Rodolfo Quarto

Repair of Large Bone Defects with the Use of Autologous Bone Marrow Stromal Cells

To the Editor: The reconstruction of large bone segments is an important clinical problem, and none of the approaches proposed thus far have proved very effective. In animals, repair and functional...

Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: Implications for their use in cell therapy

Bone marrow stromal cells (BMSC) are an attractive target for novel strategies in the gene/cell therapy of hematologic and skeletal pathologies, involving BMSC in vitro expansion/transfection and reinfusion. We investigated the effects of in vitro expansion on BMSC pluripotentiality, proliferative ability, and bone-forming efficiency in vivo. BMSC from three marrow donors were cultured to determine their growth kinetics. At each passage, their differentiation potential was verified by culture in inductive media and staining with alizarin red, alcian blue, or Sudan black, and by immunostaining for osteocalcin or collagen II. First passage cells were compared to fresh marrow for their bone-forming efficiency in vivo. Stromal cell clones were isolated from five donors and characterized for their multidifferentiation ability. The lifespan and differentiation kinetics of five of these clones were determined. After the first passage, BMSC had a markedly diminish proliferation rate and gradually lost their multiple differentiation potential. Their bone-forming efficiency in vivo was reduced by about 36 times at first confluence as compared to fresh bone marrow. Experiments on the clones yielded comparable results. Culture expansion causes BMSC to gradually lose their early progenitor properties. Both the duration and the conditions of culture could be crucial to successful clinical use of these cells and must be considered when designing novel therapeutic strategies involving stromal mesenchymal progenitor manipulation and reinfusion.

Tissue engineering and cell therapy of cartilage and bone.

Trauma and disease of bones and joints, frequently involving structural damage to both the articular cartilage surface and the subchondral bone, result in severe pain and disability for millions of people worldwide and represent major challenges for the orthopedic surgeons. Therapeutic repair of skeletal tissues by tissue engineering has raised the interest of the scientific community, providing very promising results in preclinical animal models and clinical pilot studies. In this review, we discuss this approach. The choice of a proper cell type is addressed. The use of terminally differentiated cells, as in the case of autologous chondrocyte implantation, is compared with the advantages/disadvantages of using more undifferentiated cell types, such as stem cells or early mesenchymal progenitors that retain multi-lineage and self-renewal potentials. The need for proper scaffold matrices is also examined, and we provide a brief overview of their fundamental properties. A description of the natural and biosynthetic materials currently used for reconstruction purposes, either of cartilage or bone, is given. Finally, we highlight the positive aspects and the remaining problems that will drive future research in articular cartilage and bone repair.

Ex vivo enrichment of mesenchymal cell progenitors by fibroblast growth factor 2.

Bone marrow stromal cells, obtained from postnatal bone marrow, contain progenitors able to differentiate into several mesenchymal lineages. Their use in gene and cell therapy requires their in vitro expansion and calls for the investigation of the culture conditions required to preserve these cells as a stem compartment with high differentiative potential during their life span. Here we report that fibroblast growth factor 2 (FGF-2)-supplemented bone marrow stromal cell primary cultures display an early increase in telomere size followed by a gradual decrease, whereas in control cultures telomere length steadily decreases with increasing population doublings. Together with clonogenic culture conditions, FGF-2 supplementation prolongs the life span of bone marrow stromal cells to more than 70 doublings and maintains their differentiation potential until 50 doublings. These results suggest that FGF-2 in vitro selects for the survival of a particular subset of cells enriched in pluripotent mesenchymal precursors and is useful in obtaining a large number of cells with preserved differentiation potential for mesenchymal tissue repair.

STROMAL DAMAGE AS CONSEQUENCE OF HIGH-DOSE CHEMO/RADIOTHERAPY IN BONE MARROW TRANSPLANT RECIPIENTS

Bone marrow transplant (BMT) relies on the engraftment of donor hemopoietic precursors in the host marrow space. Colony forming units-fibroblasts (CFU-f), the precursor compartment for the osteogenic lineage, are essential to hemopoietic stem cell survival, proliferation and differentiation. We have studied CFU-f in donors (aged 5 months to 62 years) and in patients who had received allogeneic BMT (aged 2 months to 63 years). In donor marrows we found an inverse correlation between CFU-f frequency and age. In BMT recipients CFU-f frequencies were reduced by 60%-90% (p < 0.05) and the numbers did not recover up to 12 years after transplant. Stromal reconstitution to normal levels was found only in patients < 5 years old. In all patients studied CFU-f post-BMT were of host origin. Patients with low CFU-f levels displayed also a decreased bone mineral density (p < 0.05) and significantly reduced levels of long-term culture-initiating cells (LTC-IC) (p < 0.05). Our study demonstrates that the marrow stromal microenvironment is seriously and irreversibly damaged after BMT. Donor cells do not contribute to reconstitute the marrow microenvironment, whose residual CFU-fs remain of host origin.

Three-dimensional perfusion culture of human bone marrow cells and generation of osteoinductive grafts

Three‐dimensional (3D) culture systems are critical to investigate cell physiology and to engineer tissue grafts. In this study, we describe a simple yet innovative bioreactor‐based approach to seed, expand, and differentiate bone marrow stromal cells (BMSCs) directly in a 3D environment, bypassing the conventional process of monolayer (two‐dimensional [2D]) expansion. The system, based on the perfusion of bone marrow–nucleated cells through porous 3D scaffolds, supported the formation of stromal‐like tissues, where BMSCs could be cocultured with hematopoietic progenitor cells in proportions dependent on the specific medium supplements. The resulting engineered constructs, when implanted ectopically in nude mice, generated bone tissue more reproducibly, uniformly, and extensively than scaffolds loaded with 2D‐expanded BMSCs. The developed system may thus be used as a 3D in vitro model of bone marrow to study interactions between BMSCs and hematopoietic cells as well as to streamline manufacture of osteoinductive grafts in the context of regenerative medicine.

Replicative aging and gene expression in long-term cultures of human bone marrow stromal cells.

Bone marrow stromal cells (BMSCs) can be easily isolated from adult marrow and contain a population of pluripotent progenitors that can give rise to different mesenchymal lineages both in vitro and in vivo. These properties make BMSCs an attractive target for cell-based therapeutic strategies for a variety of disorders. However, because of their low frequency in vivo, to obtain a sufficient number of cells for tissue engineering a step of extensive in vitro expansion is required, which could significantly alter BMSC properties. Therefore, effective therapeutic use of BMSCs requires the design of appropriate approaches for in vitro cell expansion. In this study we have investigated the biological effects of in vitro expansion on BMSC proliferative ability and on their spontaneous differentiation. Telomerase activity and telomere shortening kinetics were evaluated together with variations in osteogenic, chondrogenic, and adipogenic gene expression throughout the BMSC life span. In culture BMSCs never displayed telomerase activity and during in vitro expansion telomere length decreased. Furthermore, gene expression patterns spontaneously varied during expansion, indicating a progressive commitment of the population toward the osteogenic lineage. In conclusion, BMSCs in culture undergo progressive replicative aging and osteogenic differentiation. These observations are relevant to their successful use in clinics and should be considered when designing novel therapeutic strategies.

Design of graded biomimetic osteochondral composite scaffolds

With the ultimate goal to generate suitable materials for the repair of osteochondral defects, in this work we aimed at developing composite osteochondral scaffolds organized in different integrated layers, with features which are biomimetic for articular cartilage and subchondral bone and can differentially support formation of such tissues. A biologically inspired mineralization process was first developed to nucleate Mg-doped hydroxyapatite crystals on type I collagen fibers during their self-assembling. The resulting mineral phase was non-stoichiometric and amorphous, resembling chemico-physical features of newly deposited, natural bone matrix. A graded material was then generated, consisting of (i) a lower layer of the developed biomineralized collagen, corresponding to the subchondral bone, (ii) an upper layer of hyaluronic acid-charged collagen, mimicking the cartilaginous region, and (iii) an intermediate layer of the same nature as the biomineralized collagen, but with a lower extent of mineral, resembling the tidemark. The layers were stacked and freeze-dried to obtain an integrated monolithic composite. Culture of the material for 2 weeks after loading with articular chondrocytes yielded cartilaginous tissue formation selectively in the upper layer. Conversely, ectopic implantation in nude mice of the material after loading with bone marrow stromal cells resulted in bone formation which remained confined within the lower layer. In conclusion, we developed a composite material with cues which are biomimetic of an osteochondral tissue and with the capacity to differentially support cartilage and bone tissue generation. The results warrant testing of the material as a substitute for the repair of osteochondral lesions in orthotopic animal models.

Orderly osteochondral regeneration in a sheep model using a novel nano-composite multilayered biomaterial.

The objective of this article was to investigate the safety and regenerative potential of a newly developed biomimetic scaffold when applied to osteochondral defects in an animal model. A new multilayer gradient nano‐composite scaffold was obtained by nucleating collagen fibrils with hydroxyapatite nanoparticles. In the femoral condyles of 12 sheep, 24 osteochondral lesions were created. Animals were randomized into three treatment groups: scaffold alone, scaffold colonized in vitro with autologous chondrocytes and empty defects. Six months after surgery, the animals were sacrificed and the lesions were histologically evaluated. Histologic and gross evaluation of specimens showed good integration of the chondral surface in all groups except for the control group. Significantly better bone regeneration was observed both in the group receiving the scaffold alone and in the group with scaffold loaded with autologous chondrocytes. No difference in cartilage surface reconstruction and osteochondral defect filling was noted between cell‐seeded and cell‐free groups. In the control group, no bone or cartilage defect healing occurred, and the defects were filled with fibrous tissue. Quantitative macroscopic and histological score evaluations confirmed the qualitative trends observed. The results of the present study showed that this novel osteochondral scaffold is safe and easy to use, and may represent a suitable matrix to direct and coordinate the process of bone and hyaline‐like cartilage regeneration. The comparable regeneration process observed with or without autologous chondrocytes suggests that the main mode of action of the scaffold is based on the recruitment of local cells.

Cell Therapy for Bone Disease: A Review of Current Status

Bone marrow is a reservoir of pluripotent stem/progenitor cells for mesenchymal tissues. Upon in vitro expansion, in vivo bone‐forming efficiency of bone marrow stromal cells (BMSCs) is dramatically lower in comparison with fresh bone marrow, and their in vitro multidifferentiation potentials are gradually lost. Nevertheless, when BMSCs are isolated and expanded in the presence of fibroblast growth factor 2, the percentage of cells able to differentiate into the osteogenic, chondrogenic, and adipogenic lineages is greater. Osteogenic progenitors are not exclusive to skeletal tissues. We could also think of cells in different adult tissues as potentially capable of following an osteochondrogenic differentiation pathway, but, under normal physiological conditions, they are inhibited in this process by the environment and/or the adjacent cell populations. When, for some reason such as pathology, the environment changes dramatically and the inhibiting condition is removed, these cells could become osteoblasts. Bone is repaired via local delivery of cells within a scaffold. Bone formation was first assessed in small animal models. Large animal models were successively developed to prove the feasibility of the tissue engineering approach in a model closer to a real clinical situation. Eventually, pilot clinical studies were performed. Extremely appealing is the possibility of using mesenchymal progenitors in the therapy of genetic bone diseases via systemic infusion. There is experimental evidence to suggest that mesenchymal progenitors delivered by this route engraft with a very low efficiency and do not produce relevant and durable clinical effects. Under some conditions, where the local microenvironment is either altered (i.e., injury) or under important remodeling processes (i.e., fetal growth), engraftment of stem and progenitor cells seems to be enhanced. A better understanding of their engraftment mechanisms will, hopefully, extend the field of therapeutic applications of mesenchymal progenitors.