Regulated expression of transgene production and function is of great importance for gene therapy. Such regulation can potentially be used to monitor and control complex biological processes. We report here a regulated stem cell-based system for controlling bone regeneration, utilizing genetically engineered mesenchymal stem cells (MSCs) harboring a tetracycline-regulated expression vector encoding the osteogenic growth factor human BMP-2. We show that doxycycline (a tetracycline analogue) is able to control hBMP-2 expression and thus control MSC osteogenic differentiation both in vitro and in vivo. Following in vivo transplantation of genetically engineered MSCs, doxycycline administration controlled both bone formation and bone regeneration. Moreover, our findings showed increased angiogenesis accompanied by bone formation whenever genetically engineered MSCs were induced to express hBMP-2 in vivo. Thus, our results demonstrate that regulated gene expression in mesenchymal stem cells can be used as a means to control bone healing.

The culture expansion of human mesenchymal stem cells (hMSCs) may alter their characteristics and is a costly and timeconsuming stage. This study demonstrates for the first time that immunoisolated noncultured CD105-positive (CD105 ؉ ) hMSCs are multipotent in vitro and exhibit the capacity to form bone in vivo. hMSCs are recognized as promising tools for bone regeneration. However, the culture stage is a limiting step in the clinical setting. To establish a simple, efficient, and fast method for applying these cells for bone formation, a distinct population of CD105 ؉ hMSCs was isolated from bone marrow (BM) by using positive selection based on the expression of CD105 (endoglin). The immunoisolated CD105 ؉ cell fraction represented 2.3% ؎ 0.45% of the mononuclear cells (MNCs). Flow cytometry analysis of freshly immunoisolated CD105 ؉ cells revealed a purity of 79.7% ؎ 3.2%. In vitro, the CD105 ؉ cell fraction displayed significantly more colony-forming units-fibroblasts (CFU-Fs; 6.3 ؎ 1.4) than unseparated MNCs (1.1 ؎ 0.3; p < .05). Culture-expanded CD105 ؉ cells expressed CD105, CD44, CD29, CD90, and CD106 but not CD14, CD34, CD45, or CD31 surface antigens, and these cells were able to differentiate into osteogenic, chondrogenic, and adipogenic lineages. In addition, freshly immunoisolated CD105 ؉ cells responded in vivo to recombinant bone morphogenetic protein-2 by differentiating into chondrocytes and osteoblasts. Genetic engineering of freshly immunoisolated CD105 ؉ cells was accomplished using either adenoviral or lentiviral vectors. Based on these findings, it is proposed that noncultured BM-derived CD105 ؉ hMSCs are osteogenic cells that can be genetically engineered to induce tissue generation in vivo.

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Background Among the approximately 6.5 million fractures suffered in the United States every year, about 15% are difficult to heal. As yet, for most of these difficult cases there is no effective therapy. We have developed a mouse radial segmental defect as a model experimental system for testing the capacity of Genetically Engineered Pluripotent Mesenchymal Cells (GEPMC, C3H10T1/2 clone expressing rhBMP‐2), for gene delivery, engraftment, and induction of bone growth in regenerating bone. Methods Transfected GEPMC expressing rhBMP‐2 were further infected with a vector carrying the lacZ gene, that encodes for β‐galactosidase (β‐gal). In vitro levels of rhBMP‐2 expression and function were confirmed by immunohistochemistry, and bioassay. Differentiation was assayed using alkaline phosphatase staining. GEPMC were transplanted in vivo into a radial segmental defect. The main control groups included lacZ clones of WT‐C3H10T1/2‐LacZ, and CHO‐rhBMP‐2 cells. New bone formation was measured quantitatively via fluorescent labeling, X‐ray analysis and histomorphometry. Engrafted mesenchymal cells were localized in vivo by β‐gal expression, and double immunofluorescence. Results In vitro, GEPMC expressed rhBMP‐2, β‐gal and spontaneously differentiated into osteogenic cells expressing alkaline phosphatase. Detection of transplanted cells revealed engrafted cells that had differentiated into osteoblasts and co‐expressed β‐gal and rhBMP‐2. Analysis of new bone formation revealed that at fout to eight week post‐transplantation, GEPMS significantly enhanced segmental defect repair. Conclusions Our study shows that cell‐mediated gene transfer can be utilized for growth factor delivery to signaling receptors of transplanted cells (autocrine effect) and host mesenchymal cells (paracrine effect) suggesting the ability of GEPMC to engraft, differentiate, and stimulate bone growth. We suggest that our approach should lead to the designing of mesenchymal stem cell based gene therapy strategies for bone lesions as well as other tissues. Copyright © 1999 John Wiley & Sons, Ltd.

Epidermal pH is an indication of the skin’s physiological condition. For example, pH of wound can be correlated to angiogenesis, protease activity, bacterial infection, etc. Chronic non-healing wounds are known to have an elevated alkaline environment, while healing process occurs more readily in an acidic environment. Thus, dermal patches capable of continuous monitoring of pH can be used as point-of-care systems for monitoring skin disorder and the wound healing process. Here, we present pH-responsive hydrogel fibers that can be used for long-term monitoring of epidermal wound condition. We load pH-responsive dyes into mesoporous microparticles and incorporate them into hydrogel fibers developed through microfluidic spinning. The fabricated pH-responsive microfibers are flexible and can create conformal contact with skin. The response of pH-sensitive fibers with different compositions and thicknesses are characterized. The suggested technique is scalable and can be used to fabricate hydrogel based wound dressing with a wide range of sizes. Images of the pH-sensing fibers during real-time pH measurement can be captured with a smart phone camera for convenient readout on-site. Through image processing, a quantitative pH map of the hydrogel fibers and the underlying tissue can be extracted. The developed skin dressing can act as a point-of-care device for monitoring the wound healing process.