Nick van Gastel

Mechanisms of ectopic bone formation by human osteoprogenitor cells on CaP biomaterial carriers

Stem cell-based strategies for bone regeneration, which use calcium phosphate (CaP)-based biomaterials in combination with developmentally relevant progenitor populations, have significant potential for clinical repair of skeletal defects. However, the exact mechanism of action and the stem cell-host-material interactions are still poorly understood. We studied if pre-conditioning of human periosteum-derived cells (hPDCs) in vitro could enhance, in combination with a CaP-based biomaterial carrier, ectopic bone formation in vivo. By culturing hPDCs in a biomimetic calcium (Ca(2+)) and phosphate (P(i)) enriched culture conditions, we observed an enhanced cell proliferation, decreased expression of mesenchymal stem cell (MSC) markers and upregulation of osteogenic genes including osterix, Runx2, osteocalcin, osteopontin, and BMP-2. However, the in vitro pre-conditioning protocols were non-predictive for in vivo ectopic bone formation. Surprisingly, culturing in the presence of Ca(2+) and P(i) supplements resulted in partial or complete abrogation of in vivo ectopic bone formation. Through histological, immunohistochemical and microfocus X-ray computed tomography (μCT) analysis of the explants, we found that in situ proliferation, collagen matrix deposition and the mediation of osteoclastic activity by hPDCs are associated to their ectopic bone forming capacity. These data were validated by the multivariate analysis and partial least square regression modelling confirming the non-predictability of in vitro parameters on in vivo ectopic bone formation. Our series of experiments provided further insights on the stem cell-host-material interactions that govern in vivo ectopic bone induction driven by hPDCs on CaP-based biomaterials.

Engineering Vascularized Bone: Osteogenic and Proangiogenic Potential of Murine Periosteal Cells

One of the key challenges in bone tissue engineering is the timely formation of blood vessels that promote the survival of the implanted cells in the construct. Fracture healing largely depends on the presence of an intact periosteum but it is still unknown whether periosteum‐derived cells (PDC) are critical for bone repair only by promoting bone formation or also by inducing neovascularization. We first established a protocol to specifically isolate murine PDC (mPDC) from long bones of adult mice. Mesenchymal stem cells were abundantly present in this cell population as more than 50% of the mPDC expressed mesenchymal markers (CD73, CD90, CD105, and stem cell antigen‐1) and the cells exhibited trilineage differentiation potential (chondrogenic, osteogenic, and adipogenic). When transplanted on a collagen‐calcium phosphate scaffold in vivo, mPDC attracted numerous blood vessels and formed mature bone which comprises a hematopoiesis‐supportive stroma. We explored the proangiogenic properties of mPDC using in vitro culture systems and showed that mPDC promote the survival and proliferation of endothelial cells through the production of vascular endothelial growth factor. Coimplantation with endothelial cells demonstrated that mPDC can enhance vasculogenesis by adapting a pericyte‐like phenotype, in addition to their ability to stimulate blood vessel ingrowth from the host. In conclusion, these findings demonstrate that periosteal cells contribute to fracture repair, not only through their strong osteogenic potential but also through their proangiogenic features and thus provide an ideal cell source for bone regeneration therapies. STEM CELLS2012;30:2412–2422

Oxygen as a critical determinant of bone fracture healing - a multiscale model

A timely restoration of the ruptured blood vessel network in order to deliver oxygen and nutrients to the fracture zone is crucial for successful bone healing. Indeed, oxygen plays a key role in the aerobic metabolism of cells, in the activity of a myriad of enzymes as well as in the regulation of several (angiogenic) genes. In this paper, a previously developed model of bone fracture healing is further improved with a detailed description of the influence of oxygen on various cellular processes that occur during bone fracture healing. Oxygen ranges of the cell-specific oxygen-dependent processes were established based on the state-of-the art experimental knowledge through a rigorous literature study. The newly developed oxygen model is compared with previously published experimental and in silico results. An extensive sensitivity analysis was also performed on the newly introduced oxygen thresholds, indicating the robustness of the oxygen model. Finally, the oxygen model was applied to the challenging clinical case of a critical sized defect (3mm) where it predicted the formation of a fracture non-union. Further model analyses showed that the harsh hypoxic conditions in the central region of the callus resulted in cell death and disrupted bone healing thereby indicating the importance of a timely vascularization for the successful healing of a large bone defect. In conclusion, this work demonstrates that the oxygen model is a powerful tool to further unravel the complex spatiotemporal interplay of oxygen delivery, diffusion and consumption with the several healing steps, each occurring at distinct, optimal oxygen tensions during the bone repair process.

Expansion of Murine Periosteal Progenitor Cells with Fibroblast Growth Factor 2 Reveals an Intrinsic Endochondral Ossification Program Mediated by Bone Morphogenetic Protein 2

The preservation of the bone‐forming potential of skeletal progenitor cells during their ex vivo expansion remains one of the major challenges for cell‐based bone regeneration strategies. We report that expansion of murine periosteal cells in the presence of FGF2, a signal present during the early stages of fracture healing, is necessary and sufficient to maintain their ability to organize in vivo into a cartilage template which gives rise to mature bone. Implantation of FGF2‐primed cells in a large bone defect in mice resulted in complete healing, demonstrating the feasibility of using this approach for bone tissue engineering purposes. Mechanistically, the enhanced endochondral ossification potential of FGF2‐expanded periosteal cells is predominantly driven by an increased production of BMP2 and is additionally linked to an improved preservation of skeletal progenitor cells in the cultures. This characteristic is unique for periosteal cells, as FGF2‐primed bone marrow stromal cells formed significantly less bone and progressed exclusively through the intramembranous pathway, revealing essential differences between both cell pools. Taken together, our findings provide insight in the molecular regulation of fracture repair by identifying a unique interaction between periosteal cells and FGF2. These insights may promote the development of cell‐based therapeutic strategies for bone regeneration which are independent of the in vivo use of growth factors, thus limiting undesired side effects. Stem Cells 2014;32:2407–2418

HIF-1α Promotes Glutamine-Mediated Redox Homeostasis and Glycogen-Dependent Bioenergetics to Support Postimplantation Bone Cell Survival.

Cell-based therapy is a promising strategy in regenerative medicine, but the poor survival rate of the implanted cells remains a major challenge and limits clinical translation. We preconditioned periosteal cells to the hypoxic and ischemic environment of the bone defect site by deleting prolyl hydroxylase domain-containing protein 2 (PHD2), resulting in hypoxia-inducible factor 1 alpha (HIF-1α) stabilization. This strategy increased postimplantation cell survival and improved bone regeneration. The enhanced cell viability was angiogenesis independent but relied on combined changes in glutamine and glycogen metabolism. HIF-1α stabilization stimulated glutaminase-mediated glutathione synthesis, maintaining redox homeostasis at baseline and during oxidative or nutrient stress. Simultaneously, HIF-1α signaling increased glycogen storage, preventing an energy deficit during nutrient or oxygen deprivation. Pharmacological inhibition of PHD2 recapitulated the adaptations in glutamine and glycogen metabolism and, consequently, the beneficial effects on cell survival. Thus, targeting cellular metabolism is an appealing strategy for bone regeneration and cell-based therapy in general.

Highly proliferative primitive fetal liver hematopoietic stem cells are fueled by oxidative metabolic pathways

Hematopoietic stem cells (HSCs) in the fetal liver (FL) unlike adult bone marrow (BM) proliferate extensively, posing different metabolic demands. However, metabolic pathways responsible for the production of energy and cellular building blocks in FL HSCs have not been described. Here, we report that FL HSCs use oxygen dependent energy generating pathways significantly more than their BM counterparts. RNA-Seq analysis of E14.5 FL versus BM derived HSCs identified increased expression levels of genes involved in oxidative phosphorylation (OxPhos) and the citric acid cycle (TCA). We demonstrated that FL HSCs contain more mitochondria than BM HSCs, which resulted in increased levels of oxygen consumption and reactive oxygen species (ROS) production. Higher levels of DNA repair and antioxidant pathway gene expression may prevent ROS-mediated (geno)toxicity in FL HSCs. Thus, we here for the first time highlight the underestimated importance of oxygen dependent pathways for generating energy and building blocks in FL HSCs.

Size Does Matter: An Integrative In Vivo-In Silico Approach for the Treatment of Critical Size Bone Defects

Although bone has a unique restorative capacity, i.e., it has the potential to heal scarlessly, the conditions for spontaneous bone healing are not always present, leading to a delayed union or a non-union. In this work, we use an integrative in vivo - in silico approach to investigate the occurrence of non-unions, as well as to design possible treatment strategies thereof. The gap size of the domain geometry of a previously published mathematical model was enlarged in order to study the complex interplay of blood vessel formation, oxygen supply, growth factors and cell proliferation on the final healing outcome in large bone defects. The multiscale oxygen model was not only able to capture the essential aspects of in vivo non-unions, it also assisted in understanding the underlying mechanisms of action, i.e., the delayed vascularization of the central callus region resulted in harsh hypoxic conditions, cell death and finally disrupted bone healing. Inspired by the importance of a timely vascularization, as well as by the limited biological potential of the fracture hematoma, the influence of the host environment on the bone healing process in critical size defects was explored further. Moreover, dependent on the host environment, several treatment strategies were designed and tested for effectiveness. A qualitative correspondence between the predicted outcomes of certain treatment strategies and experimental observations was obtained, clearly illustrating the models potential. In conclusion, the results of this study demonstrate that due to the complex non-linear dynamics of blood vessel formation, oxygen supply, growth factor production and cell proliferation and the interactions thereof with the host environment, an integrative in silico-in vivo approach is a crucial tool to further unravel the occurrence and treatments of challenging critical sized bone defects.

The vasculature: a vessel for bone metastasis

Emerging evidence indicates that the interactions between tumor cells and the bone microenvironment have a crucial role in the pathogenesis of bone metastasis and that they can influence tumor cell dissemination, quiescence and tumor growth in the bone. The vasculature is known to be critical for primary tumor growth, and anti-angiogenesis drugs are approved for the treatment of certain tumor types. The role of the vasculature in bone metastasis is less well known, but recent evidence shows that blood vessels in the bone are a key component of the local microenvironment for the tumor cells and contribute to the different consecutive phases of bone metastasis. A better insight in the importance of the vasculature for bone metastasis may help develop novel treatment modalities that either slow down tumor growth or, preferably, prevent or cure bone metastasis.

Neovascularization Potential of Blood Outgrowth Endothelial Cells From Patients With Stable Ischemic Heart Failure Is Preserved

Background Blood outgrowth endothelial cells (BOECs) mediate therapeutic neovascularization in experimental models, but outgrowth characteristics and functionality of BOECs from patients with ischemic cardiomyopathy (ICMP) are unknown. We compared outgrowth efficiency and in vitro and in vivo functionality of BOECs derived from ICMP with BOECs from age‐matched (ACON) and healthy young (CON) controls. Methods and Results We isolated 3.6±0.6 BOEC colonies/100×106 mononuclear cells (MNCs) from 60‐mL blood samples of ICMP patients (n=45; age: 66±1 years; LVEF: 31±2%) versus 3.5±0.9 colonies/100×106 MNCs in ACON (n=32; age: 60±1 years) and 2.6±0.4 colonies/100×106 MNCs in CON (n=55; age: 34±1 years), P=0.29. Endothelial lineage (VEGFR2+/CD31+/CD146+) and progenitor (CD34+/CD133−) marker expression was comparable in ICMP and CON. Growth kinetics were similar between groups (P=0.38) and not affected by left ventricular systolic dysfunction, maladaptive remodeling, or presence of cardiovascular risk factors in ICMP patients. In vitro neovascularization potential, assessed by network remodeling on Matrigel and three‐dimensional spheroid sprouting, did not differ in ICMP from (A)CON. Secretome analysis showed a marked proangiogenic profile, with highest release of angiopoietin‐2 (1.4±0.3×105 pg/106 ICMP‐BOECs) and placental growth factor (5.8±1.5×103 pg/106 ICMP BOECs), independent of age or ischemic disease. Senescence‐associated β‐galactosidase staining showed comparable senescence in BOECs from ICMP (5.8±2.1%; n=17), ACON (3.9±1.1%; n=7), and CON (9.0±2.8%; n=13), P=0.19. High‐resolution microcomputed tomography analysis in the ischemic hindlimb of nude mice confirmed increased arteriogenesis in the thigh region after intramuscular injections of BOECs from ICMP (P=0.025; n=8) and CON (P=0.048; n=5) over vehicle control (n=8), both to a similar extent (P=0.831). Conclusions BOECs can be successfully culture‐expanded from patients with ICMP. In contrast to impaired functionality of ICMP‐derived bone marrow MNCs, BOECs retain a robust proangiogenic profile, both in vitro and in vivo, with therapeutic potential for targeting ischemic disease.

Endothelial Msx1 transduces hemodynamic changes into an arteriogenic remodeling response

During peripheral arterial disease, MSX1 acts downstream of BMP–SMAD signaling to transduce the arterial shear stimulus into an arteriogenic remodeling response. MSX1 activates collateral endothelium into a proinflammatory state through ICAM1/VCAM1 up-regulation, resulting in increased leukocyte infiltration and collateral remodeling.