AIM: Intrinsic tissue regeneration mechanisms are still not fully understood. The destruction/reconstruction processes are usually in fine balance; however, this can be easily destroyed, for example in the environment of chronic inflammation. One of the major proteins present at the inflammatory sites is the multifunctional protein alpha-1-antitrypsin (AAT). In this study, potential therapeutic effects of this major human antiprotease on progenitor cells are assessed.MATERIALS & METHODS: Stromal cells from human exfoliated deciduous teeth (SHEDs) were used, which are similar to the mesenchymal stromal cells isolated from other tissues. SHEDs were cultivated in the presence of subphysiological, physiological and inflammatory concentrations of AAT, and their proliferation and motility traits were assayed. Some intracellular signaling pathways, AAT internalization by SHEDs and their matrix metalloprotease profile were studied in parallel.RESULTS: Physiologic and inflammatory concentrations of AAT significantly increased the cell proliferation rate, induced phosphorylation of several key protein kinases and increased the amount of secreted active gelatinases. Moreover, cells exposed to physiologic and inflammatory levels of AAT were able to invade and migrate more efficiently. Subphysiologic AAT levels did not change cell behavior significantly.CONCLUSION: AAT at physiologic and inflammatory concentrations positively modulates the proliferation and motility of SHEDs in vitro. These results suggest the importance of AAT in the maintenance and regulation of tissue progenitor cells in vivo.
This paper describes a study conducted to test the hypothesis that aging will result in decreased bone formation during distraction osteogenesis (DO). DO is a unique clinical method for the stimulation of new bone formation and subsequent bone lengthening. When applied to other species DO reflects the clinical situation in which older DO patients demonstrate significant delays in mineralization. Given the considerable value of mouse genetics for studying the mechanism(s) of bone formation, we have developed a murine DO model and utilized it to investigate the effect of age on bone formation. Four- and 12-month-old CB57BL/6 male mice ( n = 10 per group) underwent DO. External fixators were placed on the left tibiae, and mid-diaphyseal tibial osteotomies were performed immediately following fixator placement. Distraction, which began 6 days after surgery at 0.075 mm twice a day (0.15 mm/day) for 14 days, resulted in a total lengthening of 2.1 mm. Following distraction, the distracted tibiae were removed for high-resolution radiography and histological evaluation. Analysis of radiographs and representative histological sections was performed by video microscopy. Radiographic analysis demonstrated a significant decrease in the mineralized area of distraction gaps of 12- (33.5 ± 4.8%) versus 4-month-old (51.4 ± 5.4%) mice ( p < 0.039). Histological analysis of representative specimens confirmed the decrease in bone formation observed in the radiographs ( p < 0.001). Endosteal new bone was predominantly intramembranous and appeared highly oriented toward the distraction axis. These results suggest that 12-month-old mice have a relative deficit in endosteal bone formation compared with that in younger mice. The application of this murine DO model to genetically manipulated mice may provide critical insights into the mechanisms of bone formation, repair, and regeneration in a geriatric setting.
Directly turning a somatic cell type into another (a process referred to as transdifferentiation) would be highly beneficial for producing replacement cells for therapeutic purposes. Adult stem cells have been shown to display a broader differentiation potential than anticipated and may contribute to tissues other than those in which they reside. In addition, novel transdifferentiation strategies are being developed. We report here studies on a functional reprogramming of a somatic cell using a nuclear and cytoplasmic extract from another somatic cell type. Reprogramming of human 293T fibroblasts in an extract from a human T cell line is illustrated by nuclear uptake and assembly of transcription factors, induction of activity of a chromatin remodeling complex, changes in chromatin composition, and activation of lymphoid cell-specific genes. The reprogrammed cells expressed T cell-specific surface antigens and a complex intracellular regulatory function. These studies open the door to new possibilities for producing isogenic replacement cells for therapeutic applications.
Human therapeutic cloning requires the reprogramming of a somatic cell by nuclear transfer to generate autologous totipotent stem cells. We have parthenogenetically activated 22 human eggs and also performed nuclear transfer in 17 metaphase II eggs. Cleavage beyond the eight-cell stage was obtained in the parthenogenetic-activated eggs, and blastocoele cavities were observed in six. Three somatic cell-derived embryos developed beyond the pronuclear stage up to the six-cell stage. The ability to create autologous embryos represents the first step towards generating immune-compatible stem cells that could be used to overcome the problem of immune rejection in regenerative medicine.
Powerful recent developments in the multidisciplinary field of tissue engineering have yielded a novel set of tissue replacement parts and implementation strategies. Scientific advances in biomaterials, stem cells, growth and differentiation factors, and biomimetic environments have created unique opportunities to fabricate tissues in the laboratory from combinations of engineered extracellular matrices ("scaffolds"), cells, and biologically active molecules. Among the major challenges now facing tissue engineering is the need for more complex functionality, as well as both functional and biomechanical stability in laboratory-grown tissues destined for transplantation. The continued success of tissue engineering, and the eventual development of true human replacement parts, will grow from the convergence of engineering and basic research advances in tissue, matrix, growth factor, stem cell, and developmental biology, as well as materials science and bioinformatics.
Growth and differentiation factor-5 (GDF-5) is a divergent member of the TGF-β/bone morphogenetic protein (BMP) superfamily that is required for proper skeletal patterning and joint development in the vertebrate limb. Based on the homology of GDF-5 to other bone-inducing BMP family members, the inductive activity of a recombinant form of human GDF5 (rhGDF-5), and the influence of the extracellular matrix (ECM) on this inductive activity was evaluated in a series of well-defined in vitro assays. Fetal rat calvarial (FRC) cells were plated on various purified extracellular matrix proteins in the presence of rhGDF-5 (100 ng/mL) for 3 weeks and scored for differentiation at the level of morphology, overall proteoglycan synthesis and deposition, aggrecan and Type II collagen mRNA and protein expressions. Results show that GDF-5 stimulated chondrogenic nodule formation by FRC cells plated on Type I collagen but to a lesser extent on tissue culture plastic or fibronectin. These chondrogenic nodules stained heavily with Alcian blue and expressed chondrogenic markers such as Type II collagen and aggrecan, as judged by immunohistochemical and RT-PCR analyses, respectively. Cells in the monolayer that surrounded the nodules did not express the chondrogenic markers. The molecular signaling mechanism by which GDF-5 induces chondrogenesis in FRC cells in the presence of Type I collagen was investigated using well-characterized modulators of intracellular signaling mediators. Results show that the ligand-dependent chondrogenesis was inhibited by the calcium ionophore A23187, rapamycin but not by dibutyryl-cAMP, Na 3 /VO 4 , or EGTA. The known effects of A23187 and rapamycin on intracellular signaling pathway suggest that the GDF-5/Type I collagen-induced chondrogenesis is mediated through modulation of intracellular calcium concentration accompanied by activation of the p70 S6 kinase (p70 s6k ) signaling pathway. Together, these results indicate that cellular interaction with Type I collagen significantly enhances the differentiating activity of GDF-5. This effect is likely mediated by the convergence of downstream matrix and growth factor receptor signaling pathways.
Articular cartilage is a highly organized tissue that performs many essential functions in the musculoskeletal system. Damage to articular cartilage often requires surgical intervention due to its limited capacity for self-repair. Tissue engineering strategies using biodegradable polymers as scaffolds for cell transplantation provide many advantages over current therapies. Various design requirements of a successful polymer scaffold for cartilage tissue engineering are discussed. The use of preformed or injectable scaffolds depends on the intended application. Both natural and synthetic polymers that may serve as a scaffold for cartilage regeneration are summarized.