VEGF Over-Expression by Engineered BMSC Accelerates Functional Perfusion, Improving Tissue Density and In-Growth in Clinical-Size Osteogenic Grafts.

The first choice for reconstruction of clinical-size bone defects consists of autologous bone flaps, which often lack the required mechanical strength and cause significant donor-site morbidity. We have previously developed biological substitutes in a rabbit model by combining bone tissue engineering and flap pre-fabrication. However, spontaneous vascularization was insufficient to ensure progenitor survival in the core of the constructs. Here, we hypothesized that increased angiogenic stimulation within constructs by exogenous VEGF can significantly accelerate early vascularization and tissue in-growth. Bone marrow stromal cells from NZW rabbits (rBMSC) were transduced with a retroviral vector to express rabbit VEGF linked to a truncated version of rabbit CD4 as a cell-surface marker. Autologous cells were seeded in clinical-size 5.5 cm3 HA scaffolds wrapped in a panniculus carnosus flap to provide an ample vascular supply, and implanted ectopically. Constructs seeded with VEGF-expressing rBMSC showed significantly increased progenitor survivival, depth of tissue ingrowth and amount of mineralized tissue. Contrast-enhanced MRI after 1 week in vivo showed significantly improved tissue perfusion in the inner layer of the grafts compared to controls. Interestingly, grafts containing VEGF-expressing rBMSC displayed a hierarchically organized functional vascular tree, composed of dense capillary networks in the inner layers connected to large-caliber feeding vessels entering the constructs at the periphery. These data constitute proof of principle that providing sustained VEGF signaling, independently of cells experiencing hypoxia, is effective to drive rapid vascularization and increase early perfusion in clinical-size osteogenic grafts, leading to improved tissue formation deeper in the constructs.

Spontaneous In Vivo Chondrogenesis of Bone Marrow-Derived Mesenchymal Progenitor Cells by Blocking Vascular Endothelial Growth Factor Signaling.

: Chondrogenic differentiation of bone marrow-derived mesenchymal stromal/stem cells (MSCs) can be induced by presenting morphogenetic factors or soluble signals but typically suffers from limited efficiency, reproducibility across primary batches, and maintenance of phenotypic stability. Considering the avascular and hypoxic milieu of articular cartilage, we hypothesized that sole inhibition of angiogenesis can provide physiological cues to direct in vivo differentiation of uncommitted MSCs to stable cartilage formation. Human MSCs were retrovirally transduced to express a decoy soluble vascular endothelial growth factor (VEGF) receptor-2 (sFlk1), which efficiently sequesters endogenous VEGF in vivo, seeded on collagen sponges and immediately implanted ectopically in nude mice. Although naïve cells formed vascularized fibrous tissue, sFlk1-MSCs abolished vascular ingrowth into engineered constructs, which efficiently and reproducibly developed into hyaline cartilage. The generated cartilage was phenotypically stable and showed no sign of hypertrophic evolution up to 12 weeks. In vitro analyses indicated that spontaneous chondrogenic differentiation by blockade of angiogenesis was related to the generation of a hypoxic environment, in turn activating the transforming growth factor-ß pathway. These findings suggest that VEGF blockade is a robust strategy to enhance cartilage repair by endogenous or grafted mesenchymal progenitors. This article outlines the general paradigm of controlling the fate of implanted stem/progenitor cells by engineering their ability to establish specific microenvironmental conditions rather than directly providing individual morphogenic cues. SIGNIFICANCE: Chondrogenic differentiation of mesenchymal stromal/stem cells (MSCs) is typically targeted by morphogen delivery, which is often associated with limited efficiency, stability, and robustness. This article proposes a strategy to engineer MSCs with the capacity to establish specific microenvironmental conditions, supporting their own targeted differentiation program. Sole blockade of angiogenesis mediated by transduction for sFlk-1, without delivery of additional morphogens, is sufficient for inducing MSC chondrogenic differentiation. The findings represent a relevant step forward in the field because the method allowed reducing interdonor variability in MSC differentiation efficiency and, importantly, onset of a stable, nonhypertrophic chondrocyte phenotype.

Cartilage graft engineering by co-culturing primary human articular chondrocytes with human bone marrow stromal cells.

Co-culture of mesenchymal stromal cells (MSCs) with articular chondrocytes (ACs) has been reported to improve the efficiency of utilization of a small number of ACs for the engineering of implantable cartilaginous tissues. However, the use of cells of animal origin and the generation of small-scale micromass tissues limit the clinical relevance of previous studies. Here we investigated the in vitro and in vivo chondrogenic capacities of scaffold-based constructs generated by combining primary human ACs with human bone marrow MSCs (BM-MSCs). The two cell types were cultured in collagen sponges (2 × 6 mm disks) at the BM-MSCs:ACs ratios: 100:0, 95:5, 75:25 and 0:100 for 3 weeks. Scaffolds freshly seeded or further precultured in vitro for 2 weeks were also implanted subcutaneously in nude mice and harvested after 8 or 6 weeks, respectively. Static co-culture of ACs (25%) with BM-MSCs (75%) in scaffolds resulted in up to 1.4-fold higher glycosaminoglycan (GAG) content than what would be expected based on the relative percentages of the different cell types. In vivo GAG induction was drastically enhanced by the in vitro preculture and maximal at the ratio 95:5 (3.8-fold higher). Immunostaining analyses revealed enhanced accumulation of type II collagen and reduced accumulation of type X collagen with increasing ACs percentage. Constructs generated in the perfusion bioreactor system were homogeneously cellularized. In summary, human cartilage grafts were successfully generated, culturing BM-MSCs with a relatively low fraction of non-expanded ACs in porous scaffolds. The proposed co-culture strategy is directly relevant towards a single-stage surgical procedure for cartilage repair.

Osteoblastic differentiation of Wharton jelly biopsy specimens and their mesenchymal stromal cells after serum-free culture.

BACKGROUND: Cleft lip and cleft palate are increasingly being detected by prenatal ultrasound, which raises the opportunity of using the patient's own osteogenicity from umbilical cord mesenchymal cells for bony repair. The authors address the growth of the cells under a fully defined and regulated protocol. METHODS: Wharton jelly-derived mesenchymal stromal cells were isolated and expanded as a monolayer with defined serum-free medium. Osteoblastic differentiation was tested in the cells and in the entire Wharton jelly biopsy specimens. The serum-free-cultured cells were included in hydroxyapatite granule-fibrin constructs and, without predifferentiation, subcutaneously implanted into immunoincompetent mice. RESULTS: Isolation and expansion of Wharton jelly-derived mesenchymal stromal cells were consistently successful under serum-free conditions, and the cells expressed standard mesenchymal stromal cell markers. The serum-free-cultivated cells produced a mineralized extracellular matrix under osteogenic differentiation, with a significant increase of osteoblastic lineage gene expression (Hox-A10 and Runx2) and an up-regulation of downstream osteogenic genes (OSX, OCN, ALPL, and BSP2). In vivo, they formed a dense matrix adjacent to the granules after 8 weeks, but no lamellar bone. serum-free-cultivated entire Wharton jelly biopsy specimens produced a mineralized extracellular matrix within the collagen matrix of the Wharton jelly. CONCLUSIONS: The osteogenic differentiation potential of Wharton jelly-derived mesenchymal stromal cells was maintained under serum-free isolation and expansion techniques. The cells without predifferentiation form a dense collagen matrix but not bone in vivo. Moreover, entire Wharton jelly biopsy specimens showed periosteal-like mineralization under osteogenic differentiation, which offers new options for autologous bone tissue engineering, including cleft palate surgery.

Non-adherent mesenchymal progenitors from adipose tissue stromal vascular fraction.

In primary human bone marrow cultures, the initial adherent cell fraction has been shown to provide a microenvironment for self-renewal of primitive non-adherent mesenchymal progenitors (non-adherent progenitors of bone marrow stroma [BM-NAMP]), with increased differentiation potential compared to adherent colony-forming units-fibroblast (CFU-f). The present study investigates whether NAMP exist also in cultures of stromal vascular fraction (SVF) cells derived from human adipose tissue. Adipose-tissue NAMP (AT-NAMP) were shown to be stably non-adherent and their number correlated with the number of the initial adhering CFU-f. Unlike BM-NAMP, AT-NAMP did not propagate in suspension in serial replating experiments and the number of colonies steadily decreased with each replating step. However, when AT-NAMP were kept on the initially adhering SVF cells, they could significantly expand without loss of clonogenic, proliferation, and differentiation potential. Although AT-NAMP progeny differentiated into mesodermal lineages similar to that of adherent CFU-f, it was enriched in early mesenchymal progenitor populations, characterized by increased expression of SSEA-4 and CD146. Furthermore, FGF-2 supported AT-NAMP survival and could not be replaced by another mitogenic factor, such as platelet derived growth factor BB. In conclusion, these data suggest that the SVF adherent fraction provides niche signals that regulate the expansion of adipose non-adherent mesenchymal progenitors with the maintenance of their potency. The biological differences described between BM- and AT-NAMP further qualify the properties of the stroma from different tissues and will be relevant for the selection of a cell source for specific regeneration strategies.