Mesenchymal progenitors are a powerful tool in regenerative medicine, but suffer from a rapid loss of differentiation potential during in vitro expansion. The recent discovery that well-characterized stem cells, like HSC, maintain their stemness during self-renewal through the interaction with specialized microenvironments, called stem cell niches, prompted us to investigate the existence of a niche compartment for also mesenchymal progenitors.
In Chapter 4 of this thesis we described the establishment of a niche/progenitor system in vitro for bone marrow mesenchymal stem cells (MSC). We asked whether the non-adherent fraction of human bone marrow cultures contained early progenitors which can constitute a reservoir for the mesenchymal compartment and whether the adherent cells, instead, could provide a niche function for the maintenance and regulation of these progenitors.
Replating the non-adherent fraction in a new dish at the first medium change, we found that a population of bone marrow non-adherent mesenchymal progenitors (BM-NAMP) was present and their number was 20.43.6% of the initial CFU-f. However, further investigation showed that, when serially replated in new dishes, BM-NAMP were able to steadily increase in number, self-renewing as non-adherent progenitors while generating at the same time adherent colonies. The diameter size evaluation showed that BM-NAMP could produce colonies with 2-fold larger diameter, indicating a significantly higher proliferation capacity. However, the colonies produced in the following replating steps were progressively smaller, indicating a gradual loss of BM-NAMP proliferation potential. Together with increased proliferation, first-replated BM-NAMP progeny cells displayed a higher differentiation potential compared to standard CFU-f both in vitro and in vivo. Taken together, these data indicate together that BM-NAMP show features of earlier progenitor features and suggest a biological difference between BM-NAMP and the initially adhrering CFU-f.
Serial replating experiments performed with serum alone showed that BM-NAMP critically required FGF-2 for their initial selection and maintenance in culture. Interestingly, blocking receptor experiments showed that the maintenance of BM-NAMP in culture was mediated through FGFR2c signaling, which has been shown to be involved in vivo in the balance between proliferation and differentiation of skeletal progenitors.
We also hypothesized that BM-NAMP were in close interaction with the adherent cells, and that these provide a niche function for them. BM-NAMP were not able to survive when replated either on agarose-coated dishes or on human fibroblasts. This suggests that BM-NAMP required specific signals from the adherent progeny and that this fraction constitutes a unique environment for BM-NAMP survival and self-renewal. In fact, when kept in contact with initial CFU-f progeny for 14 days instead of being serially replated, BM-NAMP were able to produce 3-fold more colonies. Furthermore, the colony diameter analysis showed that, unlike the serial replating which caused a gradual loss of BM-NAMP proliferative activity, the continuous culture in the primary plate could preserve BM-NAMP proliferation potential. Furthermore, if kept in the original plate, BM-NAMP could generate a progeny that also displayed a higher differentiation capacity. Taken together, these results suggest together that CFU-f progeny provides a niche function for BM-NAMP.
In Chapter 5 we sought at investigating the presence of a class of non-adherent progenitors in human adipose tissue stromal vascular fraction (SVF), which constitute an abundant source of mesenchymal progenitors, to determine whether the NAMP compartment was specific to bone marrow or they could constitute a reservoir also in other tissues.
NAMP were present in adipose tissue SVF cultures (AT-NAMP) with a similar frequency as observed in the bone marrow and the replating of the non-adherent fraction in the same dish revealed that they were stably non-adherent. The main difference compared to BM-NAMP was the inability of AT-NAMP to self-renew as non-adherent progenitors upon serial replating, since only few colonies were present in the last replating step. However, these colonies had a significantly increased diameter. This suggests that, when serially replated, AT-NAMP do not undergo proliferation but rather a selection for the very rare progenitors with the highest proliferation ability. Similarly to BM-NAMP, when kept in contact with the initially adhering CFU-f, AT-NAMP could proliferate without loss of their proliferation capacity. This suggests that, as for bone marrow cells, adherent CFU-f provide a niche function for the non-adherent progenitors, regulating the maintenance of their early-progenitor properties.
In conclusion, these data show that, although displaying important tissue-specific biological differences, NAMP are present in the mesenchymal progenitor compartment of different tissues and they represent a reservoir of earlier progenitors compared to standard CFU-f.
Despite major advances in medical, catheter-based or surgical treatment, cardiovascular diseases such as peripheral artery disease and coronary artery disease still cause significant morbidity and mortality. Furthermore, many patients do not qualify for catheter-based treatment or bypass surgery because of advanced disease or surgical risk. There is therefore an urgent need for novel treatment strategies. Therapeutic angiogenesis aims to restore blood flow to ischaemic tissue by stimulating the growth of new blood vessels through the local delivery of angiogenic factors, and may thus be an attractive treatment alternative for these patients. Angiogenesis is a complex process and the growth of normal, stable and functional vasculature depends on the coordinated interplay of different cell types and growth factors. Vascular endothelial growth factor-A (VEGF) is the fundamental regulator of vascular growth and the key target of therapeutic angiogenesis approaches. However, first-generation clinical trials of VEGF gene therapy have been disappointing, and a clear clinical benefit has yet to be established. In particular, VEGF delivery (a) appears to have a very limited therapeutic window in vivo: low doses are safe but mostly inefficient, whereas higher doses become rapidly unsafe; and (b) requires a sustained expression in vivo of at least about four weeks to achieve stable vessels that persist after cessation of the angiogenic stimulus. Here we will review the current understanding of how VEGF induces the growth of normal or pathological blood vessels, what limitations for the controlled induction of safe and efficient angiogenesis are intrinsically linked to the biological properties of VEGF, and how this knowledge can guide the design of more effective strategies for therapeutic angiogenesis.
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.
Vascular endothelial growth factor-A (VEGF) physiologically regulates both angiogenesis and osteogenesis, but its application in bone tissue engineering led to contradictory outcomes. A poorly understood aspect is how VEGF dose impacts the coordination between these two processes. Taking advantage of a unique and highly tunable platform, here we dissected the effects of VEGF dose over a 1,000-fold range in the context of tissue-engineered osteogenic grafts. We found that osteo-angiogenic coupling is exquisitely dependent on VEGF dose and that only a tightly defined dose range could stimulate both vascular invasion and osteogenic commitment of progenitors, with significant improvement in bone formation. Further, VEGF dose regulated Notch1 activation and the induction of a specific pro-osteogenic endothelial phenotype, independently of the promotion of vascular invasion. Therefore, in a therapeutic perspective, fine-tuning of VEGF dose in the signaling microenvironment is key to ensure physiological coupling of accelerated vascular invasion and improved bone formation.
Bone regeneration is a complex process requiring highly orchestrated interactions between different cells and signals to form new mineralized tissue. Blood vessels serve as a structural template around which bone development takes place and also bring together in the osteogenic microenvironment the key elements for bone homeostasis, including minerals, growth-factors and osteogenic progenitor cells. Vascular Endothelial Growth Factor (VEGF) is the master regulator of vascular growth and it is required for effective coupling of angiogenesis and osteogenesis during both skeletal development and postnatal bone repair. Here we will review the current state of knowledge on the molecular cross-talk between angiogenesis and osteogenesis. In particular, we will focus on the role of VEGF in coupling these two processes and how VEGF dose can control the outcome, addressing in particular: (1) the direct influence of VEGF on osteogenic differentiation of mesenchymal progenitors; (2) the angiocrine functions of endothelium to regulate osteoprogenitors; (3) the role of immune cells, e.g. myeloid cells and osteoclast precursors, recruited by VEGF to the osteogenic microenvironment. Finally, we will discuss emerging strategies, based on the current biological understanding, to ensure rapid vascularization and efficient bone formation in regenerative medicine.
Abstract Stromal vascular fraction (SVF) cells of human adipose tissue have the capacity to generate osteogenic grafts with intrinsic vasculogenic properties. However, adipose-derived stromal/stem cells (ASC), even after minimal monolayer expansion, display poor osteogenic capacity in vivo . We investigated whether ASC bone-forming capacity may be maintained by culture within a self-produced extracellular matrix (ECM) that recapitulates the native environment. SVF cells expanded without passaging up to 28 days (Unpass-ASC) deposited a fibronectin-rich extracellular matrix and displayed greater clonogenicity and differentiation potential in vitro compared to ASC expanded only for 6 days (P0-ASC) or for 28 days with regular passaging (Pass-ASC). When implanted subcutaneously, Unpass-ASC produced bone tissue similarly to SVF cells, in contrast to P0- and Pass-ASC, which mainly formed fibrous tissue. Interestingly, clonogenic progenitors from native SVF and Unpass-ASC expressed low levels of the fibronectin receptor α 5 integrin (CD49e), which was instead upregulated in P0- and Pass-ASC. Mechanistically, induced activation of α 5 β 1 integrin in Unpass-ASC led to a significant loss of bone formation in vivo . This study shows that ECM and regulation of α 5 β 1 -integrin signaling preserve ASC progenitor properties, including bone tissue-forming capacity, during in vitro expansion.
Aim: This study wants to investigate whether the administration of stromal stem cells (SSC) in a platelet-rich plasma (PRP) scaffold could promote angiogenesis which resulted in a better allograft integration. Methods: surgery: A monolateral resection of 3cm segment of the metatarsus, was perfomed in 10 adult cross-breed sheep (3–4 years old), weighting 60–70 kg. Isolation and ex-vivo expansion of SSC: nucleated cells were isolated with density gradient and expanded ex-vivo with alpha-MEM containing 20% FCS. Radiographic and histomorphometric analysis: Radiographs were made after surgery and after 1, 2 and 4 months. Histomorphometric studies were carried out to study the defect and the new bone formation at the implant site Results: Union had occurred in all the 5 animals of the SSC group after 4 months as observed radiographically and morphologically, while in the control group the osteotomy line was still visible. Histomorphometric analysis demonstrated a higher % of new-bone formation in both the host (%section quadrant) and the grafted bone in SSC animals. Conclusions: Results presented suggest that SSC in PRP-based scaffold have improved allograft integration. In conclusion the application of this surgical approach may result in an increased and accelerated bone graft integration, reducing the time required for bone healing and increasing the chances of a successful bone implant.
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