No apparent role for the Wari insulator in transcriptional regulation of the endogenous white gene of Drosophila melanogaster.

Chromatin insulators have been proposed to play an important role in chromosome organization and local regulatory interactions. In Drosophila , one of these insulators is known as Wari. It is located immediately downstream of the 3' end of the white transcription unit. Wari has been proposed to interact with the white promoter region, thereby facilitating recycling of the RNA polymerase machinery. We have tested this model by deleting the Wari insulator at the endogenous white locus and could not detect a significant effect on eye pigmentation.

T-cadherin is a novel regulator of pericyte function during angiogenesis.

Pericytes are mural cells that play an important role in regulation of angiogenesis and endothelial function. Cadherins are a superfamily of adhesion molecules mediating Ca2+-dependent homophilic cell-cell interactions that control morphogenesis and tissue remodeling. To date, classical N-cadherin is the only cadherin described on pericytes. Here, we demonstrate that pericytes also express T-cadherin (H-cadherin, CDH13), an atypical glycosyl-phosphatidylinositol (GPI)-anchored member of the superfamily that has previously been implicated in regulation of neurite guidance, endothelial angiogenic behavior, and smooth muscle cell differentiation and progression of cardiovascular disease. The aim of the study was to investigate T-cadherin function in pericytes. Expression of T-cadherin in pericytes from different tissues was performed by immunofluorescence analysis. Using lentivirus-mediated gain-of-function and loss-of-function in cultured human pericytes, we demonstrate that T-cadherin regulates pericyte proliferation, migration, invasion, and interactions with endothelial cells during angiogenesis in vitro and in vivo. T-cadherin effects are associated with the reorganization of the cytoskeleton, modulation of cyclin D1, α-smooth muscle actin (αSMA), integrin ß3, metalloprotease MMP1, and collagen expression levels, and involve Akt/GSK3ß and ROCK intracellular signaling pathways. We also report the development of a novel multiwell 3-D microchannel slide for easy analysis of sprouting angiogenesis from a bioengineered microvessel in vitro. In conclusion, our data identify T-cadherin as a novel regulator of pericyte function and support that it is required for pericyte proliferation and invasion during active phase of angiogenesis, while T-cadherin loss shifts pericytes toward the myofibroblast state rendering them unable to control endothelial angiogenic behavior.

Mini- and macro-scale direct perfusion bioreactors with optimized flow for engineering 3D tissues.

Bioreactors enabling direct perfusion of cell suspensions or culture media through the pores of 3D scaffolds have long been used in tissue engineering to improve cell seeding efficiency as well as uniformity of cell distribution and tissue development. A macro-scale U-shaped bioreactor for cell culture under perfusion (U-CUP) has been previously developed. In that system, the geometry of the perfusion chamber results in rather uniform flow through most of the scaffold volume, but not in the peripheral regions. Here, the design of the perfusion chamber has been optimized to provide a more homogenous perfusion flow through the scaffold. Then, the design of this macro-scale flow-optimized perfusion bioreactor (macro-Flopper) has been miniaturized to create a mini-scale device (mini-Flopper) compatible with medium-throughput assays. Computational fluid dynamic (CFD) modeling of the new chamber design, including a porous scaffold structure, revealed that Flopper bioreactors provide highly homogenous flow speed, pressure, and shear stress. Finally, a proof-of-principle of the functionality of the Flopper systems by engineering endothelialized stromal tissues using human adipose tissue-derived stromal vascular fraction (SVF) cells has been offered. Preliminary evidence showing that flow optimization improves cell maintenance in the engineered tissues will have to be confirmed in future studies. In summary, two bioreactor models with optimized perfusion flow and complementary sizes have been proposed that might be exploited to engineer homogenous tissues and, in the case of the mini-Flopper, for drug testing assays with a limited amount of biological material.

A composite, off-the-shelf osteoinductive material for large, vascularized bone flap prefabrication.

We previously described an immortalized, genetically-engineered human Mesenchymal stromal cell line to generate BMP2-enriched Chondrogenic Matrices (MB-CM), which after devitalization and storage could efficiently induce ectopic bone tissue by endochondral ossification. In order to increase the efficiency of MB-CM utilization towards engineering scaled-up bone structures, here we hypothesized that MB-CM can retain osteoinductive properties when combined with an osteoconductive material. We first tested different volumetric ratios of MB-CM:SmartBone® (as clinically used, osteoconductive reference material) and assessed the bone formation capacity of the resulting composites following ectopic mouse implantation. After 8 weeks, as little as 25% of MB-CM was sufficient to induce bone formation and fusion across SmartBone® granules, generating large interconnected bony structures. The same composite percentage was then further assessed in a scaled-up model, namely within an axially-vascularized, confined, ectopically prefabricated flap (0.8 cm3) in rats. The material efficiently induced the formation of new bone (31% of the cross-sectional area after 8 weeks), including bone marrow and vascular elements, throughout the flap volume. Our findings outline a strategy for efficient use of MB-CM as part of a composite material, thereby reducing the amount required to fill large spaces and enabling utilization in critically sized grafts, to address challenging clinical scenarios in bone reconstruction. STATEMENT OF SIGNIFICANCE: Most bone repair strategies rely either on osteconductive properties of ceramics and devitalized bone, or osteoinductive properties of growth factors and extracellular matrices (ECM). Here we designed a composite material made of a clinically accepted osteoconductive material and an off-the-shelf tissue engineered human cartilage ECM with strong osteoinductive properties. We showed that low amount of osteoinductive ECM potentiated host cells recruitment to form large vascularized bone structures in two different animal models, one being a challenging prefabricated bone-flap model targeting challenging clinical bone repair. Overall, this study highlights the use of a promising human off-the-shelf material for accelerated healing towards clinical applications.

Robust coupling of angiogenesis and osteogenesis by VEGF-decorated matrices for bone regeneration.

Rapid vascularization of clinical-size bone grafts is an unsolved challenge in regenerative medicine. Vascular endothelial growth factor-A (VEGF) is the master regulator of angiogenesis. Its over-expression by genetically modified human osteoprogenitors has been previously evaluated to drive vascularization in osteogenic grafts, but has been observed to cause paradoxical bone loss through excessive osteoclast recruitment. However, during bone development angiogenesis and osteogenesis are physiologically coupled by VEGF expression. Here we investigated whether the mode of VEGF delivery may be a key to recapitulate its physiological function. VEGF activity requires binding to the extracellular matrix, and heterogeneous levels of expression lead to localized microenvironments of excessive dose. Therefore we hypothesized that a homogeneous distribution of matrix-associated factor in the microenvironment may enable efficient coupling of angiogenesis and bone formation. This was achieved by decorating fibrin matrices with a cross-linkable engineered version of VEGF (TG-VEGF) that is released only by enzymatic cleavage by invading cells. In ectopic grafts, both TG-VEGF and VEGF-expressing progenitors similarly improved vascularization within the first week, but efficient bone formation was possible only in the factor-decorated matrices, whereas heterogenous, cell-based VEGF expression caused significant bone loss. In critical-size orthotopic calvaria defects, TG-VEGF effectively improved early vascular invasion, osteoprogenitor survival and differentiation, as well as bone repair compared to both controls and VEGF-expressing progenitors. In conclusion, homogenous distribution of matrix-associated VEGF protein preserves the physiological coupling of angiogenesis and osteogenesis, providing an attractive and clinically applicable strategy to engineer vascularized bone. STATEMENT OF SIGNIFICANCE: The therapeutic regeneration of vascularized bone is an unsolved challenge in regenerative medicine. Stimulation of blood vessel growth by over-expression of VEGF has been associated with paradoxical bone loss, whereas angiogenesis and osteogenesis are physiologically coupled by VEGF during development. Here we found that controlling the distribution of VEGF dose in an osteogenic graft is key to recapitulate its physiological function. In fact, homogeneous decoration of fibrin matrices with engineered VEGF could improve both vascularization and bone formation in orthotopic critical-size defects, dispensing with the need for combined osteogenic factor delivery. VEGF-decorated fibrin matrices provide a readily translatable platform for engineering a controlled microenvironment for bone regeneration.

Computed tomography osteoabsorptiometry for imaging of degenerative disc disease.

BACKGROUND: Lower back pain is a common condition with significant morbidity and economic impact. The pathophysiology is poorly understood but is in part attributable to degenerative disc disease (DDD). The healthy intervertebral disc ensures spine functionality by transferring the perceived load to the caudally adjacent vertebrae. The exposure to recurring mechanical load is mirrored in the mineralization pattern of the subchondral bone plate (SBP), where increased bone density is a sign of repetitive localized high stress. Computed tomography -osteoabsorptiometry (CT-OAM) is a technique based on conventional CT scans that displays the mineral density distribution in the SBP as a surface-color map. The objective of this study was to measure and analyze the SBP mineral density patterns of healthy lumbar intervertebral disc (IVDs) and those suffering DDD using CT-OAM densitograms. These findings should provide in vitro insight into the long-term morphological properties of the IVD and how these differ in the state of disc degeneration. METHODS: The CT-data sets of spines from 17 healthy individuals and 18 patients displaying DDD in the lumbar spine were acquired. Individual vertebrae of both cohorts were 3D reconstructed, processed using image analysis software, and compared to one another. Maximum intensity projection of the subchondral mineralization provided surface densitograms of the SBP. The relative calcium concentration, the local maxima of mineralization, and a mean surface projection of level-defined SBPs were calculated from the densitogram and statistically compared. RESULTS: The inferior SBP, adjacent to degenerating disc, display an 18-41 % higher relative calcium concentration than their healthy counterparts. In the opposing superior SBPs the relative calcium content is significantly increased. Whereas it is reasonably consistent for L1-L3 (L1: 132 %, L2: 127 %, L3: 120 %), the increase grows in caudal direction (L4: 131 %, L5: 148 %, S1: 152 %). Furthermore, a change in the areal distribution of excessive mineralization can be differentiated between healthy and diseased motion segments. CONCLUSIONS: The acquired data provide in vitro proof of the mechanical and anatomical properties of the SBP in relation to the state of disc degeneration. In conjunction with the diagnostic use of CT-osteoabsorptiometry, our data provide a basis for a non-invasive and sensitive technique that correlates with disc functionality. This could be promising in various cases, from early identification of early stages of DDD, tracking disease progression, and assessing the repercussions of surgical procedures or experimental therapies.

Repair of a Rat Mandibular Bone Defect by Hypertrophic Cartilage Grafts Engineered From Human Fractionated Adipose Tissue.

Background: Devitalized bone matrix (DBM) is currently the gold standard alternative to autologous bone grafting in maxillofacial surgery. However, it fully relies on its osteoconductive properties and therefore requires defects with healthy bone surrounding. Fractionated human adipose tissue, when differentiated into hypertrophic cartilage in vitro, was proven reproducibly osteogenic in vivo, by recapitulating endochondral ossification (ECO). Both types of bone substitutes were thus compared in an orthotopic, preclinical mandibular defect model in rat. Methods: Human adipose tissue samples were collected and cultured in vitro to generate disks of hypertrophic cartilage. After hypertrophic induction, eight samples from two donors were implanted into a mandible defect in rats, in parallel to Bio-Oss® DBM granules. After 12 weeks, the mandible samples were harvested and evaluated by Micro-CT and histology. Results: Micro-CT demonstrated reproducible ECO and complete restoration of the mandibular geometry with adipose-based disks, with continuous bone inside and around the defect, part of which was of human (donor) origin. In the Bio-Oss® group, instead, osteoconduction from the border of the defect was observed but no direct connection of the granules with the surrounding bone was evidenced. Adipose-based grafts generated significantly higher mineralized tissue volume (0.57 ± 0.10 vs. 0.38 ± 0.07, n = 4, p = 0.03) and newly formed bone (18.9 ± 3.4% of surface area with bone tissue vs. 3 ± 0.7%, p < 0.01) than Bio-Oss®. Conclusion: Our results provide a proof-of-concept that adipose-based hypertrophic cartilage grafts outperform clinical standard biomaterials in maxillofacial surgery.

Engineering of fully humanized and vascularized 3D bone marrow niches sustaining undifferentiated human cord blood hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cells (HSPCs) are frequently located around the bone marrow (BM) vasculature. These so-called perivascular niches regulate HSC function both in health and disease, but they have been poorly studied in humans due to the scarcity of models integrating complete human vascular structures. Herein, we propose the stromal vascular fraction (SVF) derived from human adipose tissue as a cell source to vascularize 3D osteoblastic BM niches engineered in perfusion bioreactors. We show that SVF cells form self-assembled capillary structures, composed by endothelial and perivascular cells, that add to the osteogenic matrix secreted by BM mesenchymal stromal cells in these engineered niches. In comparison to avascular osteoblastic niches, vascularized BM niches better maintain immunophenotypically-defined cord blood (CB) HSCs without affecting cell proliferation. In contrast, HSPCs cultured in vascularized BM niches showed increased CFU-granulocyte-erythrocyte-monocyte-megakaryocyte (CFU-GEMM) numbers. The vascularization also contributed to better preserve osteogenic gene expression in the niche, demonstrating that niche vascularization has an influence on both hematopoietic and stromal compartments. In summary, we have engineered a fully humanized and vascularized 3D BM tissue to model native human endosteal perivascular niches and revealed functional implications of this vascularization in sustaining undifferentiated CB HSPCs. This system provides a unique modular platform to explore hemato-vascular interactions in human healthy/pathological hematopoiesis.

Culturing patient-derived malignant hematopoietic stem cells in engineered and fully humanized 3D niches.

Human malignant hematopoietic stem and progenitor cells (HSPCs) reside in bone marrow (BM) niches, which remain challenging to explore due to limited in vivo accessibility and constraints with humanized animal models. Several in vitro systems have been established to culture patient-derived HSPCs in specific microenvironments, but they do not fully recapitulate the complex features of native bone marrow. Our group previously reported that human osteoblastic BM niches (O-N), engineered by culturing mesenchymal stromal cells within three-dimensional (3D) porous scaffolds under perfusion flow in a bioreactor system, are capable of maintaining, expanding, and functionally regulating healthy human cord blood-derived HSPCs. Here, we first demonstrate that this 3D O-N can sustain malignant CD34+ cells from acute myeloid leukemia (AML) and myeloproliferative neoplasm patients for up to 3 wk. Human malignant cells distributed in the bioreactor system mimicking the spatial distribution found in native BM tissue, where most HSPCs remain linked to the niches and mature cells are released to the circulation. Using human adipose tissue-derived stromal vascular fraction cells, we then generated a stromal-vascular niche and demonstrated that O-N and stromal-vascular niche differentially regulate leukemic UCSD-AML1 cell expansion, immunophenotype, and response to chemotherapy. The developed system offers a unique platform to investigate human leukemogenesis and response to drugs in customized environments, mimicking defined features of native hematopoietic niches and compatible with the establishment of personalized settings.

Natural Polymeric Scaffolds in Bone Regeneration.

Despite considerable advances in microsurgical techniques over the past decades, bone tissue remains a challenging arena to obtain a satisfying functional and structural restoration after damage. Through the production of substituting materials mimicking the physical and biological properties of the healthy tissue, tissue engineering strategies address an urgent clinical need for therapeutic alternatives to bone autografts. By virtue of their structural versatility, polymers have a predominant role in generating the biodegradable matrices that hold the cells in situ to sustain the growth of new tissue until integration into the transplantation area (i.e., scaffolds). As compared to synthetic ones, polymers of natural origin generally present superior biocompatibility and bioactivity. Their assembly and further engineering give rise to a wide plethora of advanced supporting materials, accounting for systems based on hydrogels or scaffolds with either fibrous or porous architecture. The present review offers an overview of the various types of natural polymers currently adopted in bone tissue engineering, describing their manufacturing techniques and procedures of functionalization with active biomolecules, and listing the advantages and disadvantages in their respective use in order to critically compare their actual applicability potential. Their combination to other classes of materials (such as micro and nanomaterials) and other innovative strategies to reproduce physiological bone microenvironments in a more faithful way are also illustrated. The regeneration outcomes achieved in vitro and in vivo when the scaffolds are enriched with different cell types, as well as the preliminary clinical applications are presented, before the prospects in this research field are finally discussed. The collection of studies herein considered confirms that advances in natural polymer research will be determinant in designing translatable materials for efficient tissue regeneration with forthcoming impact expected in the treatment of bone defects.

Use of nanoparticles in skeletal tissue regeneration and engineering.

Bone and osteochondral defects represent one of the major causes of disabilities in the world. Derived from traumas and degenerative pathologies, these lesions cause severe pain, joint deformity, and loss of joint motion. The standard treatments in clinical practice present several limitations. By producing functional substitutes for damaged tissues, tissue engineering has emerged as an alternative in the treatment of defects in the skeletal system. Despite promising preliminary clinical outcomes, several limitations remain. Nanotechnologies could offer new solutions to overcome those limitations, generating materials more closely mimicking the structures present in naturally occurring systems. Nanostructures comparable in size to those appearing in natural bone and cartilage have thus become relevant in skeletal tissue engineering. In particular, nanoparticles allow for a unique combination of approaches (e.g. cell labelling, scaffold modification or drug and gene delivery) inside single integrated systems for optimized tissue regeneration. In the present review, the main types of nanoparticles and the current strategies for their application to skeletal tissue engineering are described. The collection of studies herein considered confirms that advanced nanomaterials will be determinant in the design of regenerative therapeutic protocols for skeletal lesions in the future.

Magnetic nanocomposite hydrogels and static magnetic field stimulate the osteoblastic and vasculogenic profile of adipose-derived cells.

Exposure of cells to externally applied magnetic fields or to scaffolding materials with intrinsic magnetic properties (magnetic actuation) can regulate several biological responses. Here, we generated novel magnetized nanocomposite hydrogels by incorporation of magnetic nanoparticles (MNPs) into polyethylene glycol (PEG)-based hydrogels containing cells from the stromal vascular fraction (SVF) of human adipose tissue. We then investigated the effects of an external Static Magnetic Field (SMF) on the stimulation of osteoblastic and vasculogenic properties of the constructs, with MNPs or SMF alone used as controls. MNPs migrated freely through and out of the material following the magnetic gradient. Magnetically actuated cells displayed increased metabolic activity. After 1 week, the enzymatic activity of Alkaline Phosphatase (ALP), the expression of osteogenic markers (Runx2, Collagen I, Osterix), and the mineralized matrix deposition were all augmented as compared to controls. With magnetic actuation, strong activation of endothelial, pericytic and perivascular genes paralleled increased levels of VEGF and an enrichment in the CD31+ cells population. The stimulation of signaling pathways involved in the mechanotransduction, like MAPK8 or Erk, at gene and protein levels suggested an effect mediated through the mechanical stimulation. Upon subcutaneous implantation in mice, magnetically actuated constructs exhibited denser, more mineralized and faster vascularized tissues, as revealed by histological and micro-computed tomographic analyses. The present study suggests that magnetic actuation can stimulate both the osteoblastic and vasculogenic potentials of engineered bone tissue grafts, likely at least partially by mechanically stimulating the function of progenitor cells.

Delivery of cellular factors to regulate bone healing.

Bone tissue has a strong intrinsic regenerative capacity, thanks to a delicate and complex interplay of cellular and molecular processes, which tightly involve the immune system. Pathological settings of anatomical, biomechanical or inflammatory nature may lead to impaired bone healing. Innovative strategies to enhance bone repair, including the delivery of osteoprogenitor cells or of potent cytokines/morphogens, indicate the potential of 'orthobiologics', but are not fully satisfactory. Here, we review different approaches based on the delivery of regenerative cues produced by cells but in cell-free, possibly off-the-shelf configurations. Such strategies exploit the paracrine effect of the secretome of mesenchymal stem/stromal cells, presented in soluble form, shuttled through extracellular vesicles, or embedded within the network of extracellular matrix molecules. In addition to osteoinductive molecules, attention is given to factors targeting the resident immune cells, to reshape inflammatory and immunity processes from scarring to regenerative patterns.