have inconsistent cosmetic outcomes, a high risk of extrusion, or other surgical complications. Engineering ear cartilage to emulate native auricular tissue is an appealing approach, but often the cell-seeded scaffolds are susceptible to shrinkage and architectural changes when placed in vivo. The aim of this study was to assess the most favorable conditions for in vitro pre-culture of cell-seeded type I collagen scaffolds prior to in vivo implantation. METHODS: Sheep auricular chondrocytes were seeded into this type I collagen scaffold. The cell-seeded constructs were cultured in either static or dynamic conditions for two days or two weeks and then implanted into nude mice for another six weeks. The harvested constructs were evaluated histologically, immunohistochemically, and biochemically. RESULTS: Robust neo-cartilage formation was found in these collagen scaffolds seeded with auricular chondrocytes, which was comparable to native cartilage morphologically, histologically, and biochemically. Culture under dynamic conditions prior to implantation improved the neo-cartilage formation histologically and biochemically. CONCLUSION: Dynamic culture of this cell-seeded fibrous collagen material could permit predictable engineered auricular cartilage and a promising approach for external ear reconstruction.

Ovine model for auricular reconstruction: porous polyethylene implants.

OBJECTIVES: We developed a large animal model for auricular reconstruction with engineered cartilage frameworks and evaluated the performance of porous polyethylene auricular implants in this model. METHODS: Eighteen high-density porous polyethylene auricular frameworks were implanted subcutaneously in the infra-auricular areas of 9 sheep. The implants were harvested 17 weeks later for gross and histologic examination. The perioperative and postoperative courses were carefully documented. RESULTS: Five implants became exposed, and 2 implants needed to be removed at 7 weeks. Additionally, 1 infected implant was removed at 2 weeks. Seromas developed in 2 implants because of drain failures and were drained successfully during the first postoperative week. There were no other surgical site complications. The remaining 10 implants had an acceptable cosmetic appearance at 17 weeks. CONCLUSIONS: The perioperative complication rate in the ovine porous polyethylene auricular implant model was higher than that reported for auricular reconstructions in humans. The implant exposures were likely caused by ischemia and excessive stress on the thin overlying skin, because vascularized flap coverage was not used. The histologic findings were comparable to the results reported for other animal models. This large animal model is appropriate for auricular reconstruction experiments, including engineered constructs.

Combined surface micropatterning and reactive chemistry maximizes tissue adhesion with minimal inflammation.

The use of tissue adhesives for internal clinical applications is limited due to a lack of materials that balance strong adhesion with biocompatibility. The use of substrate topography is explored to reduce the volume of a highly reactive and toxic glue without compromising adhesive strength. Micro-textured patches coated with a thin layer of cyanoacrylate glue achieve similar adhesion levels to patches employing large amounts of adhesive, and is superior to the level of adhesion achieved when a thin coating is applied to a non-textured patch. In vivo studies demonstrate reduced tissue inflammation and necrosis for patterned patches with a thinly coated layer of reactive glue, thus overcoming a significant challenge with existing tissue adhesives such as cyanoacrylate. Closure of surgical stomach and colon defects in a rat model is achieved without abdominal adhesions. Harnessing the synergy between surface topography and reactive chemistry enables controlled tissue adhesion with an improved biocompatibility profile without requiring changes in the chemical composition of reactive tissue glues.

Successful creation of tissue-engineered autologous auricular cartilage in an immunocompetent large animal model.

Tissue-engineered cartilage has historically been an attractive alternative treatment option for auricular reconstruction. However, the ability to reliably generate autologous auricular neocartilage in an immunocompetent preclinical model should first be established. The objectives of this study were to demonstrate engineered autologous auricular cartilage in the immunologically aggressive subcutaneous environment of an immunocompetent animal model, and to determine the impact of in vitro culture duration of chondrocyte-seeded constructs on the quality of neocartilage maturation in vivo. Auricular cartilage was harvested from eight adult sheep; chondrocytes were isolated, expanded in vitro, and seeded onto fibrous collagen scaffolds. Constructs were cultured in vitro for 2, 6, and 12 weeks, and then implanted autologously in sheep and in control nude mice for 6 and 12 weeks. Explanted tissue was stained with hematoxylin and eosin, safranin O, toluidine blue, collagen type II, and elastin. DNA and glycosaminoglycans (GAGs) were quantified. The quality of cartilage engineered in sheep decreased with prolonged in vitro culture time. Superior cartilage formation was demonstrated after 2 weeks of in vitro culture; the neocartilage quality improved with increased implantation time. In nude mice, neocartilage resembled native sheep auricular cartilage regardless of the in vitro culture length, with the exception of elastin expression. The DNA quantification was similar in all engineered and native cartilage (p>0.1). All cartilage engineered in sheep had significantly less GAG than native cartilage (p<0.02); significantly more GAG was observed with increased implantation time (p<0.02). In mice, the GAG content was similar to that of native cartilage and became significantly higher with increased in vitro or in vivo durations (p<0.02). Autologous auricular cartilage was successfully engineered in the subcutaneous environment of an ovine model using expanded chondrocytes seeded on a fibrous collagen scaffold after a 2-week in vitro culture period.

Extensively Expanded Auricular Chondrocytes Form Neocartilage In Vivo.

OBJECTIVE: Our goal was to engineer cartilage in vivo using auricular chondrocytes that underwent clinically relevant expansion and using methodologies that could be easily translated into health care practice. DESIGN: Sheep and human chondrocytes were isolated from auricular cartilage biopsies and expanded in vitro. To reverse dedifferentiation, expanded cells were either mixed with cryopreserved P0 chondrocytes at the time of seeding onto porous collagen scaffolds or proliferated with basic fibroblast growth factor (bFGF). After 2-week in vitro incubation, seeded scaffolds were implanted subcutaneously in nude mice for 6 weeks. The neocartilage quality was evaluated histologically; DNA and glycosaminoglycans were quantified. Cell proliferation rates and collagen gene expression profiles were assessed. RESULTS: Clinically sufficient over 500-fold chondrocyte expansion was achieved at passage 3 (P3); cell dedifferentiation was confirmed by the simultaneous COL1A1/3A1 gene upregulation and COL2A1 downregulation. The chondrogenic phenotype of sheep but not human P3 cells was rescued by addition of cryopreserved P0 chondrocytes. With bFGF supplementation, chondrocytes achieved clinically sufficient expansion at P2; COL2A1 expression was not rescued but COL1A1/3A1genes were downregulated. Although bFGF failed to rescue COL2A1 expression during chondrocyte expansion in vitro, elastic neocartilage with obvious collagen II expression was observed on porous collagen scaffolds after implantation in mice for 6 weeks. CONCLUSIONS: Both animal and human auricular chondrocytes expanded with low-concentration bFGF supplementation formed high-quality elastic neocartilage on porous collagen scaffolds in vivo.

Design of composite scaffolds and three-dimensional shape analysis for tissue-engineered ear.

Engineered cartilage is a promising option for auricular reconstruction. We have previously demonstrated that a titanium wire framework within a composite collagen ear-shaped scaffold helped to maintain the gross dimensions of the engineered ear after implantation, resisting the deformation forces encountered during neocartilage maturation and wound healing. The ear geometry was redesigned to achieve a more accurate aesthetic result when implanted subcutaneously in a nude rat model. A non-invasive method was developed to assess size and shape changes of the engineered ear in three dimensions. Computer models of the titanium framework were obtained from CT scans before and after implantation. Several parameters were measured including the overall length, width and depth, the minimum intrahelical distance and overall curvature values for each beam section within the framework. Local curvature values were measured to gain understanding of the bending forces experienced by the framework structure in situ. Length and width changed by less than 2%, whereas the depth decreased by approximately 8% and the minimum intrahelical distance changed by approximately 12%. Overall curvature changes identified regions most susceptible to deformation. Eighty-nine per cent of local curvature measurements experienced a bending moment less than 50 µN-m owing to deformation forces during implantation. These quantitative shape analysis results have identified opportunities to improve shape fidelity of engineered ear constructs.

Biologic properties of surgical scaffold materials derived from dermal ECM.

Surgical scaffold materials manufactured from donor human or animal tissue are increasingly being used to promote soft tissue repair and regeneration. The clinical product consists of the residual extracellular matrix remaining after a rigorous decellularization process. Optimally, the material provides both structural support during the repair period and cell guidance cues for effective incorporation into the regenerating tissue. Surgical scaffold materials are available from several companies and are unique products manufactured by proprietary methodology. A significant need exists for a more thorough understanding of scaffold properties that impact the early steps of host cell recruitment and infiltration. In this study, a panel of in vitro assays was used to make direct comparisons of several similar, commercially-available materials: Alloderm, Medeor Matrix, Permacol, and Strattice. Differences in the materials were detected for both cell signaling and scaffold architecture-dependent cell invasion. Material-conditioned media studies found Medeor Matrix to have the greatest positive effect upon cell proliferation and induction of migration. Strattice provided the greatest chemotaxis signaling and best suppressed apoptotic induction. Among assays measuring structure-dependent properties, Medeor Matrix was superior for cell attachment, followed by Permacol. Only Alloderm and Medeor Matrix supported chemotaxis-driven cell invasion beyond the most superficial zone. Medeor Matrix was the only material in the chorioallantoic membrane assay to support substantial cell invasion. These results indicate that both biologic and structural properties need to be carefully assessed in the considerable ongoing efforts to develop new uses and products in this important class of biomaterials.

The tissue-engineered auricle: past, present, and future.

The reconstruction, repair, and regeneration of the external auricular framework continue to be one of the greatest challenges in the field of tissue engineering. To replace like with like, we should emulate the native structure and composition of auricular cartilage by combining a suitable chondrogenic cell source with an appropriate scaffold under optimal in vitro and in vivo conditions. Due to the fact that a suitable and reliable substitute for auricular cartilage has yet to be engineered, hand-carved autologous costal cartilage grafts and ear-shaped porous polyethylene implants are the current treatment modalities for auricular reconstruction. However, over the last decade, significant advances have been made in the field of regenerative medicine and tissue engineering. A variety of scaffolds and innovative approaches have been investigated as alternatives to using autologous carved costal cartilage or porous polyethylene implants. A review of recent developments and the current state of the art and science is presented, focusing on scaffolds, cell sources, seeding densities, and mechanical characteristics of tissue-engineered auricular cartilage.

The role of fibroblasts in self-assembled skeletal muscle.

Small facial skeletal muscles often have no autologous donor source to effect surgical reconstruction. Autologously derived muscles could be engineered for replacement tissue, but must be vascularized and innervated to be functional. As a critical step, engineered muscle must mimic the morphology, protein and gene expression, and function of native muscle. This study utilized a self-assembly process to engineer three-dimensional (3D) muscle from a statically strained muscle cell monolayer. Primary mouse myoblasts (PMMs) and mouse embryonic fibroblasts (MEFs) were separately proliferated and coseeded on a fibrin sheet with anchored sutures. Within 10 days of initiating PMM differentiation, the cell-gel layer contracted, lifted, and rolled into a cylindrical 3D structure around the tendon-like suture anchors; the myotubes longitudinally aligned along the lines of tensile force. The objectives of this study were to characterize these engineered muscles and to elucidate the role of the fibroblasts in the self-assembly process. Fibroblasts maintained myotube viability, mediated fibrin degradation, and assisted in muscle self-assembly. The optimal 1:1 PMM:MEF ratio resulted in tissue morphology remarkably similar to native muscle. Through gene and protein expression assays, the development and maturation of the engineered muscle tissue was demonstrated to recapitulate normal skeletal muscle development.

Engineering ear constructs with a composite scaffold to maintain dimensions.

Engineered cartilage composed of a patient's own cells can become a feasible option for auricular reconstruction. However, distortion and shrinkage of ear-shaped constructs during scaffold degradation and neocartilage maturation in vivo have hindered the field. Scaffolds made of synthetic polymers often generate degradation products that cause an inflammatory reaction and negatively affect neocartilage formation in vivo. Porous collagen, a natural material, is a promising candidate; however, it cannot withstand the contractile forces exerted by skin and surrounding tissue during normal wound healing. We hypothesised that a permanent support in the form of a coiled wire embedded into a porous collagen scaffold will maintain the construct's size and ear-specific shape. Half-sized human adult ear-shaped fibrous collagen scaffolds with and without embedded coiled titanium wire were seeded with sheep auricular chondrocytes, cultured in vitro for up to 2 weeks, and implanted subcutaneously on the backs of nude mice. After 6 weeks, the dimensional changes in all implants with wire support were minimal (2.0% in length and 4.1% in width), whereas significant reduction in size occurred in the constructs without embedded wire (14.4% in length and 16.5% in width). No gross distortion occurred over the in vivo study period. There were no adverse effects on neocartilage formation from the embedded wire. Histologically, mature neocartilage extracellular matrix was observed throughout all implants. The amount of DNA, glycosaminoglycan, and hydroxyproline in the engineered cartilage were similar to that of native sheep ear cartilage. The embedded wire support was essential for avoiding shrinkage of the ear-shaped porous collagen constructs.

Poly(glycerol sebacate)-engineered plugs to repair chronic tympanic membrane perforations in a chinchilla model.

OBJECTIVE: To evaluate the degree of neovascularization and efficacy of repair of chronic tympanic membrane perforations in a chinchilla model using poly(glycerol sebacate) (PGS), a novel bioengineered scaffold material. STUDY DESIGN: A feasibility study in which chinchilla ears with chronic perforations were randomly assigned to repair with PGS plugs or Gelfilm overlay myringoplasty. SETTING: Interventions were performed in the animal care facility of a tertiary care academic institution. SUBJECTS AND METHODS: Sixteen adult female chinchillas. Perforations were established under microscopic visualization with thermal cautery. The animals were examined six weeks later, and those ears with stable perforations were randomly assigned to repair with PGS or Gelfilm. All ears were evaluated six weeks after repair, and resected membranes underwent histological evaluation. RESULTS: Chronic perforations were established in 22 of 32 (69%) chinchilla tympanic membranes. Nineteen tympanic membranes were included in the study group (3 ears were excluded secondary to death from anesthesia during the repair); 11 were implanted with PGS, and eight underwent Gelfilm myringoplasty. Of the 11 tympanic membranes implanted with PGS, 10 were healed at six weeks, while six of the eight tympanic membranes repaired with Gelfilm had healed at six weeks. Imaging of the medial mucosal and lateral epithelial surfaces of the tympanic membranes revealed PGS plug incorporation with neovascularization. Histology demonstrated a confluent cell layer on both sides of the graft. CONCLUSIONS: PGS plugs are easily placed and allow for perforation closure and graft neovascularization in a chinchilla model.