Short Report Engineering recombinant growth factors: a tool for in vitro mesenchymal cell commitment JOSÉ BECERRA*, MANUEL CIFUENTES, PILAR ARRABAL, JESÚS A. SANTAMARÍA and JOSÉ A. ANDRADES Department of Cell Biology and Genetics, Faculty of Sciences, University of Málaga, Campus Univesitario de Teatinos, Málaga, Spain Int. J. Dev. Biol. 45 (S1): S107-S108 (2001) *Address correspondence to: José Becerra. Department of Cell Biology and Genetics, Faculty of Sciences, University of Málaga, Campus Univesitario de Teatinos, 29071 Málaga, Spain. e-mail: becerra@uma.es ABSTRACT Growth factors (GFs) are part of a large number of polypeptides that transmit signals affecting cellular activities. Cells may communicate with each other through direct molecular interac- tions involving their cell membranes, as a result of the movement of certain molecules as GFs. They are produced by the cells and can act within the cell or in vicinal or remote cells to modulate their activities by reacting with specific receptors through either autocrine, paracrine, endocrine, juxtacrine, intracrine or extracellular matrix mediated. In the last recent years, our group have been working on genetically engineering GFs which incorporate a collagen-binding domain de- rived from the von Willebrand Factor (vWF). These fused proteins exhibit functional properties that do not exist in nature and have allow us to investigate their potential important implications in terms of developing strategic biomatrices for tissue repair. Tissue regeneration is a complex process that consolidates the efforts of a variety of cells which are already present or recruited at the site of injury (Nimni, 1997). The sequence of events is guided by a series of GFs, cytokines, cell-cell and cell-matrix interactions. Therefore, inductive signals, responding stem cells and the extracel- lular matrix are the three key ingredientes for tissue repair (Reddi, 2000). Some carriers are effective in releasing GFs at the required rate and do not interfere with the normal process of tissue repair (Hecht et al., 1999) whereas others do. In general, only a few approaches provide practical modalities for delivery in a clinical setting (Groeneveld and Burger, 2000). We will focus on collagen and hydroxyapatite matrices, which have been used in our laboratory, and have received high acceptance both in experimental animal models and in ongoing clinical trials. Since fibroblast growth factor (FGF) is a pleotropic agent which can stimulate, inhibit, and modulate cellular events in a time- and concentration-dependent manner, it is important to control its deliv- ery and bioavailability to advance its use as a potential therapeutic agent. Basic FGF (FGF-2) is a potent in vitro mitogen for capillary endothelial cells, stimulates angiogenesis in vivo, and participates in tissue repair. We have performed the genetic engineering of a recombinant human FGF-2 (rhFGF-2) fusion protein which, by virtue of an auxiliary vWF-derived collagen-binding domain (rhFGF-2-F2), exhibits a high affinity for collagen matrices (Fig. 1). rhFGF-2-F2 protein can be expressed at high levels in bacteria transformed with plasmids bearing the T7 polymerase system, BL21 (DE3) strain. In the presence of 1 mM IPTG, the levels of protein expression show a representative induction of the 16 kDa rhFGF-2-F1 (commercial) polypeptide (Fig. 2, lane 3) and 17 kDa for the rhFGF-2-F2 construct bearing the auxiliary vWF-derived decapeptide (Fig. 2, lane 6). After stringent washing with urea the purified proteins were finally eluted (Fig. 2, lanes 4 and 7). We also demonstrated the practical utility of rhFGF-2-F2 in the management of wound healing, since a local application of rhFGF-2-F2 mixed with type I collagen sponge into cutaneous wounds was sufficient to accelerate wound closure in normal and diabetic rats (Andrades et al., 2001). Homeostasis of bone tissue results from the right balance among it cell components. Tight metabolic regulation of osteoblasts, osteocytes and osteoclasts activity gives rise to a synthesis/resorption equilibrium of bone matrix, resulting in the bony architecture main- tenance. Specific GFs have the ability to stimulate stem cells along the chondrogenic and osteogenic pathways. The differentiation and maturation of osteoprogenitor cells into osteoblasts and these into osteocytes are thought to be modulated by transforming growth factors-beta (TGF-ß1 and TGF-ß2), and TGF-ß-related bone mor- phogenetic proteins (BMPs). In order to capture and expand a population of cells with chondro-osteogenic potential, we have used type I collagen to develop an in vitro culture system for bone marrow (BM) mesenchymal progenitor cells, which includes a mesenchymal cell compatible collagen-gel impregnated with a rhTGF-ß1-collagen binding domain (rhTGF-ß1-F2). As mentioned before, such a struc- ture bestows on the factor the ability to bind to type I collagen specifically. The results suggest that rhTGF-ß1-F2 applied to a bovine collagen matrix as vehicle and delivery system could be of advantage in promoting the in vitro survival, proliferation, differentia- tion, and colony mineralization of the osteogenic precursor cell population. Differences between rhTGF-ß1-F2 and the commercial rhTGF-ß1 may be due to the slow release of the collagen binding GF from the collagen fibrillar network to which it is bound, resulting in a longer half-life and its better availability to the target cells (Andrades et al., 1999). When the in vitro treated cells where placed into inactivated demineralized bone matrix (iDBM) implants or diffusion chambers and implanted subdermically on the back of old rats for 28 days, they were able to form large amount of cartilage and osteoid matrix (diffusion chambers) and bone tissue (iDBM implants) (Figs. 3, 4). BMPs (BMP-2 and BMP-7) have been also tested under that experimental system. Because of it sensitivity to endogenous proteases, BMPs have to be either protected or delivered at a continuous rate at the site of action, if they are going to initiate and sustain the cascade of events that lead endochondral or intramem- branous bone formation (Bax et al., 1999). Although BMPs can induce bone formation when added as a solution, the dose needed to induce endochondral bone formation can be greatly reduced when BMPs are combined with an appropriate carrier (Andrades and