Tissue engineering最新文献

Mesenchymal stem cells (MSCs) are used extensively in cartilage tissue engineering. We have developed a photopolymerizable poly(ethylene glycol diacrylate) (PEGDA) and poly(ethylene glycol) (PEG) semi-interpenetrating network that facilitates the in vitro chondrogenesis of human MSCs (hMSCs). Network parameters were altered and tested for their effects on subsequent matrix elaboration. The mesh size, calculated for each network based on equilibrium swelling ratios, was larger with lower PEGDA:PEG ratios and with higher PEGDA molecular weight. Changes in xi correlated with changes in extracellular matrix content and deposition in hMSC-seeded networks cultured in vitro for 6 weeks in defined chondrogenic medium. Networks constructed with PEGDA (6 kDa) and PEG (88 kDa) at 1:2 displayed intercellular deposition of proteoglycan. Furthermore, their proteoglycan contents were significantly higher than with PEGDA (6 kDa) hydrogels constructed without the PEG component and those constructed at a PEGDA:PEG ratio of 2:1, which both exhibited pericellular proteoglycan deposition. However, networks constructed with PEGDA (12 and 20 kDa) and PEG (88 kDa) exhibited intercellular deposition of proteoglycan regardless of the ratio employed. Collagen content was lower in networks constructed with PEGDA (12 and 20 kDa) and PEG (88 kDa) at a ratio of 1:2 than in those fabricated at the same PEGDA molecular weights at a ratio of 2:1. This study demonstrated that semi-interpenetrating network parameters influence not only extracellular matrix content, but also the deposition of the matrix molecules by hMSCs undergoing chondrogenesis. It is important that these parameters be considered carefully when creating scaffolds for tissue-engineered cartilage.

We have recently shown that Matrigel-filled chambers containing fibroblast growth factor-2 (FGF2) and placed around an epigastric pedicle in the mouse were highly adipogenic. Contact of this construct with pre-existing tissue or a free adipose graft was required. To further investigate the mechanisms underpinning formation of new adipose tissue, we seeded these chambers with human adipose biopsies and human adipose-derived cell populations in severe combined immunodeficient mice and assessed the origin of the resultant adipose tissue after 6 weeks using species-specific probes. The tissues were negative for human-specific vimentin labeling, suggesting that the fat originates from the murine host rather than the human graft. This was supported by the strong presence of mouse-specific Cot-1 deoxyribonucleic acid labeling, and the absence of human Cot-1 labeling in the new fat. Even chambers seeded with FGF2/Matrigel containing cultured human stromal-vascular fraction (SVF) labeled strongly only for human vimentin in cells that did not have a mature adipocyte phenotype; the newly formed fat tissue was negative for human vimentin. These findings indicate that grafts placed in the chamber have an inductive function for neo-adipogenesis, rather than supplying adipocyte-precursor cells to generate the new fat tissue, and preliminary observations implicate the SVF in producing inductive factors. This surprising finding opens the door for refinement of current adipose tissue-engineering approaches.

The use of either a hydrogel or a solid polymeric scaffold alone is often associated with distinct drawbacks in many tissue engineering applications. Therefore, in this study, we investigated the potential of a combination of long-term stable fibrin gels and polyurethane scaffolds for cartilage engineering. Primary bovine chondrocytes were suspended in fibrin gel and subsequently injected into a polycaprolactone-based polyurethane scaffold. Cells were homogeneously distributed within this composite system and produced high amounts of cartilage-specific extracellular matrix (ECM) components, namely glycosaminoglycans (GAGs) and collagen type II, within 4 weeks of in vitro culture. In contrast, cells seeded directly onto the scaffold without fibrin resulted in a lower seeding efficiency and distinctly less homogeneous matrix distribution. Cell-fibrin-scaffold constructs implanted into the back of nude mice promoted the formation of adequate engineered cartilaginous tissue within the scaffold after 1, 3, and 6 months in vivo, containing evenly distributed ECM components, such as GAGs and collagen. Again, in constructs seeded without fibrin, histology showed an inhomogeneous and, thus, not adequate ECM distribution compared to seeding with fibrin, even after 6 months in vivo. Strikingly, a precultivation for 1 week in vitro elicited similar results in vivo compared to precultivation for 4 weeks; that is, a precultivation for longer than 1 week did not enhance tissue development. The presented composite system is suggested as a promising alternative toward clinical application of engineered cartilaginous tissue for plastic and reconstructive surgery.

Tissue-engineered constructs offer a new hope to patients suffering from functional impairment after nerve injury. An effort has been made to focus on delivery, regulation, and "molecular shutoff" of nerve growth factor (NGF) in tissue-engineered constructs. We have previously demonstrated that human embryonic kidney (HEK-293) cells can be genetically modified to secrete NGF at varying time points upon up regulation with Ponasterone A (PonA) both in vitro and in vivo. In the present study, HEK-293 cells that stably and inducibly produce NGF were further stably transfected with herpes simplex virus-thymidine kinase gene as a suicide gene (hNGF-EcR-293-TK) in order to shut off the NGF secretion and kill the cells upon treatment with ganciclovir (GCV). These cells following induction with PonA secreted NGF levels of 6659.2 +/- 489.4 pg/mL at day 10 postbooster dose at day 5, which was significantly higher than the control noninduced cells. The NGF secreted by these cells was bioactive as determined by a rat adrenal pheochromocytoma (PC-12) cell bioassay. Treatment of these cells with GCV significantly reduced the NGF levels to 645.3 +/- 16.2 pg/mL at day 10 and live cell numbers dropped to 7.95 x 10(3) +/- 278 compared to 2.73 x 10(5) +/- 6.1 x 10(4). GCV-treated cell media when transferred to the PC-12 cell bioassay demonstrated less than 10% cells differentiating into neurite-like extensions. We conclude that hNGF-EcR-293-TK cells can inducibly secrete bioactive NGF when treated with the inducing agent and can also be killed upon treatment with GCV. This double-gene transfection for gene expression and molecular shutoff mechanism will be a useful tool in tissue-engineered nerve constructs.

Using a self-assembly (SA), scaffoldless method, five high-density co-cultures with varied ratios of meniscal fibrochondrocytes (MFCs) and articular chondrocytes (ACs) were seeded into novel meniscus-specific, ring-shaped agarose wells. The following ratios of MFCs to ACs were used: 0% MFC, 25% MFC, 50% MFC, 75% MFC, and 100% MFC. Over 4 weeks, all ratios of cells self-assembled into three-dimensional constructs with varying mechanobiological and morphological properties. All groups stained for collagen II (Col II), and all groups except the 0% MFC group stained for collagen I (Col I). It was found that the tensile modulus was proportional to the percentage of MFCs employed. The 100% MFC group yielded the greatest mechanical stiffness with 432.2 +/- 47 kPa tensile modulus and an ultimate tensile strength of 23.7 +/- 2.4 kPa. On gross inspection, the 50% MFC constructs were the most similar to our idealized meniscus shape, our primary criterion. A second experiment was performed to examine the anisotropy of constructs as well as to directly compare the scaffoldless, SA method with a poly-glycolic acid (PGA) scaffold-based construct. When compared to PGA constructs, the SA groups were 2-4 times stiffer and stronger in tension. Further, at 8 weeks, SA groups exhibited circumferential fiber bundles similar to native tissue. When pulled in the circumferential direction, the SA group had significantly higher tensile modulus (226 +/- 76 kPa) than when pulled in the radial direction (67 +/- 32 kPa). The PGA constructs had neither a directional collagen fiber orientation nor differences in mechanical properties in the radial or circumferential direction. It is suggested that the geometric constraint imposed by the ring-shaped, nonadhesive mold guides collagen fibril directionality and, thus, alters mechanical properties. Co-culturing ACs and MFCs in this manner appears to be a promising new method for tissue engineering fibrocartilaginous tissues exhibiting a spectrum of mechanical and biomechanical properties.

Deciphering the cellular signals leading to cardiac muscle assembly is a major challenge in ex vivo tissue regeneration. For the first time, we demonstrate that pulsatile interstitial fluid flow in three-dimensional neonatal cardiac cell constructs can activate ERK1/2 sixfold, as compared to static-cultivated constructs. Activation of ERK1/2 was attained under physiological shear stress conditions, without activating the p38 cell death signal above its basic level. Activation of the ERK1/2 signaling cascade induced synthesis of high levels of contractile and cell-cell contact proteins by the cardiomyocytes, while its inhibition diminished the inducing effects of pulsatile flow. The pulsed medium-induced cardiac cell constructs showed improved cellularity and viability, while the regenerated cardiac tissue demonstrated some ultra-structural features of the adult myocardium. The cardiomyocytes were elongated and aligned into myofibers with defined Z-lines and multiple high-ordered sarcomeres. Numerous intercalated disks were positioned between adjacent cardiomyocytes, and deposits of collagen fibers surrounded the myofibrils. The regenerated cardiac tissue exhibited high density of connexin 43, a major protein involved in electrical cellular connections. Our research thus demonstrates that by judiciously applying fluid shear stress, cell signaling cascades can be augmented with subsequent profound effects on cardiac tissue regeneration.

Articular cartilage is recalcitrant to repair and regeneration. Tissue engineering and regenerative medicine are potential strategies to treat the damage to articular cartilage. A thorough understanding of the gene expression profiles in articular cartilage and growth plate chondrocytes will be an important prerequisite for tissue engineering of cartilage. Regeneration is a recapitulation of embryonic development and morphogenesis. We used laser capture microdissection to capture the surface articular chondrocytes and the resting zone chondrocytes of growth plate from 14-day-old C57BL/6J mice. Total RNA was individually purified, pooled, and amplified by two rounds of in vitro transcription. Labeled cRNA probes were analyzed using the Affymetrix GeneChip Mouse Genome 430 2.0 Array. We identified 107 genes that were highly expressed by the surface articular chondrocytes and 130 genes that were highly expressed by the resting zone chondrocytes of growth plate (> or = fivefold). The expression of major matrix proteins aggrecan and collagen II were similar, while several morphogens and growth factors were differentially expressed by the surface articular chondrocytes and the resting zone chondrocytes of growth plate. The results of this investigation will be of use in the evaluation of tissue engineered cartilage.

Vascularization caused by encapsulated cells engineered to secrete vascular endothelial growth factor (VEGF) improved the in vivo survival of the encapsulated cells in a syngeneic mouse Matrigel plug model. Murine fibroblast cells (L929) were engineered to secrete recombinant human vascular endothelial growth factor (rhVEGF(165)). Transfected and nontransfected L929 cells were microencapsulated in a 75:25 hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA) copolymer. Capsules containing transfected cells induced vascularization in vivo at 1 and 3 weeks postimplantation. In histological sections, a significant positive correlation was seen between the number of capsules and blood vessel density for VEGF-secreting cell capsule implants. New vessels, many positively stained for smooth muscle cells and pericytes, were seen surrounding these VEGF-secreting cell capsule explants. Few vessels were seen in nontransfected L929 capsule implants. The viability of transfected and nontransfected encapsulated cells was assessed on explantation. Although the viability of all encapsulated cells decreased at both 1 and 3 weeks, encapsulated VEGF-secreting cells retained more of the viability than did encapsulated nontransfected control cells. Genetically modified cells promoted vascularization in this context and appeared to enhance the viability of the encapsulated cells, although the extent of the functional benefit was less than expected. Additional effort is required to enhance the benefit, to quantify it, and to understand further the host response to HEMA-MMA microencapsulated cells and tissue constructs, more generally.

Cell-based regenerative medicine, including tissue engineering, is a novel approach to reconstituting tissues that do not spontaneously heal, such as damaged cartilage, and to curing diseases caused by malfunctioning cells. Typically, manufacturing processes to generate cartilage for replacement therapies involve isolation and expansion of cells from cartilage biopsies. A challenge in the field is potential contamination by other cell types (e.g., fibroblast-like cells), which can overgrow the desired cells during culturing and may ultimately compromise clinical efficacy. No standard analytical system has been absolutely effective in ensuring the identity of these cell-based products. Therefore, we tested deoxyribonucleic acid methylation analysis as a quality assessment tool, applying it to Genzyme's Carticel product, a chondrocyte implant that the Food and Drug Administration has approved. We identified 7 potent discriminators by assaying candidate genomic regions derived from methylation discovery approaches and literature searches regarding a functional role of genes in chondrocyte biology. Using a support vector machine, we trained an optimal cell type classifier that was absolutely effective in discriminating chondrocytes from synovial membrane derived cells, the major potential contaminant of chondrocyte cultures. The abundant marker availability and high quality of this assay format also suggest it as a potential quality control test for other cell types grown or manipulated in vitro.