3D printing of human ear pinna using cartilage specific ink.

Biofabrication of a complex structure such as ear pinna is not precise with currently available techniques. Auricular deformities (e.g. microtia) can cause physical, social as well as psychological impacts on a patient's wellbeing. Currently available surgical techniques and transplantation methods have many limitations that can be overcome with the help of 3D bioprinting technology. Printable bioink enriched with cartilage-specific extracellular matrix (ECM) synthesis was done by digesting goat ear pinna cartilage and polymerized by adding polyvinyl alcohol and gelatine. Rheological analysis and Fourier-transform infrared spectroscopy were used for the characterization of bioink to get desired viscosity and polymerization. Human ear pinna was printed using extrusion method and computer-aided design, stereolithography software which facilitated the automated printing in relatively less time without continuous monitoring. Thermal degradation of pinna was checked by thermal gravimetric analysis. Biodegradability and swelling of ear pinna were observed for understanding the nature of pinna and the impact of external factors. Reconstructed pinna's biocompatibility was proved byin ovoandin vivostudies. The occurrence of angiogenesis in the grafted ear manifested the capacity of proliferation and engraftment of cartilage cells. Histology and SEM analysis revealed the recellularization and the synthesis of ECM components such as glycosaminoglycan and collagen in transplanted 3D printed ear pinna. The expression of CD90+ which indicated newly synthesized cartilage in the transplanted 3D printed ear pinna. The absence expression of CD14+ also indicated acceptance of xenogenic transplanted 3D printed ear pinna. Transplantation of 3D ear pinna was successful in an animal model and can be utilized as tissue engineered ear bank.

Development and Evaluation of Topical Delivery of Microemulsions Containing Adapalene (MEs-Ap) for Acne.

The main objective of the study was to prepare the microemulsions containing adapalene (MEs-Ap) to enhance epidermal penetration, dermal retention, and local bioavailability compared with the commercial preparation. The optimal formulations were selected by solubility experiments, pseudo-ternary phase diagram, and percutaneous permeation experiments and the physiochemical properties were also investigated. Then, the study of permeability, retention, safety, pharmacodynamics, and pharmacokinetics in the skin for MEs-Ap compared with the commercial preparation were researched. The optimized formulation was developed as follows: the ratio of AP, isopropyl myristate, polyoxyethylene hydrogenated castor oil, ethanol, and water was 0.01:1:1.25:3.75:4 (w/w). The globule size and average viscosity of the optimized MEs-Ap were 99.34 nm and 1.7 mPa·s, respectively, which was oil-in-water microemulsion without serious irritation or allergy for skin. The Js, Qn, and Qretention of MEs-Ap (0.81 ± 0.19 µg/cm2/h, 24.73 ± 4.24 µg/cm2, 2.08 ± 0.18 µg/cm2) were apparently higher than Differin® (0.022 ± 0.009 µg/cm2/h, 0.536 ± 0.103 µg/cm2, and 0.523 ± 0.130 µg/cm2) respectively. The local bioavailability study showed that the AUC0 → 36h of the MEs-Ap in the dermal (19.6 ± 1.22 µg/cm2) was significantly improved comparing to Differin® (13.9 ± 1.73 µg/cm2) (p < 0.01). The pharmacodynamics study showed that the therapeutic effect of MEs-Ap was better than that of Differin® in the acne model of rabbit auricle. These results suggested that the MEs-Ap could be considered as a having higher epidermal penetrability, dermal retention, local bioavailability, efficacy, and safety topical preparations for acne. Graphical abstract.

Protein profile of ear auricle cartilage and the important role of ITGB1/PTK2 in microtia.

BACKGROUND: Microtia is a congenital malformation of the external ear that involves anything from a small reduction in size to a complete absence. The external ear is composed of elastic cartilage which is also the important skeleton of the outer ear. However no previous study explored the difference between abnormal elastic cartilage and normal cartilage in the molecular level. METHODS: Microtia cartilage and normal cartilage tissue samples from patients subjected to autologous costal cartilage reconstruction were obtained in surgery. Total proteins were extracted and purified, and then proteomic analyzed via LC-MS/MS using DDA/DIA data collection methods. Proteins were also isolated with lysis beads and then analyzed via antibody chip. Differentially expressed proteins were identified in both experiments and further analyzed with functional enrichment analysis and KEGG pathway analysis. Valuable regulatory gene expression level was verified by RT-PCR. RESULTS: A total of 4178 protein types were identified in the DDA experiment. A total of 2154 proteins were quantified, 172 of which were significantly upregulated and 82 downregulated in the microtia group (P < 0.05). Antibody chip detection allowed identification of 584 protein phosphorylation sites with 102 upregulation sites and 9 downregulation sites (P < 0.05). Differentially altered proteins were annotated to 143 KEGG pathways, while differentiated phosphate site-associated genes were annotated into 21 KEGG pathways. Two intersecting pathways, the PI3K/AKT/mTOR pathway and the focal adhesion pathway, may paly important role on ear auricle cartilage development. One item is significant in both differential protein expression and phosphorylation. Integrin beta-1, that is downregulated in protein quantification of the microtia group. The mean ITGB1 mRNA level of the microtia patient group was significantly lower than in the healthy control group (P = 0.0007 < 0.05). And the gene expression of downstream gene PTK2 was also decreased. (P = 0.0288 < 0.05). CONCLUSION: The research locates the key protein Integrin Beta-1, and verified it at the mRNA level. The increasing level of ITGB1 and decreasing of PTK2 may play an important role in congenital ear deformity. This research will inspire more otolaryngologists and orthopedics doctors to pay attention to the etiology and mechanism of microtia.

Duplication in ECR Near HMX1 and a SNP in GATA6 Genes Regulate Microtia in Awassi Sheep.

Microtia and anotia are hereditary traits characterized by an underdevelopment or complete absence of the outer ear. These congenital malformations observed in many species can exist as part of various syndromes or as an isolated trait as seen in the fat-tailed Awassi sheep breed. Our study aims to identify the genetic mutations causing microtia in Awassi sheep by DNA sequencing. DNA was extracted from blood samples randomly collected from 84 Awassi sheep (16 earless, 41 short ear and 27 normal ear) across different farms. GATA6 exons 1, 2, 4, 6 and 7, CLRN1 intron 3, DCC intron 2, ECR near HMX1 and the intergenic region between GATA6 and MIB1 genes were screened, amplified and sequenced. Allele and genotype frequencies were calculated by direct counting. Association was performed using chi-squared test for goodness-of-fit. Results showed mutations in only two genes significantly associated with microtia in Awassi: duplication in part of ECR near HMX1 (6:114293121-6:114293196) and a SNP at GATA6 exon 7 (23:34498242). Association results revealed that the ECR locus accounts for the microtia phenotype, while GATA6 exon 7 acts as a modifier gene. Genetic screening for these loci can be used to improve selection against microtia in Awassi sheep.

Effects of Zoledronate on Local and Systemic Production of IL-1ß, IL-18, and TNF-α in Mice and Augmentation by Lipopolysaccharide.

Bisphosphonates (BPs) containing nitrogen (N-BPs) exhibit far stronger anti-bone-resorptive effects than non-N-BPs. However, repeated administration of N-BPs causes osteonecrosis selectively in jawbones. As BPs accumulate in large amounts within inflamed bones, any N-BP released from the pool accumulated within jawbones might directly act on cells in the surrounding soft-tissues and induce inflammation or necrosis. Here, we examined the local and systemic effects of zoledronate (the most potent N-BP with the highest incidence of jawbone-necrosis) on inflammatory cytokines in mice. Locally within ear-pinnas: (i) zoledronate induced long-lasting accumulation of interleuikin-1ß (IL-1ß) and IL-18, but not tumor necrosis factor-α (TNF-α), (ii) zoledronate and lipopolysaccharide (LPS, a cell-wall component of Gram-negative bacteria) mutually augmented the productions of IL-1ß, IL-18, and TNF-α, and (iii) oxidronate (a toxic non-N-BP) by itself produced not only IL-1ß and IL-18, but also TNF-α. In systemic experiments using intraperitoneal injection of zoledronate and/or LPS, (i) zoledronate by itself increased none of the above cytokines in serum, and (ii) in mice pretreated (3 d before) with zoledronate, the LPS-induced increases in serum IL-1ß and IL-18 were greatly augmented with a delayed slight TNF-α augmentation. These results, together with previous ones, suggest that (a) pro-IL-1ß and pro-IL-18 accumulate within cells in soft-tissues exposed to N-BPs, and infection may augment not only their production, but also the release of their mature forms, (b) IL-1ß and IL-18 (possibly together with TNF-α) may play important roles in N-BP-induced inflammation and/or necrosis, and (c) mechanisms underlying the cytotoxic effects of BPs may differ between N-BPs and non-N-BPs.

Peri-arterial Autonomic Innervation of the Human Ear.

Auricular vasomotor responses are considered to be signs of clinical conditions including migraine. The mechanisms of auricular vasomotor control are still debatable. This study aimed at investigating perivascular co-transmitters of vasomotor control in the auricle. Another aim was to provide three-dimensional arterial maps of the auricle, as a proxy of periarterial autonomic innervation. Twelve paired human auricles were used to visualize the arteries following Spalteholz clearing and µ-CT-based reconstruction. Perivascular innervation staining was conducted using anti-tyrosine hydroxylase (TH), anti-neuropeptide Y (NPY), anti-vasoactive intestinal peptide (VIP) and anti-choline acetyl transferase (ChAT). The combined Spalteholz technique and µ-CT revealed a highly consistent arrangement of the auricular vasculature. The superficial temporal (STA) and posterior auricular artery (PAA) supply the helical rim arcade and arcade, with the STA mainly forming the superior and the PAA forming the middle and inferior auricular artery. Co-existence of sympathetic NPY+ and TH+ terminals mediating vasoconstriction, and VIP+ and ACh+ indicating cholinergic vasodilatation, was found in the perivascular zone. The presence of both sympathetic vasoconstriction and cholinergic co-innervation for active vasodilatation was shown in the perivascular auricular zone. Assuming that the highly-consistent vasculature gives way to these terminals, this periarterial innervation may be found spread out across the helix.

Gene expression profiles in auricle skin as a possible additional endpoint for determination of sensitizers: A multi-endpoint evaluation of the local lymph node assay.

The murine local lymph node assay (LLNA) is widely used to test chemicals to induce skin sensitization. Exposure of mouse auricle skin to a sensitizer results in proliferation of local lymph node T cells, which has been measured by in vivo incorporation of H3-methyl thymidine or 5-bromo-2'-deoxyuridine (BrdU). The stimulation index (SI), the ratio of the mean proliferation in each treated group to that in the concurrent vehicle control group, is frequently used as a regulatory-authorized endpoint for LLNA. However, some non-sensitizing irritants, such as sodium dodecyl sulfate (SDS) or methyl salicylate (MS), have been reported as false-positives by this endpoint. In search of a potential endpoint to enhance the specificity of existing endpoints, we evaluated 3 contact sensitizers; (hexyl cinnamic aldehyde [HCA], oxazolone [OXA], and 2,4-dinitrochlorobenzene [DNCB]), 1 respiratory sensitizer (toluene 2,4-diisocyanate [TDI]), and 2 non-sensitizing irritants (MS and SDS) by several endpoints in LLNA. Each test substance was applied to both ears of female CBA/Ca mice daily for 3 consecutive days. The ears and auricle lymph node cells were analyzed on day 5 for endpoints including the SI value, lymph node cell count, cytokine release from lymph node cells, and histopathological changes and gene expression profiles in auricle skin. The SI values indicated that all the test substances induced significant proliferation of lymph node cells. The lymph node cell counts showed no significant changes by the non-sensitizers assessed. The inflammatory findings of histopathology were similar among the auricle skins treated by sensitizers and irritants. Gene expression profiles of cytokines IFN-γ, IL-4, and IL-17 in auricle skin were similar to the cytokine release profiles in draining lymph node cells. In addition, the gene expression of the chemokine CXCL1 and/or CXCL2 showed that it has the potential to discriminate sensitizers and non-sensitizing irritants. Our results suggest that multi-endpoint analysis in the LLNA leads to a better determination of the sensitizing potential of test substances. We also show that the gene expression of CXCL1 and/or CXCL2, which is involved in elicitation of contact hypersensitivity (CHS), can be a possible additional endpoint for discrimination of sensitizing compounds in LLNA.

A distal 594†bp ECR specifies Hmx1 expression in pinna and lateral facial morphogenesis and is regulated by the Hox-Pbx-Meis complex.

Hmx1 encodes a homeodomain transcription factor expressed in the developing lateral craniofacial mesenchyme, retina and sensory ganglia. Mutation or mis-regulation of Hmx1 underlies malformations of the eye and external ear in multiple species. Deletion or insertional duplication of an evolutionarily conserved region (ECR) downstream of Hmx1 has recently been described in rat and cow, respectively. Here, we demonstrate that the impact of Hmx1 loss is greater than previously appreciated, with a variety of lateral cranioskeletal defects, auriculofacial nerve deficits, and duplication of the caudal region of the external ear. Using a transgenic approach, we demonstrate that a 594 bp sequence encompassing the ECR recapitulates specific aspects of the endogenous Hmx1 lateral facial expression pattern. Moreover, we show that Hoxa2, Meis and Pbx proteins act cooperatively on the ECR, via a core 32 bp sequence, to regulate Hmx1 expression. These studies highlight the conserved role for Hmx1 in BA2-derived tissues and provide an entry point for improved understanding of the causes of the frequent lateral facial birth defects in humans.

La utilidad de la nitroglicerina tópica al 0, 2% en la condrodermatitis nodular del hélix / The Usefulness of 0. 2% Topical Nitroglycerin for Chondrodermatitis Nodularis Helicis

INTRODUCCIÓN: La condrodermatitis nodular del hélix (CNH) es un proceso idiopático, degenerativo y doloroso que afecta a la piel y al cartílago del hélix o del antéhelix. Recientemente se ha descrito la utilidad de la nitroglicerina (NTG) tópica a 2% en el tratamiento de la CNH con buenos resultados, aunque con una tasa de efectos secundarios en el 17% de los casos. Es probable que a una concentración menor se pueda mantener el mismo efecto mejorando la tolerancia. Nuestra finalidad fue evaluar la efectividad y seguridad de la NTG tópica al 0,2% para el tratamiento de la CNH. MATERIAL Y MÉTODOS: Se llevó a cabo un estudio observacional retrospectivo entre los años 2012 y 2014 en 2 centros hospitalarios españoles. La efectividad se determinó a través de la evaluación clínica, realizada mediante seguimiento fotográfico, y de los síntomas de la lesión, medido mediante una escala numérica verbal. RESULTADOS: Veintinueve pacientes recibieron el tratamiento, de los cuales el 93% manifestaron una mejoría clínica con una duración media del tratamiento de 1,8 meses y un tiempo de seguimiento medio en los pacientes respondedores de 5,9 meses. La tolerancia fue buena en general en todos los casos. CONCLUSIÓN: La NTG tópica al 0,2% se plantea como una opción conservadora, efectiva y bien tolerada para el tratamiento de la condrodermatitis nodular del hélix que mejora tanto la apariencia clínica como la sintomatología en la mayoría de los pacientes

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

Shikonin extracted from medicinal Chinese herbs exerts anti-inflammatory effect via proteasome inhibition.

Shikonin, extracted from medicinal Chinese herb (Lithospermum erythrorhizo), was reported to exert anti-inflammatory and anti-cancer effects both in vitro and in vivo. We have found that proteasome was a molecular target of shikonin in tumor cells, but whether shikonin targets macrophage proteasome needs to be investigated. In the current study, we report that shikonin inhibited inflammation in mouse models as efficiently as dexamethasone. Shikonin at 4 µM reduced the Lipopolysaccharides (LPS)-mediated TNFα release in rat primary macrophage cultures, and blocked the translocation of p65-NF-κB from the cytoplasm to the nucleus, associated with decreased proteasomal activity. Consistently, shikonin accumulated IκB-α, an inhibitor of NF-κB, and ubiquitinated proteins in rat primary macrophage cultures, demonstrating that the proteasome is a target of shikonin under inflammatory conditions. Shikonin also induced macrophage cell apoptosis and cell death. These results demonstrate for the first time that proteasome inhibition by shikonin contributes to its anti-inflammatory effect. The novel finding about macrophage proteasome as a target of shikonin suggests that this medicinal compound has great potential to be developed into an anti-inflammatory agent.

Induction of inflammatory reactions by lipopolysaccharide in hamster sebaceous glands and pilosebaceous units in vivo and in vitro.

Lipopolysaccharide (LPS) from Gram-negative bacteria has been reported to exert inflammatory reactions in epidermis, dermis, and sebaceous glands. Here, we demonstrated that the intradermal administration of Escherichia coli-derived LPS, three times a week for 4 weeks, to hamster auricle skin did not influence sebaceous morphology or sebum accumulation in sebaceous glands but in fact induced epidermal thickness. In addition, the administration of LPS, once a day for 2 days, augmented the production of cyclooxygenase 2 (COX-2) in sebaceous glands. Furthermore, LPS increased the production of prostaglandin F(2α) (PGF(2α) ) in hamster sebocytes. Moreover, the production of progelatinase A/promatrix metalloproteinase 2 (proMMP-2) was transcriptionally augmented by LPS and PGF(2α) in hamster sebocytes. Therefore, these results suggest that LPS directly increases inflammation by augmenting COX-2, PGF(2α) , and the PGF(2α) -mediated proMMP-2 production in sebaceous glands as well as epidermal inflammatory events in skin disorders including acne and folliculitis.

Synthesis of anthraquinone-ibuprofen prodrugs with hydroxyapatite affinity and anti-inflammatory activity characteristics.

The synthesis and pharmacological activities of anthraquinone-ibuprofen prodrugs for finding new anti-inflammatory drugs specifically targeting osseous tissues were studied. Two hydrolytically activated anti-inflammatory prodrugs containing anthraquinone moiety and ibuprofen moiety were designed and synthesized. Rhein was chosen as bone-targeting agent and potentially active drug, which was linked chemically with ibuprofen through glycol ester as bone-targeting anti-inflammatory prodrugs. The chemical structures of the new compounds were confirmed by IR, 1H NMR, 13C NMR, MS and elemental analysis. The studies of bioactivities demonstrated that both prodrugs showed significant binding capability to hydroxyapatite (HAP), the major component of bone, and were hydrolytically activated under physiological conditions in vitro and better anti-inflammatory activity in vivo.

Induction of eosinophil- and Th2-attracting epidermal chemokines and cutaneous late-phase reaction in tape-stripped skin.

Skin barrier damage induces various harmful or even protective reactions in the skin, as represented by enhancement of keratinocyte cytokine production. To investigate whether acute removal of stratum corneum modulates the production of chemokines by epidermal cells, we treated ears of BALB/c and C57BL/6 mice by tape-stripping, or acetone-rubbing as a control of acute barrier disruption procedure. There was no difference between the tape-stripped and acetone-rubbed skin sites in the increased and recovered levels of transepidermal water loss. The mRNA expression levels of all the chemokines tested, including Th1 chemokines (CXCL10, CXCL9 and CXCL11), Th2 chemokines (CCL17 and CCL22) and eosinophil chemoattractant (CCL5), were higher in the epidermal cells from BALB/c than in those of C57BL/6 mice. In particular, CCL17, CCL22 and CCL5 were remarkably elevated in BALB/c mice and augmented by tape-stripping more markedly than acetone-rubbing, whereas Th1 chemokines were enhanced by acetone-rubbing more remarkably. Tape-stripping induced dermal infiltration of eosinophils in BALB/c but not C57BL/6 mice. In a contact hypersensitivity model, where BALB/c mice were sensitized on the abdomen and challenged on the ears with fluorescein isothiocyanate, mice exhibited higher ear swelling responses at the late-phase as well as delayed-type reactions, when challenged via the tape-stripped skin. The challenge via tape-stripped skin augmented the expression of IL-4 and CCR4 in the skin homogenated samples, indicating infiltration of Th2 cells. These findings suggest that acute barrier removal induces the expression of Th2 and eosinophil chemokines by epidermal cells and easily evokes the late phase reaction upon challenge with antigen.

Modification of the picryl chloride-induced allergic dermatitis model in mouse ear lobes by 12-O-tetradecanoylphorbol 13-acetate, and analysis of the role of histamine in the modified model.

BACKGROUND: In atopic dermatitis, inflammation induced by antigen-nonspecific stimuli further enhances the allergic inflammation. However, there is no experimental model in which allergic dermatitis is evoked where the inflammation has been induced by antigen-nonspecific stimuli. Here, we established a novel dermatitis model in mice and analyzed the role of histamine. METHODS: After sensitization with picryl chloride (PiCl) by painting on ear lobes of cyclophosphamide-treated mice, 12-O-tetradecanoylphorbol 13-acetate (TPA) was painted twice at the same site, and then allergic inflammation was induced by painting PiCl. Histamine antagonists and cyclosporine A (CsA) were administered intravenously. RESULTS: The application of TPA shifted the PiCl-induced allergic inflammation from a delayed-type response to a biphasic response, increased the infiltration of eosinophils and mast cells at the inflammatory site, shifted the cytokine milieu from Th1 to Th2 and induced the expression of thymic stromal lymphopoietin in the ear lobes. The PiCl-induced increase in the thickness of the ear lobe in the immediate phase was suppressed by the H1 antagonist pyrilamine. In contrast, the increase in the swelling in the late phase and the infiltration of eosinophils were suppressed by the H3/H4 antagonist thioperamide. The inhibitory effect of the combined treatment with pyrilamine and thioperamide on the TPA-modified contact dermatitis was as potent as that of CsA. CONCLUSION: Induction of the antigen-nonspecific inflammation by TPA enhanced the PiCl-induced allergic inflammation. Histamine plays significant roles in the early-phase swelling via H1 receptors, and the late-phase swelling via H3/H4 receptors in this TPA-modified allergic dermatitis model.

Intracellular expression of cytokines and granzyme B in auricular lymph nodes draining skin exposed to irritants and sensitizers.

The murine local lymph node assay (LLNA) has been extensively utilized to evaluate sensitizing chemicals. However, there have been some concerns that its use to discriminate between classes of chemicals is minimal. It is thus desirable to identify better or alternative immune endpoints with in LLNA itself. Here, we evaluated the protein and/or mRNA levels of cytokines and granzyme B (GzmB), a cytotoxic lymphocyte product, to discriminate between sensitizers and irritants and to characterize the chemical sensitizers when used as supplemental indicators in LLNA endpoints. For this, CBA/N mice were topically treated daily with a well-known chemical sensitizer such as a strong contact sensitizer 1-chloro-2,4-dinitrobenzene (DNCB), a skin contact sensitizer 2-phenyl-4-ethoxymethylene-5-oxazolone (OXA), and a skin or respiratory sensitizer toluene 2,4-diisocyanate (TDI), and the non-sensitizing irritants, croton oil (CRO) and nonanoic acid (NA), for 3 consecutive days. The protein and/or mRNA levels in auricular lymph nodes draining the ear skin were then analyzed by real-time RT-PCR and immunoassay. The sensitizers, but not the irritants, evoked pronounced interleukin (IL)-2, IL-3 and IL-4 or interferon (IFN)-gamma. Significantly, different sensitizers evoked different cytokine patterns of IL-4 and IFN-gamma, as DNCB strongly up-regulated both IFN-gamma and IL-4, OXA up-regulated IFN-gamma strongly but IL-4 weakly, and TDI up-regulated IL-4 strongly but IFN-gamma weakly. The sensitizers also strongly up-regulated GzmB mRNA, while the irritants had a much weaker effect. Thus, these cytokines and GzmB mRNA may be useful as additional endpoints for discriminating between irritants and sensitizers or contact and respiratory sensitizers in the LLNA.

Differential behavior of auricular and articular chondrocytes in hyaluronic acid hydrogels.

Chondrocytes isolated from a variety of sources, including auricular (AU) and articular (AR) cartilage, can differ in cell behavior, growth, and extracellular matrix (ECM) production, which can impact neocartilage properties in tissue engineering approaches. This behavior is also affected by the surrounding microenvironment, including soluble factors, biomaterials, and mechanical loading. The objective of this study was to investigate differences in juvenile AU and AR chondrocyte behavior when encapsulated in radically polymerized hyaluronic acid hydrogels. When implanted in vivo, differences in macroscopic appearance, mechanical properties, glycosaminoglycan content, and collagen content were observed depending on the chondrocyte type encapsulated. Specifically, AU constructs exhibited construct growth and neocartilage formation with increases in aggregate modulus and ECM accumulation with culture, whereas AR constructs retained their construct size and remained translucent with only a minimal increase in the compressive modulus. When cultured in vitro, both cell types remained viable and differences in gene expression were observed for type I and II collagens. Likewise, differences in gene expression were noted after dynamic mechanical loading, where stimulated AR constructs exhibited 2.3- and 1.5-fold increases in type II collagen and aggrecan over free-swelling controls, while AU samples exhibited smaller fold increases of 1.4- and 1.3-fold, respectively. Thus, these data indicate that the specific cell source, cell/material interactions, and loading environment are important in the final properties of tissue-engineered products.

Analysis of differential gene expression in auricular lymph nodes draining skin exposed to sensitizers and irritants.

There has been some concern that certain non-sensitizing irritants may yield false positive results in the murine local lymph node assay (LLNA). This study compared gene expression profiles in lymph nodes draining skin following exposure to sensitizers and irritants, to identify gene transcripts that could distinguish sensitizers from irritants. After treating CBA/N mouse ears for 3 days with the sensitizers 1-chloro-2,4-dinitrobenzene, 2-phenyl-4-ethoxymethylene-5-oxazolone, or toluene-2,4-diisocyanate or the non-sensitizing irritants croton oil or nonanoic acid, auricular lymph nodes and ear tissues were excised. Sensitizer-induced changes in parameters such as ear thickness, lymph node weight, and cell count also occurred in irritant-treated mouse tissues. However, gene transcripts such as Ifi27, Il12rb1, Ifng, and Zbp1, which are related to T-cell activation, were shown by gene expression microarrays and real-time RT-PCR analyses to be up-regulated in auricular lymph nodes by sensitizers exclusively. These findings suggest that gene expression analysis may enable distinction between sensitizing chemicals and non-sensitizing irritants.