The field of tissue engineering is developing rapidly. Given its ultimate importance to clinical care, the time is appropriate to assess the field's strategic directions to optimize research and development activities. To characterize strategic directions in tissue engineering, a distant but reachable clinical goal was proposed and a worldwide body of 24 leaders in tissue engineering was queried systematically to determine the best paths toward that goal. Using a modified Hoshin process, we identified 14 critical activity categories and then stratified them by their immediate priority for the field. The result of the analysis illustrates a highly interdependent set of activities that are dominated by the need for an understanding of angiogenesis, stem cell science, and the utilization of molecular biology and systems biology tools to enable a deeper comprehension of tissue development and control.

In vivo vascularization of implanted (bio)artificial constructs is essential for their proper function. Vascularization may rely on sprouting angiogenesis, vascular incorporation of bone marrow-derived endothelial cells (BMDECs), or both. Here we investigated the relative contribution of these 2 mechanisms to neovascularization in a mouse model of a foreign body reaction (FBR) to subcutaneously implanted Dacron and in hind limb ischemia (HLI) in relation to the molecular microenvironment at these neovascularization sites. Neovascularization was studied in C57Bl/6 mice reconstituted with enhanced green fluorescent protein (EGFP) transgenic bone marrow. Sprouting angiogenesis, detected using nuclear incorporation of bromodeoxyuridine in endothelial cells was present in both models, whereas vascular incorporation of EGFP(+) BMDECs was restricted to HLI. In HLI, the presence of a pro-angiogenic molecular microenvironment comprising vascular endothelial growth factor, fibroblast growth factor 2, and granulocyte colony-stimulating factor corroborated the importance of these factors for vascular BMDEC incorporation, whereas this microenvironment was absent in FBR. Enhanced mobilization of BMDECs by granulocyte-macrophage colony-stimulating factor administration or by combining HLI and FBR with Dacron did not induce incorporation of BMDECs in FBR neovessels. We conclude that the efficacy of BMDEC-based therapy is not generally warranted, but it depends on the molecular microenvironment in the targeted tissue.

The success of therapeutic strategies within the fields of regenerative medicine, including tissue engineering, biomaterials engineering, and cell and tissue transplantation science, relies on researchers' understanding of the complex cellular microenvironments that occur within functional tissue. Microfabricated biomedical platforms provide tools for researchers to study cellular response to various stimuli with micro- and nanoscale spatial control. Initial studies utilizing relatively passive means of microenvironmental control have provided the fundamental knowledge required to begin to design microculture platforms that closely mimic these biological systems. In this review, we discuss second-generation cell and tissue culture platforms that utilize active components, borrowed from work in the development of microelectromechanical systems (MEMS). These microsystems offer the unprecedented opportunity to fabricate culture platforms designed to match tissue-specific growth parameters. In addition, the adoption of MEMS components opens up the door for future integration with the burgeoning field of microanalytical systems, providing analytical platforms that retain the sensitivity and resolution required within low-volume, microfluidic culture technologies.

To develop biomaterials for tissue engineering, a silk-like protein inspired by mussel-adhesive proteins (MAPs) was designed and prepared. The primary structure of this silk-like protein is designed as TS[AKPSYPPTYKAS (GAGAGS)(3)](10) by combining the sequences (GAGAGS)(3), the crystalline region of Bombyx mori silk fibroin, and AKPSYPPTYK, the adhesive sequence of MAP from Mytilus edulis. This protein was synthesized by the genetic engineering method. Solid-state (13)C NMR spectra showed that this silk-like protein adopts flexible conformation due to introduction of the sequence AKPSYPPTYK. Cell assay indicated that this silk-like protein has significantly higher cell adhesion activities in response to normal human dermal fibroblasts (NHDFs) than Pronectin F, which is available as commercialized cell-adhesive silk-like protein. Thus, combination of remarkably high cell-adhesive activity from MAP with superiority of silk fibroin provides potentiality for application to the field of biomaterials.

Vacuum-assisted closure (VAC) negative pressure wound therapy (NPWT) is a highly successful and widely used treatment modality for wound healing, although no apparatus exists to monitor the effects of subatmospheric pressure application in vitro. Such an apparatus is desirable to better understand the biological effects of this therapy and potentially improve upon them. This article describes the development and validation of a novel bioreactor that permits such study. Tissue analogues consisting of 3-dimensional fibroblast-containing fibrin clots were cultured in off-the-shelf disposable cell culture inserts and multi-well plates that were integrated into the bioreactor module. Negative pressure dressings, commercialized for wound therapy, were placed on top of the culture, and subatmospheric pressure was applied to the dressing. Cultures were perfused with media at controlled physiologic wound exudate flow rates. The design of this bioreactor permits observation of the culture using an inverted microscope in brightfield and fluorescence modes and sustained incubation of the system in a 5% carbon dioxide atmosphere. This closed-system mimics the wound micro-environment under VAC NPWT. Matrix compression occurs as the subatmospheric pressure draws the dressing material down. At the contact zone, surface undulations were clearly evident on the fibroblast-containing tissue analogues at 24 h and appeared to correspond to the dressing microstructure. The bioreactor design, consisting of sterilizable machined plastics and disposable labware, can be easily scaled to multiple units. Validation experiments show that cell survival in this system is comparable with that seen in cells grown in static tissue culture. After application of VAC NPWT, cell morphology changed, with cells appearing thicker and with an organized actin cytoskeleton. The development and validation of this new culture system establishes a stable platform for in vitro investigations of subatmospheric pressure application to tissues.

Embryonic stem cell (ESC) culture is fragmented and laborious and involves operator decisions. Most protocols consist of 3 individual steps: maintenance, embryoid body (EB) formation, and differentiation. Integration will assist automation, ultimately aiding scale-up to clinically relevant numbers. These problems were addressed by encapsulating undifferentiated murine ESCs (mESCs) in 1.1% (w/v) low-viscosity alginic acid, 0.1% (v/v) porcine gelatin hydrogel beads (d = 2.3 mm). Six hundred beads containing 10,000 mESCs per bead were cultured in a 50-mL high-aspect-ratio vessel bioreactor. Bioreactor cultures were rotated at 17.5 revolutions per min, cultured in maintenance medium containing leukemia inhibitory factor for 3 days, replaced with EB formation medium for 5 days followed by osteogenic medium containing L-ascorbate-2-phosphate (50 microg/mL), beta-glycerophosphate (10 mM), and dexamethasone (1 microM) for an additional 21 days. After 29 days, 84 times as many cells per bead were observed and mineralized matrix was formed within the alginate beads. Osteogenesis was confirmed using von Kossa, Alizarin Red S staining, alkaline phosphatase activity, immunocytochemistry for osteocalcin, OB-cadherin, collagen type I, reverse transcriptase polymerase chain reaction, microcomputed tomography (micro-computed tomography) and Fourier transform infrared spectroscopic imaging. This simplified, integrated, and potentially scaleable methodology could enable the production of 3-demensional mineralized tissue from ESCs for potential clinical applications.

The clinical need for improved blood vessel substitutes, especially in small-diameter applications, drives the field of vascular tissue engineering. The blood vessel has a well-characterized structure and function, but it is a complex tissue, and it has proven difficult to create engineered tissues that are suitable for widespread clinical use. This review is focused on approaches to vascular tissue engineering that use proteins as the primary matrix or "scaffold" material for creating fully biological blood vessel replacements. In particular, this review covers four main approaches to vascular tissue engineering: 1) cell-populated protein hydrogels, 2) cross-linked protein scaffolds, 3) decellularized native tissues, and 4) self-assembled scaffolds. Recent advances in each of these areas are discussed, along with advantages of and drawbacks to these approaches. The first fully biological engineered blood vessels have entered clinical trials, but important challenges remain before engineered vascular tissues will have a wide clinical effect. Cell sourcing and recapitulating the biological and mechanical function of the native blood vessel continue to be important outstanding hurdles. In addition, the path to commercialization for such tissues must be better defined. Continued progress in several complementary approaches to vascular tissue engineering is necessary before blood vessel substitutes can achieve their full potential in improving patient care.

Cell guidance is a new tissue engineering concept aimed at total in vivo tissue engineering without the need for cell seeding. This technique aims to create a biomimetic environment through constant delivery of cytokines to different areas of an implanted scaffold, such that site-specific homing of cells can be achieved. In this study, expression of CXCR4 on mesenchymal stem cells (MSCs) was characterized by immunohistochemistry and flow cytometry, subsequent to which chemotaxis toward stromal cell-derived factor 1 (SDF-1) was demonstrated. In a subsequent three-dimensional in vitro study, MSCs were shown to migrate within a polycaprolactone scaffold in response to SDF-1, such that polarized tissue formation could be achieved. A customized cytokine microdelivery system comprising a reservoir housing system and microneedle apparatus was fabricated to ensure constant delivery of SDF-1 to the scaffold. Following on this experiment, we demonstrated in an in vivo rat bone tissue engineering model that a cytokine combination consisting of vascular endothelial growth factor, SDF-1, and bone morphogenetic protein-6 delivered at 10-day intervals through the microneedle apparatus could lead to tissue formation through migrating cell fronts, with evidence of angiogenesis and vascularization without the need for cell seeding on scaffolds prior to implantation. In summary, cell guidance offers an advancement in cellular methodology for tissue engineering, and promises a novel, minimally invasive option for tissue regeneration.

An ideal biomaterial for urethral reconstruction has not been developed. To create a urethral graft biomaterial with optimal biodegradability and biocompatibility, a copoly(L-lactide/epsilon-caprolactone) [P(LA/CL)] fabric tube was combined with a type I collagen sponge. The P(LA/CL) fibers were knitted into a vascular stent style (Type 1) or weaved into a mesh style (Type 2) to prepare P(LA/CL) tubes. The tubes were dipped in aqueous collagen solution and lyophilyzed to prepare the P(LA/CL)-collagen sponge graft. The grafts were applied to a 1.5-cm rabbit urethral defect (n = 14 for each condition), and tissue repair was evaluated using urethrographical, urethroscopical, and histological examination 1, 3, and 6 months after surgery. Although epithelialization was observed after 1 month in all Type 1 grafts, stenoses, fistulae, or stone formation was seen in 7 of the rabbits. In some cases, P(LA/CL) fibers prolapsed into the urethral lumen, causing stone formation. Only 3 rabbits survived for 6 months, and 2 of these had stenoses. For the Type 2 graft, all urethras were patent, without fistulae or stenoses, over the entire observation period. Histologically, urethral structure was disorganized for the Type 1 graft, whereas the urethral tissue on the Type 2 graft was slightly fibrotic but completely epithelialized and supported by a regenerated smooth muscle layer at 6 months. These findings suggest that creation of a scaffold suitable for urethral tissue regeneration will depend not only on the biomaterial composition, but also on the fabrication technique.

A cell carrier made from synthetic material supporting selective growth of keratinocytes is a promising approach to avoid the phenomenon of fibroblast overgrowth during in vitro culture of skin substitutes. Therefore, we investigated polymer membranes made of polyacrylonitrile and copolymers of acrylonitrile and N-vinylpyrrolidone (NVP) for their ability to support selectively the growth of keratinocytes. It was found that a copolymer with an NVP-content of 30% (NVP30) supports growth of human keratinocyte cell line (HaCaT) cells and inhibits fibroblast growth under serum-containing conditions. Cell proliferation of HaCaT cells was measured over 14 days. If both cell types were cultured under serum-free conditions for initial adhesion over 6 h on these NVP30 polymers, they adhered to the same extent. Long-term experiments over 7 days were performed as a coculture of both cell types showing that HaCaT cells had a growth advantage that seems to be related to the paracrine activity of contaminating fibroblasts. As a result, confluent layers of HaCaT cells were obtained with small numbers of remaining fibroblasts. The new poly [acrylonitrile-co(NVP) membranes seem to be a promising culture system for the production of epidermal transplants.

The goal of this study was the development of a bioartificial nerve guide to induce axonal regeneration in the peripheral nervous system (PNS). In this in vitro study, the ability of a novel, 3-dimensional (3D), highly oriented, cross-linked porcine collagen scaffold to promote directed axonal growth has been studied. Collagen nerve guides with longitudinal guidance channels were manufactured using a series of chemical and mechanical treatments with a patented unidirectional freezing process, followed by freeze-drying (pore sizes 20-50 microm). Hemisected rat dorsal root ganglia (DRG) were positioned such that neural and non-neural elements could migrate into the collagen scaffold. After 21 days, S100-positive Schwann cells (SCs) migrated into the scaffold and aligned within the guidance channels in a columnar fashion, resembling "Bands of Büngner." Neurofilament-positive axons (mean length +/- SD 756 microm +/- 318 microm, maximum 1496 microm) from DRG neurons entered the scaffold where the growth within the guidance channels was closely associated with the oriented SCs. This study confirmed the importance of SCs in the regeneration process (neurotrophic theory). The alignment of SCs within the guidance channels supported directional axonal growth (contact guidance theory). The microstructural properties of the scaffold (open, porous, longitudinal pore channels) and the in vitro data after DRG loading (axonal regeneration along migrated and columnar-aligned SCs resembling "Band of Büngner") suggest that this novel oriented 3D collagen scaffold serves as a basis for future experimental regeneration studies in the PNS.

Because engineered tissues are designed for clinical applications in humans, a major problem is the contamination of cocultures and tissues by allogenic molecules used to grow stem cells in vitro. The protocols that are commonly applied to generate epidermal equivalents in vitro require the use of irradiated murine fibroblasts as a feeder layer for keratinocytes. In this study, we report a simple procedure for growing human keratinocytes, isolated from adult skin, to generate an epidermal construct on a collagen layer alone. In this model, no human or murine feeder layers were used to amplify cell growth, and isolated keratinocytes were seeded directly at high cell density on the collagen-coated flasks or coverslips in an epithelial growth medium containing low calcium concentration. Morphological, immunochemical, and cytokinetic features of epithelial colonies grown on the collagen layer were typical of keratinocytes and were comparable with those reported for keratinocytes grown on a feeder layer. The stratification of keratinocytes generated 3-dimensional synthetic constructs displaying a tissue architecture comparable with that of natural epidermis. Epithelial cells expressed specific markers of keratinocyte terminal differentiation, including involucrin and filaggrin. Nevertheless, the number of cell layers was lower than in natural skin, and electron microscopical analysis revealed that the overall organization of these layers was poor compared with natural epidermis, including the formation of junctional complexes, basement membrane, and keratinization. The lack of epithelial-mesenchymal interactions that occur during skin histogenesis may account for such an incomplete maturation of epidermal constructs.

It has been proven that mesenchymal stromal cells (MSCs) can differentiate into tenocytes. Attempts to repair tendon lesions have been performed, mainly using scaffold carriers in experimental settings. In this article, we describe the clinical use of undifferentiated MSCs in racehorses. Significant clinical recovery was achieved in 9 of 11 horses evaluated using ultrasound analysis and their ability to return to racing. Our results show that the suspension of a small number of undifferentiated MSCs may be sufficient to repair damaged tendons without the use of scaffold support. Ultrasound scanning showed that fibers were correctly oriented. By using undifferentiated cells, no ectopic bone deposition occurred. A sufficient number of cells was recovered for therapeutic purposes in all but 1 case. We suggest that the use of autologous MSCs is a safe therapeutic method for treating incompletely (i.e., not full-thickness) damaged tendons.