Ethanol treatment of nanoPGA/PCL composite scaffolds enhances human chondrocyte development in the cellular microenvironment of tissue-engineered auricle constructs.

A major obstacle for tissue engineering ear-shaped cartilage is poorly developed tissue comprising cell-scaffold constructs. To address this issue, bioresorbable scaffolds of poly-ε-caprolactone (PCL) and polyglycolic acid nanofibers (nanoPGA) were evaluated using an ethanol treatment step before auricular chondrocyte scaffold seeding, an approach considered to enhance scaffold hydrophilicity and cartilage regeneration. Auricular chondrocytes were isolated from canine ears and human surgical samples discarded during otoplasty, including microtia reconstruction. Canine chondrocytes were seeded onto PCL and nanoPGA sheets either with or without ethanol treatment to examine cellular adhesion in vitro. Human chondrocytes were seeded onto three-dimensional bioresorbable composite scaffolds (PCL with surface coverage of nanoPGA) either with or without ethanol treatment and then implanted into athymic mice for 10 and 20 weeks. On construct retrieval, scanning electron microscopy showed canine auricular chondrocytes seeded onto ethanol-treated scaffolds in vitro developed extended cell processes contacting scaffold surfaces, a result suggesting cell-scaffold adhesion and a favorable microenvironment compared to the same cells with limited processes over untreated scaffolds. Adhesion of canine chondrocytes was statistically significantly greater (p ≤ 0.05) for ethanol-treated compared to untreated scaffold sheets. After implantation for 10 weeks, constructs of human auricular chondrocytes seeded onto ethanol-treated scaffolds were covered with glossy cartilage while constructs consisting of the same cells seeded onto untreated scaffolds revealed sparse connective tissue and cartilage regeneration. Following 10 weeks of implantation, RT-qPCR analyses of chondrocytes grown on ethanol-treated scaffolds showed greater expression levels for several cartilage-related genes compared to cells developed on untreated scaffolds with statistically significantly increased SRY-box transcription factor 5 (SOX5) and decreased interleukin-1α (inflammation-related) expression levels (p ≤ 0.05). Ethanol treatment of scaffolds led to increased cartilage production for 20- compared to 10-week constructs. While hydrophilicity of scaffolds was not assessed directly in the present findings, a possible factor supporting the summary data is that hydrophilicity may be enhanced for ethanol-treated nanoPGA/PCL scaffolds, an effect leading to improvement of chondrocyte adhesion, the cellular microenvironment and cartilage regeneration in tissue-engineered auricle constructs.

An analytical study of neocartilage from microtia and otoplasty surgical remnants: A possible application for BMP7 in microtia development and regeneration.

To investigate auricular reconstruction by tissue engineering means, this study compared cartilage regenerated from human chondrocytes obtained from either microtia or normal (conchal) tissues discarded from otoplasties. Isolated cells were expanded in vitro, seeded onto nanopolyglycolic acid (nanoPGA) sheets with or without addition of bone morphogenetic protein-7 (BMP7), and implanted in nude mice for 10 weeks. On specimen harvest, cartilage development was assessed by gross morphology, histology, and RT-qPCR and microarray analyses. Neocartilages from normal and microtia surgical tissues were found equivalent in their dimensions, qualitative degree of proteoglycan and elastic fiber staining, and quantitative gene expression levels of types II and III collagen, elastin, and SOX5. Microarray analysis, applied for the first time for normal and microtia neocartilage comparison, yielded no genes that were statistically significantly different in expression between these two sample groups. These results support use of microtia tissue as a cell source for normal auricular reconstruction. Comparison of normal and microtia cells, each seeded on nanoPGA and supplemented with BMP7 in a slow-release hydrogel, showed statistically significant differences in certain genes identified by microarray analysis. Such differences were also noted in several analyses comparing counterpart seeded cells without BMP7. Summary data suggest a possible application for BMP7 in microtia cartilage regeneration and encourage further studies to elucidate whether such genotypic differences translate to phenotypic characteristics of the human microtic ear. The present work advances understanding relevant to the potential clinical use of microtia surgical remnants as a suitable cell source for tissue engineering of the pinna.

Utilidad del diltiazem tópico al 2% en la condrodermatitis nodular del hélix: descripción de 2 casos / Usefulness of 2% Topical Diltiazem in Chondrodermatitis Nodularis Helicis: A Report of 2 Cases

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Inhibition of apoptosis signal-regulating kinase 1 alters the wound epidermis and enhances auricular cartilage regeneration.

Why regeneration does not occur in mammals remains elusive. In lower vertebrates, epimorphic regeneration of the limb is directed by the wound epidermis, which controls blastema formation to promote regrowth of the appendage. Herein, we report that knockout (KO) or inhibition of Apoptosis Signal-regulated Kinase-1 (ASK1), also known as mitogen-activated protein kinase kinase kinase 5 (MAP3K5), after full thickness ear punch in mice prolongs keratinocyte activation within the wound epidermis and promotes regeneration of auricular cartilage. Histological analysis showed the ASK1 KO ears displayed enhanced protein markers associated with blastema formation, hole closure and regeneration of auricular cartilage. At seven days after punch, the wound epidermis morphology was markedly different in the KO, showing a thickened stratum corneum with rounded cell morphology and a reduction of both the granular cell layer and decreased expression of filament aggregating protein. In addition, cytokeratin 6 was expressed in the stratum spinosum and granulosum. Topical application of inhibitors of ASK1 (NQDI-1), the upstream ASK1 activator, calcium activated mitogen kinase 2 (KN93), or the downstream target, c-Jun N-terminal kinase (SP600125) also resulted in enhanced regeneration; whereas inhibition of the other downstream target, the p38 α/ß isoforms, (SB203580) had no effect. The results of this investigation indicate ASK1 inhibition prolongs keratinocyte and blastemal cell activation leading to ear regeneration.

Plasticity of Auricular Cartilage in Response to Hormone Therapy.

INTRODUCTION: Correction of auricular deformities can be accomplished through splinting within the first few weeks of life. This is hypothesized to be due to retained circulating maternal estrogens decreasing the structural density of collagen; however, this has not been fully tested. Cartilage elasticity is dependent on the concentration of the proteoglycan aggregate, and hyaluronic acid, a constituent of proteoglycan aggregate, is increased by estrogens. Nonsurgical correction of these deformities in more developed patients has the potential to change clinical practice and eliminate surgical risks. Previous studies have demonstrated preliminary promise with the use of injectable estrogen to treat auricular deformities. For this study, we have validated an animal model and demonstrated the feasibility of a more therapeutically appropriate topical estrogen treatment in restoring neonatal plasticity of auricular cartilage. METHODS: Ears of 12 New Zealand rabbits were folded and splinted, and assigned an experimental group (estrogen, placebo, and untreated control) (n = 8 ears). Treatment ears received topical estrogen or placebo cream daily for 4 weeks, whereas controls received no treatment. The splints were removed following 2 additional weeks, and photographs were taken to calculate the retained fold angle. Biopsies were also taken for histologic analysis. RESULTS: The 8 control ears showed a statistically increased angle from a folded orientation of 46.6 degrees to return of ear position to a normal upright position of 151.2 degrees by the fourth day after splint removal. Both the estrogen-treated and placebo-treated ears responded to splinting with maintained folding (36.6 degrees and 32.5 degrees, respectively). Auricular cartilage thickness trended toward thicker in ears treated with estrogen, consistent with increased matrix components. CONCLUSIONS: Estrogen and placebo treatment with splinting of ears lead to a significant change to the cartilage configuration, validating the model. The results of this study are very encouraging and provide the foundation for a noninvasive therapeutic approach for correcting auricular deformities. Future work will include a more detailed mechanistic study evaluating the dosing of estrogen and the efficiency of dermal penetration as well as evaluating the long-term outcomes and molecular mechanism-associated cartilaginous responses to estrogen.

A Biodesigned Nanocomposite Biomaterial for Auricular Cartilage Reconstruction.

Current biomaterials for auricular replacement are associated with high rates of infection and extrusion. The development of new auricular biomaterials that mimic the mechanical properties of native tissue and promote desirable cellular interactions may prevent implant failure. A porous 3D nanocomposite scaffold (NS) based on POSS-PCU (polyhedral oligomeric silsesquioxane nanocage into polycarbonate based urea-urethane) is developed with an elastic modulus similar to native ear. In vitro biological interactions on this NS reveal greater protein adsorption, increased fibroblast adhesion, proliferation, and collagen production compared with Medpor (the current synthetic auricular implant). In vivo, the POSS-PCU with larger pores (NS2; 150-250 µm) have greater tissue ingrowth (≈5.8× and ≈1.4 × increase) than the POSS-PCU with smaller pores (NS1; 100-50 µm) and when compared to Medpor (>100 µm). The NS2 with the larger pores demonstrates a reduced fibrotic encapsulation compared with NS1 and Medpor (≈4.1× and ≈1.6×, respectively; P < 0.05). Porosity also influences the amount of neovascularization within the implants, with no blood vessel observed in NS1 (12 weeks postimplantation). The lack of chronic inflammatory response for all materials may indicate that the elastic modulus and pore size of the implant scaffold could be important design considerations for influencing fibrotic responses to auricular and other soft tissue implants.

The Usefulness of 0.2% Topical Nitroglycerin for Chondrodermatitis Nodularis Helicis.

BACKGROUND AND OBJECTIVE: Chondrodermatitis nodularis helicis (CNH) is a painful idiopathic degenerative condition involving the skin and cartilage of the helix or antihelix of the ear. Topical nitroglycerin 2% is a relatively recent treatment option for CNH that has produced good results, although with adverse effects (17% of cases). The use of a lower concentration would probably achieve similar results with fewer adverse effects. The aim of this study was to evaluate the effectiveness and safety of topical nitroglycerin 0.2% in the treatment of CNH. MATERIAL AND METHODS: We performed a retrospective observational study of patients treated in 2 Spanish hospitals between 2012 and 2014. The effectiveness of treatment was determined by clinical photography and assessment of symptoms using a verbal numerical rating scale. RESULTS: Of the 29 patients treated, 93% showed clinical improvement. In the group of responders, mean treatment duration was 1.8 months and mean follow-up was 5.9 months. Overall tolerance was good in all cases. CONCLUSION: Topical nitroglycerin 0.2% is an effective and well-tolerated conservative treatment option that improves the appearance of lesions and provides symptomatic relief in the majority of patients with CNH.

Role of insulin-transferrin-selenium in auricular chondrocyte proliferation and engineered cartilage formation in vitro.

The goal of this study is to determine the effects of Insulin-Transferrin-Selenium (ITS) on proliferation of auricular chondrocytes and formation of engineered cartilage in vitro. Pig auricular monolayer chondrocytes and chondrocyte pellets were cultured in media containing 1% ITS at different concentrations of fetal bovine serum (FBS, 10%, 6%, 2%, 0%), or 10% FBS alone as a control for four weeks. Parameters including cell proliferation in monolayer, wet weight, collagen type I/II/X (Col I, II, X) and glycosaminoglycan (GAG) expression, GAG content of pellets and gene expression associated with cartilage formation/dedifferentiation (lost cartilage phenotype)/hypertrophy within the chondrocyte pellets were assessed. The results showed that chondrocytes proliferation rates increased when FBS concentrations increased (2%, 6%, 10% FBS) in ITS supplemented groups. In addition, 1% ITS plus 10% FBS significantly promoted cell proliferation than 10% FBS alone. No chondrocytes grew in ITS alone medium. 1% ITS plus 10% FBS enhanced cartilage formation in terms of size, wet weight, cartilage specific matrices, and homogeneity, compared to 10% FBS alone group. Furthermore, ITS prevented engineered cartilage from dedifferentiation (i.e., higher index of Col II/Col I mRNA expression and expression of aggrecan) and hypertrophy (i.e., lower mRNA expression of Col X and MMP13). In conclusion, our results indicated that ITS efficiently enhanced auricular chondrocytes proliferation, retained chondrogenic phenotypes, and promoted engineered cartilage formation when combined with FBS, which is potentially used as key supplementation in auricular chondrocytes and engineered cartilage culture.

Recombinant human midkine stimulates proliferation and decreases dedifferentiation of auricular chondrocytes in vitro.

Autologous chondrocyte implantation (ACI) is widely used for the repair of cartilage defects. However, due to the lack of chondrocyte growth factor and dedifferentiation of the cultured primary chondrocytes, cell source has limited the clinical potential of ACI. Auricular cartilage is an attractive potential source of cells for cartilage tissue engineering. Here we demonstrated that recombinant human midkine (rhMK) significantly promoted proliferation of rat primary auricular chondrocytes cultured and passaged in monolayer, which was mediated by the activation of mitogen-activated protein kinase and phosphoinositide 3-kinase pathways. Furthermore, rhMK attenuated the dedifferentiation of cultured chondrocytes by maintaining the expression of chondrocyte-specific matrix proteins during culture expansion and passage. Importantly, rhMK-expanded chondrocytes reserved their full chondrogenic potential and redifferentiated into elastic chondrocytes after being cultured in high density. The results suggest that rhMK may be used for the preparation of chondrocytes in cartilage tissue engineering.

Esterified hyaluronic acid improves cartilage viability in experimental tracheal reconstruction with an auricular graft.

OBJECTIVE: The aim of this study was to determine the efficacy of esterified hyaluronic acid (HYAFF) on the vitality of auricular cartilage grafts used for tracheoplasty, with respect to macroscopic and microscopic parameters. STUDY DESIGN: Prospective, controlled. SETTING: Academic research laboratory. SUBJECTS AND METHODS: The study included 14 New Zealand rabbits acquired specifically for the study. The rabbits were divided into two groups: the control group, in which free cartilage grafts were not exposed to any materials or additional procedures (n = 7), and the hyaluronic acid (HA) treatment group, in which auricular grafts and anastomosis lines were covered with HYAFF (n = 7). Free auricular cartilage grafts used for the reconstruction of experimentally created tracheal defects were anastomosed extraluminally. All the rabbits were sacrificed two months post surgery. Samples were collected and examined histopathologically. The sections were stained with hematoxylin-eosin, Masson's trichrome, and inducible nitric oxide synthase (iNOS) and examined under a light microscope. RESULTS: Fibrosis and cartilage mass significantly differed between the control and HYAFF group (P < 0.05). Immunohistochemical examination showed that more chondrocytes stained with iNOS in the control group than in the HYAFF group, according to histologists' observations. CONCLUSION: HYAFF catalyzed wound healing with less fibrous tissue formation, had chondroprotective and stimulatory effects on chondrocyte metabolism, and decreased nitric oxide production and apoptosis via improving the nourishment of free auricular cartilage grafts, subsequently preventing hypoxia and oxidative stress, particularly in the early postimplantation period.

CCN family 2/connective tissue growth factor (CCN2/CTGF) stimulates proliferation and differentiation of auricular chondrocytes.

OBJECTIVES: CCN family 2/connective tissue growth factor (CCN2/CTGF) is an atypical growth factor for growth plate chondrocytes. It plays an important role in their proliferation and differentiation in vitro, but does not stimulate hypertrophy or calcification of articular chondrocytes. We herein report for the first time that CCN2/CTGF promotes growth and differentiation of auricular chondrocytes and maintains their molecular phenotype in vitro and in vivo. METHODS: Auricular chondrocytes were isolated from rabbit auricular cartilage by trypsin-collagenase treatment, and treated with human recombinant CCN2/CTGF or infected with adenovirus harboring the ccn2/ctgf gene. Cell proliferation was measured by [(3)H] thymidine incorporation and MTS assay, and changes in gene expression of auricular chondrocyte markers were monitored by real-time polymerase chain reaction, Northern hybridization, and histological analysis. For in vivo studies, auricular chondrocytes were cultured as pellets and implanted subcutaneously after treatment of recombinant human CCN2/CTGF. Ectopically formed cartilage was subjected to histological analysis. Cell death was monitored by in situ TUNEL analysis. RESULTS: CCN2/CTGF stimulated proliferation, differentiation and synthesis of elastin and proteoglycans of rabbit primary auricular chondrocytes in a dose-dependent manner. CCN2/CTGF caused a 2.5-fold increase in the expression of elastin in comparison to the control, resulting in enhanced deposition of elastin fibers in a monolayer culture of auricular chondrocytes. Mineralization was not induced; in contrast, CCN2/CTGF stimulated expression of matrix gla protein which is known to impair mineralization. Furthermore, pretreatment of pellets of auricular chondrocytes with CCN2/CTGF and subcutaneous implantation significantly enhanced the growth of ectopic auricular cartilage pieces expressing phenotypic markers of auricular chondrocytes including type II and X collagen. Notably, chondrocyte apoptosis was impaired by CCN2/CTGF. CONCLUSIONS: These findings show that CCN2/CTGF may be a suitable agent for promoting differentiation and growth of auricular chondrocytes, while preventing mineralization and apoptosis, and suggests that CCN2/CTGF may be useful for the repair or reconstruction of elastic cartilage.

In vitro enzymatic treatment and carbon dioxide laser beam irradiation of morphologic cartilage specimens.

OBJECTIVES: To determine the role of the main cartilage components in the internal system of interlocked stresses and to clarify the effect of laser beam irradiation on cartilage. DESIGN: Control and experimental series. SUBJECTS: Rabbit ear cartilage. INTERVENTION: Rabbit ear cartilage strips incubated in collagenase and hyaluronidase enzyme solutions for specific periods were examined, and the observed changes in shape, strength, and elasticity were recorded, as well as the effect of carbon dioxide laser irradiation. Laser-pretreated cartilage strips were also incubated in the enzyme solutions to determine whether the laser-provoked changes were susceptible to enzymatic action. All cartilage pieces were examined by light and electron microscopy. RESULTS: Collagenase-treated cartilage strips gradually lost their interlocked stresses, while hyaluronidase-treated strips mostly maintained their shape and their physical characteristics. Hyaluronidase-incubated cartilage strips altered their shape when they were laser treated. Collagenase-treated cartilages did not modify their shape when they were laser treated. Laser-pretreated cartilage pieces lost their new form in collagenase solutions but kept their laser-evoked shape when put in hyaluronidase solutions. CONCLUSION: The macroscopic observations combined with light and electron microscopy findings argue for the distinct role of the collagen network in morphologic cartilage shape and tensile strength preservation and provide a probable mechanism of cartilage transformation owing to carbon dioxide laser irradiation.

Repair of cartilage defects with periosteal grafts.

Alternative sources for repair of cartilage defects are limited and donor sites are associated with morbidity. It is known that cartilage development from periosteal grafts is possible. Various factors have been found positively to affect this process in experimental settings. However, all of these studies were limited to joint cartilage. We conducted an experimental study in rabbits for the investigation of the elastic cartilage regeneration from perichondrial and periosteal grafts together with the effects of hyaluronan on this process. 1 x 1 cm(2) cartilage defects were created on the elastic ear cartilage of rabbits. Four experimental groups with 18 ears in each group were created: Group 1 (repair with perichondrium graft), group 2 (repair with periosteum graft), group 3 (repair with periosteum graft+hyaluronan), group 4 (defect-only control group). Macroscopic and microscopic evaluations were done on the 4th, 8th and 12th weeks. Cellular morphology of the regenerated cartilage and its integration and similarity with adjacent cartilage were evaluated. Cartilage regeneration groups 1, 2 and 3 were found to be statistically different from the control group. There was not a significant difference between groups 1 and 2 or 2 and 3. There is no significant difference between perichondrial and periosteal grafts in cartilage regeneration, and hyaluronan has no beneficial effect on this process.

Cell yield, proliferation, and postexpansion differentiation capacity of human ear, nasal, and rib chondrocytes.

Human ear, nasal, and rib chondrocytes were compared with respect to their suitability to generate autologous cartilage grafts for nonarticular reconstructive surgery. Cells were expanded for two passages in medium containing 10% fetal bovine serum without (control) or with transforming growth factor beta(1) (TGF-beta(1)), fibroblast growth factor 2 (FGF-2), and platelet-derived growth factor bb (PDGF-bb) (TFP). Expanded cells were cultured as three-dimensional pellets in chondrogenic serum-free medium containing insulin, dexamethasone, and TGF-beta(1). Chondrocytes from all three sources were successfully isolated, increased their proliferation rate in response to TFP, and dedifferentiated during passaging. Redifferentiation by ear and nasal, but not rib, chondrocytes was enhanced after TFP expansion, as assessed by the significant increase in glycosaminoglycan (GAG)/DNA content and collagen type II mRNA expression in the resulting pellets. TFP-expanded ear and nasal chondrocytes generated pellets of better quality than rib chondrocytes, as assessed by the significantly higher GAG/DNA content and collagen type II mRNA expression, and by the relative stain intensities for GAG and collagen types I and II. In conclusion, postexpansion cell yields suggest that all three sources investigated could be used to generate autologous grafts of clinically relevant size. However, ear and nasal chondrocytes, if expanded with TFP, display superior postexpansion chondrogenic potential and may be a preferred cell source for cartilage tissue engineering.

Stimulation of in vivo angiogenesis by cytokine-loaded hyaluronic acid hydrogel implants.

Crosslinked hyaluronic acid (HA) hydrogels were evaluated for their ability to elicit new microvessel growth in vivo when preloaded with one of two cytokines, vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF). HA film samples were surgically implanted in the ear pinnas of mice, and the ears retrieved 7 or 14 days post implantation. Histologic analysis showed that all groups receiving an implant demonstrated significantly more microvessel density than control ears undergoing surgery but receiving no implant (p < 0.01). Moreover, aqueous administration of either growth factor produced substantially more vessel growth than an HA implant with no cytokine. However, the most striking result obtained was a dramatic synergistic interaction between HA and VEGF. Presentation of VEGF in crosslinked HA generated vessel density of NI = 6.7 at day 14, where NI is a neovascularization index defined below, more than twice the effect of the sum of HA alone (NI = 1.8) plus VEGF alone (NI=1.3). This was twice the vessel density generated by co-addition of HA and bFGF (NI=3.4, p<0.001). New therapeutic approaches for numerous pathologies could be notably enhanced by the localized, synergistic angiogenic response produced by release of VEGF from crosslinked HA films.

Effects of transforming growth factor Beta and insulinlike growth factor 1 on the biomechanical and histologic properties of tissue-engineered cartilage.

OBJECTIVE: To investigate the histologic and biomechanical properties of rabbit tissue-engineered cartilage exposed to insulinlike growth factor 1 and transforming growth factor beta. DESIGN: Controlled study. SUBJECTS: New Zealand white rabbits aged 3 to 4 weeks. INTERVENTION: A mean of 3.42 million rabbit chondrocytes were placed onto 2 x 1-cm polyglycolic/poly-L-lactic acid mesh templates. One group (n = 21) was placed in complete medium for 4 days. The experimental group (n = 19) was placed into complete medium with insulinlike growth factor 1 (50 ng/mL) and transforming growth factor beta (1 ng/mL). After 96 hours the templates were removed and implanted into the dorsum of the donor rabbit. The templates were harvested after 8 weeks and subjected to gross, histologic, and biomechanical testing. RESULTS: All samples showed histologic characteristics consistent with normal cartilage. No statistically significant differences were found with biomechanical testing between the control and experimental groups. CONCLUSION: In spite of more promising results from earlier studies, these results do not support improved histologic features or mechanical performance with the addition of insulinlike growth factor 1 and transforming growth factor beta to the chondrocyte/template complex.

Survival of composite chondrocutaneous grafts by vessel implantation: a study in the rabbit ear model.

Composite chondrocutaneous graft reconstruction or reattachment has limited applicability, is traditionally restricted to small segmental losses, and is dependent on the status of the recipient bed and graft periphery for successful revascularization. Surgical enhancement of composite graft survival was experimentally investigated in the rabbit ear model through transposition and appositional placement of an adjacent vascular pedicle. Fluorescein-derived surface-survival determinations, microangiographic vessel-counting methods, and histologic analysis were used to study the effects of vascular augmentation, pedicle design variations, and angiogenic substance in sixty 8-cm2, full-thickness auricular grafts. A statistically significant survival advantage was demonstrated for the implanted grafts, secondary to perivascular angiogenesis from the implanted pedicle.