Tissue engineering最新文献

There is a clinical need for new bone replacement materials that combine long implant life with complete integration and appropriate mechanical properties. We have used human mesenchymal stem cells (MSCs) to populate porous apatite-wollastonite (A-W) glass-ceramic scaffolds produced by the layer manufacturing technique, selective laser sintering, to create custom-built bone replacements. Confocal and scanning electron microscopy were used to determine optimal seeding densities and to demonstrate that MSCs adhered and retained viability on the surface of A-W scaffolds over a culture period of 21 days. We found a significant increase in the number of MSCs growing on the scaffolds over 7 days. Using bromodeoxyuridine incorporation we demonstrated that MSCs proliferated on the scaffolds. Using real-time PCR we analyzed the expression of the osteogenic markers alkaline phosphatase, collagen type-I, Cbfa-1, osteocalcin, osteonectin, and osteopontin by MSCs cultured in the absence of osteogenic supplements. The expression of the osteogenic markers by MSCs was equivalent to or significantly greater on A-W scaffolds than on tissue culture plastic. We also identified significantly higher alkaline phosphatase activity on A-W compared to a commercial calcium phosphate scaffold. These results indicate for the first time the biocompatibility and osteo-supportive capacity of A-W scaffolds and their potential as patient-specific bone replacement materials.

Changes in dimensional and mechanical properties of degradable sheaths in poly lactic-co glycolic acid (PLGA) have been researched extensively. Composite PLGA having variable resorption rates in multiple layers under physiological loading has not been reported. Our novel design of a PLGA sheath is composed of 3 layers with different degradation rates (i.e., the innermost layer degrades the fastest, followed by the middle, while the outer layer degrades the slowest). In the presence of physiological luminal pressure, diameter is greater, thickness is less, resorption rate is greater, pore size is greater, and incremental modulus is greater than in nonpressurized sheaths. Furthermore, the ratio of the pore size to the sheath radius affects the dimensional changes of the sheath in the radial direction. In addition to changing the pore size-to-sheath radius ratio, the dimensional changes can be manipulated by choosing different glycolic and lactic acid ratios for the different layers. The application of this novel PLGA design for gradual arterialization of vein grafts is contemplated.

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 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.

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.

Cartilage is an avascular tissue, and chondrocytes in vivo experience a severely hypoxic environment. Using a defined in vitro model of early chondrogenesis, we attempted to enrich for cells with an enhanced ability for chondrogenic differentiation by pre-exposure of mouse adipose-derived adult stromal cells (ADASs) to a hypoxic (2% oxygen) environment. ADASs were subsequently expanded in 2% or 21% oxygen environments, resulting in 2 groups of cells, and then early chondrogenic differentiation was induced at 21% oxygen tension using a 3-dimensional micromass culture system. ADAS chondrogenesis was assessed using Alcian Blue staining for proteoglycans and quantification of sulfated glycosaminoglycans. Osteogenesis of the 2 cell groups was also studied. Two percent oxygen tension profoundly increased the proliferation of ADASs. ADASs expanded in 2% oxygen tension exhibited enhanced early chondrogenic differentiation and diminished osteogenesis, suggesting that the reduced oxygen environment may favor chondroprogenitors. Gene expression analysis suggested that matrix metalloproteinase synthesis was inhibited in cells expanded in 2% oxygen. Furthermore, re-oxygenation of the 2% oxygen-expanded ADASs before differentiation did not significantly affect early chondrogenesis. Thus, priming ADASs with 2% oxygen may have selected for chondrogenic progenitors with an enhanced ability to survive and differentiate. This study is relevant for the future application of cell-based therapies involving cartilage tissue regeneration.

Shaped cartilage grafts can be used in the restoration of injured joints and the reconstruction of deformities of the head and neck. This study describes a novel method for altering cartilage shape, based on the hypothesis that mechanical loading coupled with in vitro tissue growth and remodeling facilitates tissue reshaping. Static bending deformations were imposed on strips of immature articular cartilage, and retention of the imposed shape and structural and biochemical measures of growth were assessed after 2, 4, and 6 days of incubation. The results show that mechanical reshaping of tissue is feasible, because shape retention was greater than 86% after 6 days of culture. The imposed mechanical deformations had little effect on measures of tissue viability or growth within the 6-day culture period. The addition of cycloheximide to the culture medium only slightly reduced the ability to reshape these tissues, but cycloheximide plus a lower culture temperature of 4 degrees C markedly inhibited the reshaping response. These results suggest a limited role for chondrocyte biosynthesis but a potentially important role for metabolic reactions in the cartilage matrix in the reshaping process. The ability to modulate cartilage shape in vitro may prove useful for tissue engineering of shaped cartilage grafts.

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.