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.

Protocol for isolation of adult mouse ear pinnae-derived primary fibroblasts.

Researchers need in vitro models that mirror the biology of organisms. Primary fibroblasts play essential roles in wound healing and are present in many tissues. They are widely used in studies of cell cycle control, reprogramming, and aging. Though extraction protocols exist, alternatives that maximize use of available resources are useful. Here, we present our protocol for extracting primary fibroblasts from adult mouse ear pinnae, an often-discarded source of primary cells, which consistently yield large, pure numbers of primary fibroblasts.

Epithelial defect repair in the auricle and auditory meatus by grafting with cultured adipose-derived mesenchymal stem cell aggregate-extracellular matrix.

BACKGROUND: Several patients experience persistent otorrhea after a flawless surgical procedure because of insufficient epithelial healing. Several efforts, such as autologous tissue allograft and xenograft, have been made to halt otorrhea. However, a stable technology to induce temporal epithelial repair is yet to be established. Therefore, this study aims to investigate whether implantation of seeding adipose-derived mesenchymal stem cell (ADMSC) aggregates on extracellular matrix (ECM; herein, ADMSC aggregate-ECM) into damaged skin wound promotes skin regeneration. METHODS: ADMSC aggregate-ECM was prepared using a previously described procedure that isolated ADMSCs from rabbits and applied to the auricle and auditory meatus wound beds of New Zealand white rabbits. Wound healing was assessed by general observation and hematoxylin and eosin (H&E) staining. Secretion of growth factor of the tissue was evaluated by western blotting. Two other groups, namely, ECM and control, were used. Comparisons of three groups were conducted by one-way analysis of variance analysis. RESULTS: ADMSCs adhered tightly to the ECM and quickly formed cell sheets. At 2 weeks, general observation and H&E staining indicated that the wound healing rates in the ADMSC aggregate-ECM (69.02 ±â€Š6.36%) and ECM (59.32 ±â€Š4.10%) groups were higher than that in the control group (43.74 ±â€Š12.15%; P = 0.005, P < 0.001, respectively) in ear auricle excisional wounds. At 7 weeks, The scar elevation index was evidently reduced in the ADMSC aggregate-ECM (2.08 ±â€Š0.87) and ECM (2.31 ±â€Š0.33) groups compared with the control group (4.06 ±â€Š0.45; P < 0.001, P < 0.001, respectively). In addition, the scar elevation index of the ADMSC aggregate-ECM group reached the lowest rate 4 weeks in advance. In auditory meatus excisional wounds, the ADMSC aggregate-ECM group had the largest range of normal skin-like structure at 4 weeks. The ADMSC aggregate-ECM and ECM groups secreted increased amounts of growth factors that contributed to skin regeneration at weeks 1 and 2, respectively. CONCLUSIONS: ADMSC aggregate-ECM and ECM are effective repair materials for wound healing, especially ADMSC aggregate-ECM. This approach will provide a meaningful experimental basis for mastoid epithelium repair in subsequent clinical trials.

Facilitating In Vivo Articular Cartilage Repair by Tissue-Engineered Cartilage Grafts Produced From Auricular Chondrocytes.

BACKGROUND: Insufficient cell numbers still present a challenge for articular cartilage repair. Converting heterotopic auricular chondrocytes by extracellular matrix may be the solution. HYPOTHESIS: Specific extracellular matrix may convert the phenotype of auricular chondrocytes toward articular cartilage for repair. STUDY DESIGN: Controlled laboratory study. METHODS: For in vitro study, rabbit auricular chondrocytes were cultured in monolayer for several passages until reaching status of dedifferentiation. Later, they were transferred to chondrogenic type II collagen (Col II)-coated plates for further cell conversion. Articular chondrogenic profiles, such as glycosaminoglycan deposition, articular chondrogenic gene, and protein expression, were evaluated after 14-day cultivation. Furthermore, 3-dimensional constructs were fabricated using Col II hydrogel-associated auricular chondrocytes, and their histological and biomechanical properties were analyzed. For in vivo study, focal osteochondral defects were created in the rabbit knee joints, and auricular Col II constructs were implanted for repair. RESULTS: The auricular chondrocytes converted by a 2-step protocol expressed specific profiles of chondrogenic molecules associated with articular chondrocytes. The histological and biomechanical features of converted auricular chondrocytes became similar to those of articular chondrocytes when cultivated with Col II 3-dimensional scaffolds. In an in vivo animal model of osteochondral defects, the treated group (auricular Col II) showed better cartilage repair than did the control groups (sham, auricular cells, and Col II). Histological analyses revealed that cartilage repair was achieved in the treated groups with abundant type II collagen and glycosaminoglycans syntheses rather than elastin expression. CONCLUSION: The study confirmed the feasibility of applying heterotopic chondrocytes for cartilage repair via extracellular matrix-induced cell conversion. CLINICAL RELEVANCE: This study proposes a feasible methodology to convert heterotopic auricular chondrocytes for articular cartilage repair, which may serve as potential alternative sources for cartilage repair.

Aberration compensation for optical trapping of cells within living mice.

Optical tweezers have been used to trap and manipulate microparticles within living animals. When the optical trap is constructed with an oil-immersion objective, it suffers from spherical aberration. There have been many investigations on the influence of spherical aberration when the particles are trapped in a water medium. However, the dependence of optical force on trapping depth is still ambiguous when the trapped particles are immersed in a high refractive index medium (such as biological tissue, refractive index solution) in experiments. In this paper, the microparticles are immersed in an aqueous solution of glycerol to mimic the cells within biological tissue. As the trapping laser is focused into the specimen, spherical aberration is introduced, degrading the optical trapping performance. It is similar to trapping in water; altering the effective tube length can also compensate for the spherical aberration of the optical trap in a high refractive index medium. Finally, the cells in living mice are trapped by the optical tweezers with the help of spherical aberration compensation.

Long-Term Comparison between Human Normal Conchal and Microtia Chondrocytes Regenerated by Tissue Engineering on Nanofiber Polyglycolic Acid Scaffolds.

BACKGROUND: Previous regeneration studies of auricle-shaped cartilage by tissue engineering leave unresolved whether the chondrocyte phenotype from human auricular chondrocytes seeded onto polymeric scaffolds is retained over the long term and whether microtia remnants may be a viable cell source for auricular reconstruction. METHODS: Chondrocytes were isolated from human ears, either normal conchal ear or microtia cartilage remnants, expanded in vitro, and seeded onto nanoscale-diameter polyglycolic acid sheets. These tissue-engineered constructs were implanted into athymic mice for up to 40 weeks. At harvest times of 5, 10, 20, and 40 weeks, samples were documented by gross morphology, histology, and reverse transcription-quantitative polymerase chain reaction analysis. RESULTS: Neocartilages generated from the two types of surgical tissues were similar in appearance of their extracellular matrices and positive staining for elastin and proteoglycans. In the 5- to 40-week time interval, there was an increasing trend in gene expression for type II collagen, elastin, and sex determining region Y box 5, important to normal cartilage phenotype, and a decreasing trend in gene expression for type III collagen, a fibroblast and dedifferentiation marker. Over 40 weeks of implantation, the original nanoscale-diameter polyglycolic acid scaffold dimensions (1 cm × 1 cm × 80 µm) were generally maintained in tissue-engineered cartilage length and width, and thickness was statistically significantly increased. CONCLUSIONS: Auricular cartilage can be regenerated over the long term (40 weeks) from surgical remnants by tissue-engineering techniques incorporating nanoscale-diameter polyglycolic acid scaffolds. Based on the present assays, microtia neocartilage very closely resembles tissue-engineered cartilage regenerated from chondrocytes isolated from normal conchal cartilage.

Correlation of external ear auricle formation with staging of human embryos.

The formation of auricles in human embryos was evaluated between Carnegie stage (CS)19 and CS23, and the findings were correlated across the stages. The auricle was categorized into 11 steps according to Streeter's criteria with modifications. Mesenchyme cell condensation was observed at Step 7, and two layers of cartilage consisting of the auricle were recognized at Step 11. The representative steps at each CS shifted from Step 3 to Step 11 during CS16 and CS23, although several steps overlapped between adjacent CSs. These results indicate that observations of the auricle between CS19 and CS23 may be utilized for determining embryo staging as convincing supportive evidence of external features reflecting the internal histological structure, although other findings should also be taken into account.

Use of an in vitro model in tissue engineering to study wound repair and differentiation of blastema tissue from rabbit pinna.

Rabbit ear wound repair is an accepted model for studies of tissue regeneration, leading to scar less wound repair. It is believed that a specific tissue, blastema, is responsible for such interesting capacity of tissue regeneration. To test this idea further and to elucidate the cellular events happening during the ear wound repair, we designed some controlled experiments in vitro. Small pieces of the ear were punched and washed immediately with normal saline. The tissues were then cultured in the Dulbecco's Modified Eagle(')s Medium, supplemented with fetal bovine serum in control group. As a treatment vitamin A and C was used to evaluate the differentiation potency of the tissue. These tissues were fixed, sectioned, stained, and microscopically studied. Micrographs of electron microscopy provided evidences revealing dedifferentiation of certain cells inside the punched tissues after incubation in tissue culture medium. The histological studies revealed that cells of the tissue (i) can undergo cellular proliferation, (ii) differentiate to epithelial, condrogenic, and osteogenic tissues, and (iii) regenerate the wounds. These results could be used for interpretation of the possible events happening during tissue engineering and wound repair in vitro. An important goal of this study is to create a tissue engineering and tissue banking model, so that in the future it could be used in further blastema tissue studies at different levels.

Chondrogenesis of Human Adipose-Derived Stem Cells by In Vivo Co-graft with Auricular Chondrocytes from Microtia.

OBJECTIVE: To evaluate the efficiency of chondrogenesis of human adipose-derived stem cells (ADSCs) induced by auricular chondrocytes from microtia via subcutaneous co-graft in nude mice. METHODS: Human ADSCs and auricular chondrocytes were mixed at the ratio of 7:3 and suspended in 0.2 ml of Pluronic F-127 (5.0 × 10(7) cells/ml), and injected into Balb/c nude mice as the experimental group (Exp group). The same quantity of auricular chondrocytes (Ctr.1 group) or ADSCs (Ctr.2 group) in 0.2 ml of Pluronic F-127 was set as positive and negative control groups. The mixture of auricular chondrocytes (1.5 × 10(7) cells/ml) in 0.2 ml of Pluronic F-127 was set as the low concentration of chondrocyte control group (Ctr.3). At 8 weeks after grafting, the newly generated tissue pellets were isolated for morphological examination, haematoxylin and eosin staining, toluidine blue staining and safranin O staining of glycosaminoglycan (GAG), Masson's trichrome staining and immunohistochemical staining of type II collagen, and Verhoeff-iron-hematoxylin staining of elastic fibers. GAG content was determined by Alcian blue colorimetric method, and mRNA expression of type II collagen and aggrecan were examined by real-time PCR. RESULTS: Cartilage-like tissue with a white translucent appearance and good elasticity was generated in the Exp and Ctr.1 groups. The tissue pellets in the Ctr.2 and Ctr.3 groups were much smaller than those in the Ctr.1 group. The mature cartilage lacunas could be observed in the Exp and Ctr.1 groups, while were rarely seen in the Ctr.3 group and not observed in the Ctr.2 group. The expression of cartilage-specific extracellular matrix such as type II collagen, GAG content, aggrecan, and elastic fibers in the Exp group was similar to that in the Ctr.1 group, whereas the expression of these extracellular matrix substances was significantly lower in the Ctr.2 and Ctr.3 groups (both P < 0.01). CONCLUSION: Auricular chondrocytes from microtia can efficiently promote the chondrogenic differentiation and chondrogenesis of ADSCs by co-grafting in vivo. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors http://www.springer.com/00266 .

Study of proliferation and 3D epidermal reconstruction from foreskin, auricular and trunk keratinocytes in children.

OBJECTIVE: Severe burns in children are conventionally treated with split-thickness skin autografts or epidermal sheets. An alternative approach is to graft isolated keratinocytes. We evaluated foreskin and other anatomic sites as donor sources for autologous keratinocyte graft in children. We studied in vitro capacities of isolated keratinocytes to divide and reconstitute epidermal tissue. METHODS: Keratinocytes were isolated from foreskin, auricular skin, chest and abdominal skin by enzymatic digestion. Living cell recovery, in vitro proliferation, epidermal reconstruction capacities and differentiation status were analyzed. RESULTS: In vitro studies revealed the higher yield of living keratinocyte recovery from foreskin and higher potential in terms of proliferative capacity, regeneration and differentiation. Cultured keratinocytes from foreskin express lower amounts of differentiation markers than those isolated from trunk and ear. Histological analysis of reconstituted human epidermis derived from foreskin and inguinal keratinocytes showed a structured multilayered epithelium, whereas those obtained from ear pinna-derived keratinocytes were unstructured. CONCLUSION: Our studies highlight the potential of foreskin tissue for autograft applications in boys. A suitable alternative donor site for autologous cell transplantation in female paediatric burn patients remains an open question in our department. We tested the hypothesis that in vitro studies and RHE reconstructive capacities of cells from different body sites can be helpful to select an optimal site for keratinocyte isolation before considering graft protocols for girls.

Osteochondral articular defect repair using auricle-derived autologous chondrocytes in a rabbit model.

Hypothesizing that the implantation of non-articular (heterotopic) chondrocytes might be an alternative approach to support articular cartilage repair, we analyzed joint cartilage defect healing in the rabbit model after implantation of autologous auricle-derived (auricular) chondrocytes. Autologous lapine articular and auricular chondrocytes were cultured for 3 weeks in polyglycolic acid (PGA) scaffolds before being implanted into critical sized osteochondral defects of the rabbit knee femoropatellar groove. Cell-free PGA scaffolds and empty defects served as controls. Construct quality was determined before implantation and defect healing was monitored after 6 and 12 weeks using vitality assays, macroscopical and histological score systems. Neo-cartilage was formed in the PGA constructs seeded with both articular and auricular chondrocytes in vitro and in vivo. At the histological level, cartilage repair was slightly improved when using autologous articular chondrocyte seeded constructs compared to empty defects and was significantly superior compared to defects treated with auricular chondrocytes 6 weeks after implantation. Although only the immunohistological differences were significant, auricular chondrocyte implantation induced an inferior healing response compared with the empty defects. Elastic auricular chondrocytes might maintain some tissue-specific characteristics when implanted into joint cartilage defects which limit its repair capacity.

[Comparison study of tissue engineered cartilage constructed with chondrocytes derived from porcine auricular and articular cartilage].

OBJECTIVE: To compare the tissue engineered cartilage constructed with chondrocytes derived from auricular and articular cartilage. METHODS: Chondrocytes were isolated from porcine auricular and articular cartilage, and BMSCs were obtained from bone marrow aspirate and cultured. Each kind of chondrocytes were resuspended alone or mixed with BMSCs at a ratio of 1:1, and seeded onto PGA/PLA scaffolds to construct tissue engineered cartilage (n = 4). The constructs were cultured for 8 weeks in vitro and then subcutaneously implanted into nude mice for 6 weeks. The differences between chondrocytes monoculture from articular and auricular cartilage or between each of them co-cultured with BMSCs were evaluated by gross view, measurement of thickness and wet weight, histological examinations including H&E, Safranin O, type II collagen, and Ponceau's & Victoria blue staining, and gene expression analysis of cartilage related genes. RESULTS: No obvious differences were found histologically among the complexes constructed in vitro 8 weeks except for few elastic fibers secreted in the auricular chondrocytes + BMSCs co-culture group. Neo-cartilage is thicker in the groups of articular chondrocytes (38. 1% than the group of auricular chondrocytes, P < 0.05) and articular chondrocytes + BMSCs co-culture (19.3% than the group of auricular chondrocytes + BMSCs, P < 0.05). However, after 6 weeks in vivo the elastic fibers were found positive in the complexes constructed by auricular chondrocytes, and its staining was even stronger and more homogenous in the group of auricular chondrocytes + BMSCs co-culture. The tissues generated by articular chondrocytes alone and co-cultured with BMSCs both formed the characteristic features of three-layer structure of hyaline cartilage and ossified in vivo with significant up-regulation of COL10A1 and MMP-13. To summarize, auricular chondrocytes formed the elastic cartilage while articular chondrocytes formed the hyaline cartilage during the development of tissue engineered cartilage either by monoculture or the co-culture with BMSCs. CONCLUSIONS: The chondrogenic response of chondrocytes from different cartilage origins demonstrates that an initial chondrocyte and cartilage type recapitulates the same in later tissue-engineered development.

Auricular reconstruction using tissue-engineered alloplastic implants for improved clinical outcomes.

BACKGROUND: Alloplastic implants have been used clinically to treat congenital abnormalities and traumatic injuries. However, these implants are often associated with complications, including inflammation, infection, erosion, and dislodgment. To minimize these complications, the authors have developed a system in which tissue-engineered cartilage serves as a shell that entirely covers the implant. This system is designed to improve the structural and functional stability between the implant and recipient tissue. METHODS: Chondrocytes isolated from rabbit ear cartilage were expanded in vitro. The cells were mixed with fibrin hydrogel for spray-coating a human ear-shaped implant. The surface of the implant was modified using an oxidizing solution to provide hydrophilic characteristic; thus, the cell-fibrin suspension readily adhered onto the surface of the implants. The engineered cartilage-covered implants were implanted into the dorsal subcutaneous space of athymic mice. Histologic and gross examinations of the implants were performed at 2, 4, 8, and 12 weeks after implantation. RESULTS: None of the engineered cartilage-covered implants showed evidence of skin necrosis, implant exposure, or extrusion (n = 10). However, the control implants developed extensive necrosis following implantation (n = 10). In the experimental group, histologic evaluations showed the formation of neocartilage covering the implants. The presence of sulfated glycosaminoglycans was evident in the engineered cartilage tissue. CONCLUSIONS: These results demonstrate that engineered cartilage tissues can be used as a biological cover for an alloplastic implant. This system may improve the structural and functional interactions between the implant and the recipient's tissues and thus enhance the outcome of total auricular reconstruction.

Prefabrication of 3D cartilage contructs: towards a tissue engineered auricle--a model tested in rabbits.

The reconstruction of an auricle for congenital deformity or following trauma remains one of the greatest challenges in reconstructive surgery. Tissue-engineered (TE) three-dimensional (3D) cartilage constructs have proven to be a promising option, but problems remain with regard to cell vitality in large cell constructs. The supply of nutrients and oxygen is limited because cultured cartilage is not vascular integrated due to missing perichondrium. The consequence is necrosis and thus a loss of form stability. The micro-surgical implantation of an arteriovenous loop represents a reliable technology for neovascularization, and thus vascular integration, of three-dimensional (3D) cultivated cell constructs. Auricular cartilage biopsies were obtained from 15 rabbits and seeded in 3D scaffolds made from polycaprolactone-based polyurethane in the shape and size of a human auricle. These cartilage cell constructs were implanted subcutaneously into a skin flap (15 × 8 cm) and neovascularized by means of vascular loops implanted micro-surgically. They were then totally enhanced as 3D tissue and freely re-implanted in-situ through microsurgery. Neovascularization in the prefabricated flap and cultured cartilage construct was analyzed by microangiography. After explantation, the specimens were examined by histological and immunohistochemical methods. Cultivated 3D cartilage cell constructs with implanted vascular pedicle promoted the formation of engineered cartilaginous tissue within the scaffold in vivo. The auricles contained cartilage-specific extracellular matrix (ECM) components, such as GAGs and collagen even in the center oft the constructs. In contrast, in cultivated 3D cartilage cell constructs without vascular pedicle, ECM distribution was only detectable on the surface compared to constructs with vascular pedicle. We demonstrated, that the 3D flaps could be freely transplanted. On a microangiographic level it was evident that all the skin flaps and the implanted cultivated constructs were well neovascularized. The presented method is suggested as a promising alternative towards clinical application of engineered cartilaginous tissue for plastic and reconstructive surgery.

Epigallocatechin-3-gallate suppresses IGF-I-induced lipogenesis and cytokine expression in SZ95 sebocytes.

Acne vulgaris is the most common disease of the pilosebaceous unit. The pathogenesis of this inflammatory disease is complex, involving increased sebum production and perifollicular inflammation. To identify effective agents for factors that induce acne vulgaris, we explored the pharmacological potential of epigallocatechin-3-gallate (EGCG), which has been widely investigated as an anti-proliferative and anti-inflammatory agent. In this study, we demonstrated that topical application of EGCG to rabbit auricles reduced the size of the sebaceous glands. When applied to cultured human SZ95 sebocytes, EGCG strongly suppressed cell proliferation and lipogenesis. These actions of EGCG were reproduced in IGF-I-differentiated SZ95 sebocytes. To investigate the anti-inflammatory potential of EGCG, we evaluated pro-inflammatory cytokine synthesis in IGF-I-differentiated SZ95 sebocytes and found that expression of IL-1, IL-6, and IL-8 was decreased. These results provide early evidence that EGCG is an effective candidate for acne therapy whose mechanisms of action in IGF-I-differentiated SZ95 sebocytes include the inhibition of lipogenesis and inflammation.

Differentiation of adipose-derived stem cells into ear auricle cartilage in rabbits.

BACKGROUND: Adipose-derived stem cells have been reported as a novel candidate for the repair of cartilage injuries in vivo. METHODS: In order to assess their differentiation ability, adipose-derived stem cells isolated from rabbit fat tissue were injected into the midportion of a surgically created rabbit ear auricle cartilage defect. After several months, the auricles were resected, histopathologically assessed and compared with a control group. RESULTS: Histopathological examination of auricles removed three, four and five months after injection showed islands of new cartilage formation at the site of the surgically induced defect. Six months after injection, we observed well-formed, mature cartilaginous plates that completely filled the defect in the native cartilage. In the control group, there was no significant growth of new cartilage. CONCLUSION: The results of this study suggest the great potential of adipose-derived stem cells to repair damaged cartilage tissue in vivo.

Supercontinuum light source enables in vivo optical microangiography of capillary vessels within tissue beds.

This Letter reports on the use of a supercontinuum light source to achieve ultrahigh resolution and ultrahigh sensitive optical microangiography (OMAG) imaging of microcirculations within tissue beds in vivo. After passing through a specially designed optical filter with a passband of 120 nm centered on 800 nm, the light source is coupled into an optic-fiber-based OMAG system that provides a measured axial resolution of ∼3 µm over a ranging distance of 2 mm. Within this ranging distance, the system gives an averaged signal-to-noise ratio of 87 dB and a sensitivity roll-off of 7 dB at an A-scan rate of 70 kHz. We demonstrate the capability of the system to visualize a detailed microvascular perfusion map, including the single red blood cells within the capillaries, by imaging a mouse ear flap in vivo.

Transcutaneous immunization as preventative and therapeutic regimens to protect against experimental otitis media due to nontypeable Haemophilus influenzae.

We have developed three nontypeable Haemophilus influenzae (NTHI) adhesin-derived immunogens that are significantly efficacious against experimental otitis media (OM) due to NTHI when delivered parenterally. We now expanded our preventative immunization strategies to include transcutaneous immunization (TCI) as a less invasive, but potentially equally efficacious, regimen to prevent OM due to NTHI. Additionally, we examined the potential of TCI as a therapeutic immunization regimen to resolve ongoing experimental OM. Preventative immunization with NTHI outer membrane protein (OMP) P5- and type IV pilus-targeted immunogens, delivered with the adjuvant LT(R192G-L211A), induced significantly earlier clearance of NTHI from the nasopharynges and middle ears of challenged chinchillas compared with receipt of immunogen or adjuvant alone. Moreover, therapeutic immunization resulted in significant resolution of established NTHI biofilms from the middle ear space of animals compared with controls. These data advocate TCI with the adhesin-directed immunogens as an efficacious regimen for prevention and resolution of experimental NTHI-induced OM.

Considerations in the choice of a skin donor site for harvesting keratinocytes containing a high proportion of stem cells for culture in vitro.

The treatment of severely burned patients has benefited from the grafting of skin substitutes obtained by expansion of epithelial cells in culture. The aim of this study was to evaluate whether the anatomic site chosen for harvesting skin had an impact on the quality of the derived cell cultures. Considering that hair follicles contain epithelial stem cells, we compared hairy skin sites featuring different densities and sizes of hair follicles for their capacity to generate high quality keratinocyte cultures. Three anatomic sites from adult subjects were compared: scalp, chest skin and p-auricular (comprising pre-auricular and post-auricular) skin. Keratin (K) 19 was used as a marker to evaluate the proportion of stem cells. Keratinocytes were isolated using the two-step thermolysin and trypsin cell extraction method, and cultured in vitro. The proportion of K19-positive cells harvested from p-auricular skin was about twice that of the scalp. This K19-positive cell content also remained higher during the first subcultures. In contrast to these in vitro results, the number of K19-positive cells estimated in situ on skin sections was about double in scalp as in p-auricular skin. Chest skin had the lowest number of K19-positive cells. These results indicate that in addition to the choice of an adult anatomic site featuring a high number of stem cells in situ, the quality of the cultures greatly depends on the ability to extract stem cells from the skin biopsy.

Immunological response to tissue-engineered cartilage derived from auricular chondrocytes and a PLLA scaffold in transgenic mice.

The immune response against biomaterials in tissue-engineered constructs could potentially worsen the outcome of tissue regeneration, but immunological reactions between host and donor in tissue-engineered constructs remain to be clarified. In the present study, we syngenically transplanted tissue-engineered cartilage constructs consisting of C57BL/6 mice auricular chondrocytes and poly-l-lactic acid scaffolds (MW:200,000) into EGFP transgenic mice of C57BL/6 background, and evaluated the response by the localization of donor-derived and host-derived cells, the latter of which were distinguished by the presence of EGFP. While donor-derived cells constituted the areas of regenerated cartilage, host-derived cells were increased in number for the initial two weeks, and then decreased and excluded to non-cartilage areas thereafter. Furthermore, EGFP positivity was mostly co-localized with that of F4/80, suggesting most of the host-derived cells in the tissue-engineered constructs could be macrophages. Immunohistochemical staining of the tissue-engineered cartilage constructs revealed expression of factors related to immune privilege in chondrocytes, such as macrophage migration inhibitory factor (MIF), fas ligand (FasL) and others. Co-culture of chondrocytes and macrophages in vitro increased the expression of MIF and FasL in the chondrocytes, suggesting that chondrocytes in tissue-engineered cartilage constructs could regulate the actions of host-derived macrophages by expressing factors related to immune privilege.