Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.     

Pigmented Human Skin Equivalent: New Method of Reconstitution by Grafting an Epithelial Sheet Onto a Non-Contractile Dermal Equivalent

We have established a new protocol for reconstituting a pigmented human skin equivalent (PSE) and have evaluated its functional responses to environmental stimulus, UVB. The PSE is reconstituted by grafting an epithelial sheet consisting of keratinocytes and melanocytes onto a porous non-contractile dermal equivalent populated with mitotically and metabolically active fibroblasts. i) The PSE has a multilayered, well-differentiated epidermis with cuboidal basal cells and highly organised dermis with newly synthesised extracellular matrix components. ii) Ki67-positive proliferating keratinocytes (18.1 ± 7.4%) were detected on the basal layer of the epidermis. iii) Melanocytes located exclusively within the basal layer were detected by monoclonal antibody against tyrosinase-related protein (TRP-1). iv) After exposure to UVB (100 mJ/cm² per day) for 7 consecutive days, the intensity of TRP-1 staining was increased in the PSE, showing their functional state, whereas the number of melanocytes was not changed. This non-contractile and functioning new PSE is potentially useful as a model for studying the role of melanocyte-keratinocyte-fibroblast interactions in photoprotection of the skin in more complex cutaneous microenvironment than monolayer culture, and for developing in vitro disease models and therapeutic protocols with genetically altered cells both in epidermis and dermis.     

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one’s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.     

Skin structure in the snout of the Australian lungfish,Neoceratodus forsteri (Osteichthyes: Dipnoi)

Many fossil lungfish have a system of mineralised tubules in the dermis of the snout, branching extensively and radiating towards the epidermis. The tubules anastomose in the superficial layer of the dermis, forming a plexus consisting of two layers of vessels, with branches that expand into pore canals and flask organs, flanked by cosmine nodules where these are present. Traces of this system are found in the Australian lungfish, Neoceratodus forsteri, consisting of branching tubules in the dermis, a double plexus below the epidermis and dermal papillae entering the epidermis without reaching the surface. In N. forsteri, the tubules, the plexus and the dermal papillae consist of thick, unmineralised connective tissue, enclosing fine blood vessels packed with lymphocytes. Tissues in the epidermis and the dermis of N. forsteri are not associated with deposits of calcium, which is below detectable limits in the skin of the snout at all stages of the life cycle. Canals of the sensory line system, with mechanoreceptors, are separate from the tubules, the plexus and the dermal papillae, as are the electroreceptors in the epidermis. The system of tubules, plexus, dermal papillae and lymphatic capillaries may function to protect the tissues of the snout from infection.     

Cryopreservation of tissue-engineered dermal replacement in Me2SO: Toxicity study and effects of concentration and cooling rates on cell viability

Cryopreservation of tissue-engineered human dermal replacement plays an important role in skin tissue engineering and skin banking. With the inspection of electronic scanning microscope and viability evaluation by Trypan Blue staining assay and the tetrazolium salt, MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, this study investigated the toxicity of Me(2)SO to dermal fibroblasts and effects of cryoprotectant concentration and cooling rate on the viability of dermal replacement. The results demonstrated that the Me(2)SO toxicity to fibroblasts was affected by the exposure time, temperature, and concentration. Furthermore adding cryoprotectant solution at low temperature of 4 degrees C significantly reduced the toxic effect on the tissue-engineered dermal equivalent. An optimal cryopreservation protocol consisting of cooling rate at 1 degrees Cmin(-1) in 10% (V/V) Me(2)SO was derived, with the viability of studied dermal equivalent treated by this protocol being 75% of that of fresh control. The micrograph obtained by electronic scanning microscope also confirmed this result.     

人表皮角质形成细胞自噬功能细胞模型的构建及鉴定

目的: 建立稳定表达GFP-LC3的人永生化角质形成细胞HaCaT细胞系。方法: 将构建的pcDNA3.1-GFP-LC3 真核表达载体转入HaCaT细胞,经G418筛选稳定表达的细胞系。HaCaT细胞中GFP-LC3的表达分别用荧光显微镜与Western blot方法检测,并利用该稳定表达的细胞系观察验证Rapamycin对细胞发生自噬透射电镜超微结构的变化。结果: 获得了3株转染并经G418反复筛选的HaCaT细胞系,在倒置荧光显微镜下观察可见绿色荧光细胞的表达率在95% 以上,Western blot结果证实了GFP-LC3融合蛋白的表达。Western blot和激光共聚焦显微镜均证明Rapamycin可以诱导自噬的发生。透射电镜细胞超微结构的观察表明Rapamycin可以有效地诱导HaCaT-LC3细胞自噬的发生。结论: 成功构建GFP-LC3稳定表达的HaCaT系,该细胞系可以作为研究人角质形成细胞自噬功能的一种细胞模型。     

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH₂O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.     

Chemically-Synthesized Gene Encoding Modified Human Superoxpde Dismutase: Its Construction,Expression and Properties of the Product

The gene encoding modified human superoxide dismutax (h-SOD) with 153 amino acid residues was constructed by chemical synthesis using the phosphoramidite method. The gene was designed so as to use bacterial codons for expression in prokaryotes and to introduce several unique restriction sites for further mutagenesis by the cassette exchange method. The distance between Shine-Dalgarno sequence and initiation codon was adjusted to maximum expression by using synthesized oligonucleotide. In addition, Cys 6 of h-SOD was changed to Ala to improve instability of native h-SOD.

Synthesized structural gene of h-SOD was expressed in E. coli after induction of isopropyl β-D-thiogalactoside by inserting the gene into the expression vector pKK223–3 having tac promoter. The gene that has 10 base pairs between Shine-Dalgarno sequence and initiation codøn showed the most efficient expression. The gene produced three active SOD isomers as revealed by chromatofocusing.

The main isomer was purified to homogeneity and characterized. The h-SOD-Ala⁶ showed similar properties to those of native h-SOD with respect to molecular weight, subunit structure, absorption spectrum. but the modified SOD was more resistant to heat denaturation than was native h-SOD; half-denaturing temperature was shifted by 10°C. Thus. the exchange of Cys 6 to Ala of h-SOD increased a stability of the enzyme.     

Tooth regeneration: Implications for the use of bioengineered organs in first-wave organ replacement

Experiments with animal models have shown that the tooth crown structure can be regenerated using tissue engineering techniques that combine tooth bud cells and biodegradable materials, or by using embryonic tissue and adult stem cells. Moreover, tooth roots and periodontal tissues have been reconstructed by grafting dental stem cells, which leads to the recovery of tooth function, suggesting that tooth regeneration will become possible in humans in the near future. The present article reviews current research on tooth regeneration, discusses a model of tooth replacement that could be used clinically, and proposes a new tooth regeneration approach that overcomes the difficulties associated with the tooth replacement model. Tooth regeneration is an important stepping stone in the establishment of engineered organ transplantation, which is one of the ultimate goals of regenerative therapies.     

Developmental engineering the kidney: Leveraging principles of morphogenesis for renal regeneration

Multiple methodological approaches are currently under active development for application in tissue engineering and regenerative medicine of tubular and solid organs. Most recently, developmental engineering (TE/RM), or the leveraging of embryonic and morphological paradigms to recapitulate aspects of organ development, has been proposed as a strategy for the sequential, iterative de novo assembly of tissues and organs as discrete developmental modules ex vivo, prior to implantation in vivo. In this article, we focus on the kidney to highlight in detail how principles of developmental biology are impacting approaches to TE of this complex solid organ. Ultimately, such methodologies may facilitate the establishment of clinically relevant therapeutic strategies for regeneration of renal structure and function, greatly impacting treatment regimens for chronic kidney disease. Birth Defects Research (Part C) 96:30–38, 2012. © 2012 Wiley Periodicals, Inc.     

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact bioprinting^1-4 (extrusion, dip pen and soft lithography), contactless bioprinting^5-7 (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization⁸. It can be used for many applications such as tissue engineering^9-13, biosensor microfabrication^14-16 and as a tool to answer basic biological questions such as influences of co-culturing of different cell types¹⁷. Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches.Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 °C and a solid above its gelation temperature ~20 °C for 24.5% w/v solutions¹⁸. This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an epi-fluorescence microscope.