Журнал тканевой науки и инженерии

Transforming growth factor (TGF)-?1 activates the expression of matrix metalloproteinases (MMPs) in fibroblasts. Attachment of these cells to laminin-111 further raises the TGF-?1-induced expression of MMP-3 and MMP-10. Mesenchymal stromal cells (MSC) attach to a variety of extracellular matrix proteins during development and wound healing. We therefore investigated the TGF-?1-regulated expression of MMPs in MSC upon attachment to laminin-111 and type I collagen. The expression of MMPs was determined by quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. The TGF-?1 signalling pathways were investigated by immunoblot and by pharmacological blocking of Smad2, MEK/ERK and p38MAPK activities. Overall, TGF-?1 significantly activated the expression of mRNA encoding MMP-3 (p=0.05), MMP-13 (p=0.05) and TIMP-1 (p=0.01) in MSC. Induction of MMP-10 was not significant. In contrast to our observation on fibroblasts, the attachment of MSC to laminin-111 did not affect the TGF-?1-induced expression of MMP-3 and MMP-10. Attachment to type I collagen reduced the TGF-?1-induced secretion of MMP-3 and MMP-10 compared to cells grown on laminin-111 or tissue culture plastic dishes. The expression of MMP-3 was induced by TGF-?1 via Smad2, ERK1/2 and p38MAPK. The expression of MMP-10 was regulated by Smad2 and ERK/1/2, whereas the expression of MMP-13 was shown to be p38 MAPkinase dependent. We conclude that the regulation of MMP-3, MMP-10, and MMP-13 by TGF-?1 proceeds via distinct signalling routes. In contrast to the regulatory pathways in fibroblasts, we could not prove a co-signalling of TGF-?1- and integrin-dependent pathways for the regulation of MMP-3 and MMP-10 in MSC upon attachment to laminin-111. Therefore, MSC respond differently to TGF-?1 and extracellular matrix molecules compared to fibroblasts.

Activated platelet rich plasma (a PRP) contains supra physiologic amounts of autologous growth factors and cytokines known to enhance wound healing and tissue regeneration. Here we report the first results of electro spinning nanofibers from a PRP to create fibrous scaffolds that could be used for various tissue engineering applications. Human platelet rich plasma (PRP) was created, activated by a freeze-thaw-freeze process, and lyophilized to form a powdered preparation rich in growth factors (PRGF). It was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) at different concentrations to form fibers with average diameters of 0.3 ? 3.6 ?m. A sustained release of protein from the PRGF scaffolds was demonstrated up to 35 days, and cell interactions with the PRGF scaffolds confirmed cell infiltration after just 3 days. As electro spinning is a simple process, and PRGF contains naturally occurring growth factors in physiologic ratios, creating nanofibrous structures from PRGF has the potential to be beneficial for a variety of tissue engineering applications.

In the last few years? stem cell research contributed to gain a fundamental understanding of how organisms grow and develop and how tissues are maintained throughout adult life. Mesenchymal stem cells (MSC) are self renewing, multipotent cells that are present in many adult tissues, such as bone marrow, adipose tissue, trabecular bone and muscle. More recently they have been found also in skin, liver and other tissues. Dermal skin-derived fibroblasts exhibit mesenchymal surface antigen immunophenotype and differentiation capabilities versus the three main mesenchymal tissues (bone, fat and cartilage). Hereditary Hemorrhagic Telangiectasia affects 1 in 5000 people and leads to abnormal blood vessel formation in skin and mucous membranes. We isolated human dermal fibroblasts from patients with Hereditary Hemorrhagic Telangiectasia (HHT) and healthy controls. In order to evaluate future applications of these cells in tissue engineering we compared mesenchymal properties (self-renewal, differentiation potential) of human gingival fibroblasts isolated from healthy and HHT-affected subjects using a combination of phenotypic (flow cytometry), morphologic (senescence), and functional (in vitro differentiation, colony forming unit assay and proliferation assay) criteria. Our results suggest that HHT cells were ideal candidates for tissue engineering.

In regenerative medicine the use of stem/progenitor cells is a valuable therapeutical option for the regeneration of diseased tissues and organs. However, the secure application of cell-based therapies for the treatment of renal failure requires exact information regarding the mechanisms of parenchyma development in combination with different kinds of biomaterials. Recently, we demonstrated that application of stem/progenitor cells in combination with a polyester fleece I-7 as an artificial interstitium supported the spatial generation of tubules during perfusion culture in chemically defined Iscove`s Modified Dulbecco’s Medium containing aldosterone (1x10-7M). In the present experiments we investigated if the use of different polyester fleeces (Posi-4 and Posi-5 in comparison to I-7) has any effect on generation, differentiation and spatial development of tubules. In consequence, immunohistochemistry, transmission electron microscopy and scanning electron microscopy were performed. Since the specimens were not coated by extracellular matrix proteins, unique insights in the contact zone between the basal lamina, interstitial cells and surrounding polyester fibers can be obtained. Analyzing the specimens developing in I-7, Posi-4 and Posi-5 polyester fleeces by means of immune histochemistry and transmission electron microscopy illuminated that no cell biological differences could be observed. In contrast, scanning electron microscopy of generated tubules demonstrated that a difference in spatial distribution and different diameters of tubules could be registered. It is concluded that tested I-7, Posi-4 and Posi-5 polyester fleeces appear as promising candidates to shelter stem/progenitor cells after implantation although they exhibit different spatial growth pattern.

Objectives: Synovium-derived stem cells (SDSCs) are tissue-specific stem cells for chondrogenesis. Our aim was to evaluate whether decellularized matrix deposited by SDSCs was superior to chondrocytes in providing a stem cell microenvironment to conduct large scale expansion of high-quality cells for cartilage tissue engineering.
Materials and Methods: We generated two extracellular matrices (ECMs) deposited by either SDSCs (SECM) or chondrocytes (CECM). Passage 4 SDSCs and chondrocytes were expanded separately for two passages on three substrates: conventional plastic flasks (Plastic), SECM, or CECM. Expanded cells were incubated in a pellet culture system supplemented with serum-free chondrogenic medium for 14 days. Histology, biochemistry, real-time PCR, and western blot were used to evaluate expanded cell chondrogenic capacity.
Results: Cell proliferation was greatly improved during expansion on both ECMs, especially on SECM. ECM expansion enhanced cell chondrogenic potential, particularly for cells expanded on SECM. Collagen II and aggrecan were deposited only in CECM while collagen I and decorin existed in both ECMs. High levels of phospho-TGF-β receptor II found in chondrogenically induced cells after expansion on either ECM suggested that enhancement of chondrogenic potential might result from upregulated sensitivity in ECM-expanded cells when they are chondrogenically induced.
Conclusions: SECM is superior to CECM in promoting cell expansion and enhancing expanded cell chondrogenic potential. Decellularized stem cell matrix can serve as a novel cell expansion system for cartilage tissue engineering.