CELL THERAPY FOR BONE DISEASES

Abstract

Bone marrow is the tissue where hemopoiesis occurs in close contact with the stromal microenvironment which support hemopoietic stem cell growth and differentiation. The bone marrow stroma is composed of a variety of different cell types providing structural and functional support for hemopoiesis: endothelial cells, adipocytes, smooth muscle cells, reticular cells, osteoblasts and stromal fibroblasts. Among these cell types, stromal fibroblasts have a peculiar biologic relevance. They are in fact able to support hemopoiesis, to differentiate towards osteogenic, chondrogenic and adipogenic lineage and to form a bone structure complete of hemopoietic marrow in in vivo assays. Their in vitro clonogenic counterpart is represented by Colony Forming Units-fibroblasts (CFU-f), which in turn give rise to Bone Marrow Stromal Cells (BMSC). In vivo bone formation by BMSC has been strikingly demonstrated and therefore these cells are considered a progenitor compartment for osteoblasts, responsible for the maintenance of bone turnover throughout life.

BMSC can be easily isolated from bone marrow aspirates. Nevertheless, given the low frequency of BMSC in a marrow sample, a step of extensive in vitro expansion is required to obtain a consistent number of cells available for both reconstruction and repair of mesodermally derived tissues. Moreover, their use for gene and cell therapy of skeletal diseases requires the long-lasting engraftment of BMSC endowed with a residual proliferation potential sufficient to sustain the low, but continuous, bone turnover in adulthood. The maintenance of BMSC stemness and the possibility to reprogram their commitment is therefore a field of primary interest given their potential use in regenerative medicine. Cell therapy of bone lesions by ex vivo expanded BMSC is passing from the phase of experimental animal model to the phase of clinical trials. Bone is repaired via local delivery of cells within a scaffold. Extremely appealing is the possibility of using mesenchymal progenitors in the therapy of genetic bone diseases via systemic infusion. Under some conditions where the local microenvironment is either altered (i.e. injury) or under important remodelling processes (i.e. fetal growth), engraftment of stem and progenitor cells seems to be enhanced. A better understanding of the mechanisms controlling BMSC differentiation and engraftment is required for their exploitation in therapy of human diseases. Furthermore, a better understanding of the interactions occurring between BMSC and biomaterials used to deliver cells in vivo will hopefully extend the field of therapeutic applications of mesenchymal progenitors. In this talk we will go through our experimental evidences on: a) influence of signaling molecule; b) transplantation route and engraftment; c) biomaterials.

Growth factors are essential for a number of cellular functions. Our results show that FGF-2 supplemented BMSC primary cultures display better differentiation potential, a higher degree of osteogenicity and undergo an early increase in telomere size followed by a gradual decrease, whereas in control cultures telomere length decreases with increasing population doublings. In conjunction with clonogenic culture conditions, FGF-2 supplementation extends the life-span of BMSC to over 70 doublings and preserves their differentiation potential up to 50 doublings. All together, these data suggest that FGF-2 supplementation in vitro selects for the survival of a particular subset of cells enriched in pluripotent mesenchymal precursors and may be useful to obtain a large number of cells for mesenchymal tissue repair.

BMSC intravenous infusion has been proposed as a means to support the hematopoiesis in Bone Marrow Transplants or as a vehicle for gene therapy. However, it seems that this route of injection leads to engraftment of a small proportion of BMSC. We have transplanted human BMSC transduced with the human erythropoietin gene, either intravenously or subcutaneously in NOD/SCID mice. Efficiency of engraftment was evaluated monitoring the hematocrit levels. Systemic infusion never increased hematocrit levels, whereas subcutaneous transplantation of the same number of cells induced an important increase of the hematocrit for at least two months. To determine whether the transient effect was due to cell loss or to reduction in expression, we recovered the cells implanted into a tridimensional scaffold, after the normalization of the hematocrit, expanded them in vitro, and re-implanted them in a new group of mice. Again the hematocrit levels rose one week after the transplantation. These results demonstrate that ex-vivo expanded human BMSC are not transplantable by systemic infusion, whereas the local implantation into a 3D scaffold allows their long term engraftment.

Biomaterials for bone regeneration should have a suitable structure to allow cell adhesion and an ideal level of vascularisation, a key factor to achieve new bone formation. Furthermore, they have to be informative, driving the cells towards osteogenesis and allowing the deposition of bone extracellular matrix. Our results indicate that BMSC need a mineralized scaffold to initiate bone formation which will occur with an extent proportional to the availability of biomaterial surface.