Richard P. Visconti

Towards organ printing: engineering an intra-organ branched vascular tree

Importance of the field: Effective vascularization of thick three-dimensional engineered tissue constructs is a problem in tissue engineering. As in native organs, a tissue-engineered intra-organ vascular tree must be comprised of a network of hierarchically branched vascular segments. Despite this requirement, current tissue-engineering efforts are still focused predominantly on engineering either large-diameter macrovessels or microvascular networks. Areas covered in this review: We present the emerging concept of organ printing or robotic additive biofabrication of an intra-organ branched vascular tree, based on the ability of vascular tissue spheroids to undergo self-assembly. What the reader will gain: The feasibility and challenges of this robotic biofabrication approach to intra-organ vascularization for tissue engineering based on organ-printing technology using self-assembling vascular tissue spheroids including clinically relevantly vascular cell sources are analyzed. Take home message: It is not possible to engineer 3D thick tissue or organ constructs without effective vascularization. An effective intra-organ vascular system cannot be built by the simple connection of large-diameter vessels and microvessels. Successful engineering of functional human organs suitable for surgical implantation will require concomitant engineering of a ‘built in’ intra-organ branched vascular system. Organ printing enables biofabrication of human organ constructs with a ‘built in’ intra-organ branched vascular tree.

Transplanted Human Cord Blood Cells Give Rise to Hepatocytes in Engrafted Mice

Abstract: Recent studies suggest that rodent hepatocytes may be derived from hematopoietic stem cells. In the current study, the potential hematopoietic origin of hepatocytes was addressed using xenogeneic transplantation of human cord blood cells. CD34+ or CD45+ human cord blood cells were transplanted into “conditioned” newborn NOD/SCID/β2‐microglobulinnull mice. At 4 to 5 months post‐transplantation, livers of the recipient mice were cryosectioned and examined for evidence of human hepatocyte engraftment using RT‐PCR to detect human albumin mRNA, immunohistochemistry to detect human hepatocytic proteins, and fluorescence in situ hybridization (FISH) to detect the presence of human centromeric DNA. Analysis of the bone marrow of transplanted mice revealed that 21.0–45.9% of the cells were human CD45+ cells. FISH analysis of frozen sections of transplanted mouse liver revealed the presence of engrafted cells positive for human centromeric DNA. That engrafted human cells functioned as hepatocytes was indicated by the expression of human albumin mRNA, as judged by RT‐PCR. FISH analysis with human and mouse centromeric DNA probes excluded spontaneous cell fusion as the cause for the generation of human hepatocytes. Human cord blood cells can give rise to hepatocytes in a xenogeneic transplantation model. This model will be useful to further characterize the cord blood progenitors of hepatocytes.

Engineering alginate as bioink for bioprinting.

Recent advances in three-dimensional (3-D) printing offer an excellent opportunity to address critical challenges faced by current tissue engineering approaches. Alginate hydrogels have been used extensively as bioinks for 3-D bioprinting. However, most previous research has focused on native alginates with limited degradation. The application of oxidized alginates with controlled degradation in bioprinting has not been explored. Here, a collection of 30 different alginate hydrogels with varied oxidation percentages and concentrations was prepared to develop a bioink platform that can be applied to a multitude of tissue engineering applications. The authors systematically investigated the effects of two key material properties (i.e. viscosity and density) of alginate solutions on their printabilities to identify a suitable range of material properties of alginates to be applied to bioprinting. Further, four alginate solutions with varied biodegradability were printed with human adipose-derived stem cells (hADSCs) into lattice-structured, cell-laden hydrogels with high accuracy. Notably, these alginate-based bioinks were shown to be capable of modulating proliferation and spreading of hADSCs without affecting the structure integrity of the lattice structures (except the highly degradable one) after 8days in culture. This research lays a foundation for the development of alginate-based bioink for tissue-specific tissue engineering applications.

Scalable robotic biofabrication of tissue spheroids.

Development of methods for scalable biofabrication of uniformly sized tissue spheroids is essential for tissue spheroid-based bioprinting of large size tissue and organ constructs. The most recent scalable technique for tissue spheroid fabrication employs a micromolded recessed template prepared in a non-adhesive hydrogel, wherein the cells loaded into the template self-assemble into tissue spheroids due to gravitational force. In this study, we present an improved version of this technique. A new mold was designed to enable generation of 61 microrecessions in each well of a 96-well plate. The microrecessions were seeded with cells using an EpMotion 5070 automated pipetting machine. After 48 h of incubation, tissue spheroids formed at the bottom of each microrecession. To assess the quality of constructs generated using this technology, 600 tissue spheroids made by this method were compared with 600 spheroids generated by the conventional hanging drop method. These analyses showed that tissue spheroids fabricated by the micromolded method are more uniform in diameter. Thus, use of micromolded recessions in a non-adhesive hydrogel, combined with automated cell seeding, is a reliable method for scalable robotic fabrication of uniform-sized tissue spheroids.

Altered monocyte and fibrocyte phenotype and function in scleroderma interstitial lung disease: reversal by caveolin-1 scaffolding domain peptide

Interstitial lung disease (ILD) is a major cause of morbidity and mortality in scleroderma (systemic sclerosis, or SSc). Fibrocytes are a monocyte-derived cell population implicated in the pathogenesis of fibrosing disorders. Given the recently recognized importance of caveolin-1 in regulating function and signaling in SSc monocytes, in the present study we examined the role of caveolin-1 in the migration and/or trafficking and phenotype of monocytes and fibrocytes in fibrotic lung disease in human patients and an animal model. These studies fill a gap in our understanding of how monocytes and fibrocytes contribute to SSc-ILD pathology. We found that C-X-C chemokine receptor type 4-positive (CXCR4+)/collagen I-positive (ColI+), CD34+/ColI+ and CD45+/ColI+ cells are present in SSc-ILD lungs, but not in control lungs, with CXCR4+ cells being most prevalent. Expression of CXCR4 and its ligand, stromal cell-derived factor 1 (CXCL12), are also highly upregulated in SSc-ILD lung tissue. SSc monocytes, which lack caveolin-1 and therefore overexpress CXCR4, exhibit almost sevenfold increased migration toward CXCL12 compared to control monocytes. Restoration of caveolin-1 function by administering the caveolin scaffolding domain (CSD) peptide reverses this hypermigration. Similarly, transforming growth factor β-treated normal monocytes lose caveolin-1, overexpress CXCR4 and exhibit 15-fold increased monocyte migration that is CSD peptide-sensitive. SSc monocytes exhibit a different phenotype than normal monocytes, expressing high levels of ColI, CD14 and CD34. Because ColI+/CD14+ cells are prevalent in SSc blood, we looked for such cells in lung tissue and confirmed their presence in SSc-ILD lungs but not in normal lungs. Finally, in the bleomycin model of lung fibrosis, we show that CSD peptide diminishes fibrocyte accumulation in the lungs. Our results suggest that low caveolin-1 in SSc monocytes contributes to ILD via effects on cell migration and phenotype and that the hyperaccumulation of fibrocytes in SSc-ILD may result from the altered phenotype and migratory activity of their monocyte precursors.

3D Printing Facilitated Scaffold-free Tissue Unit Fabrication

Tissue spheroids hold great potential in tissue engineering as building blocks to assemble into functional tissues. To date, agarose molds have been extensively used to facilitate fusion process of tissue spheroids. As a molding material, agarose typically requires low temperature plates for gelation and/or heated dispenser units. Here, we proposed and developed an alginate-based, direct 3D mold-printing technology: 3D printing microdroplets of alginate solution into biocompatible, bio-inert alginate hydrogel molds for the fabrication of scaffold-free tissue engineering constructs. Specifically, we developed a 3D printing technology to deposit microdroplets of alginate solution on calcium containing substrates in a layer-by-layer fashion to prepare ring-shaped 3D hydrogel molds. Tissue spheroids composed of 50% endothelial cells and 50% smooth muscle cells were robotically placed into the 3D printed alginate molds using a 3D printer, and were found to rapidly fuse into toroid-shaped tissue units. Histological and immunofluorescence analysis indicated that the cells secreted collagen type I playing a critical role in promoting cell-cell adhesion, tissue formation and maturation.

Recruitment of bone marrow-derived valve interstitial cells is a normal homeostatic process.

Advances in understanding of the maintenance of the cardiac valves during normal cardiac function and response to injury have led to several novel findings, including that there is contribution of extra-cardiac cells to the major cellular population of the valve: the valve interstitial cell (VIC). While suggested to occur in human heart studies, we have been able to experimentally demonstrate, using a mouse model, that cells of bone marrow hematopoietic stem cell origin engraft into the valves and synthesize collagen type I. Based on these initial findings, we sought to further characterize this cell population in terms of its similarity to VICs and begin to elucidate its contribution to valve homeostasis. To accomplish this, chimeric mice whose bone marrow was repopulated with enhanced green fluorescent protein (EGFP) expressing total nucleated bone marrow cells were used to establish a profile of EGFP(+) valve cells in terms of their expression of hematopoietic antigens, progenitor markers, fibroblast- and myofibroblast-related molecules, as well as their distribution within the valves. Using this profile, we show that normal (non-irradiated, non-transplanted) mice have BM-derived cell populations that exhibit identical morphology and phenotype to those observed in transplanted mice. Collectively, our findings establish that the engraftment of bone marrow-derived cells occurs as part of normal valve homeostasis. Further, our efforts demonstrate that the use of myeloablative irradiation, which is commonly employed in studies involving bone marrow transplantation, does not elicit changes in the bone marrow-derived VIC phenotype in recipient mice.

What is regenerative medicine? Emergence of applied stem cell and developmental biology.

Regenerative medicine is an emerging, but still poorly defined, field of biomedicine. The ongoing ‘regenerative medicine revolution’ is based on a series of new exciting breakthrough discoveries in the field of stem cell biology and developmental biology. The main problem of regenerative medicine is not so much stem cell differentiation, isolation and lineage diversity, although these are very important issues, but rather stem cell mobilisation, recruitment and integration into functional tissues. The key issue in enhancing tissue and organ regeneration is how to mobilise circulating stem and progenitor cells and how to provide an appropriate environment (‘niche’) for their tissue and organo-specific recruitment, ‘homing’ and complete functional integration. We need to know more about basic tissue biology, tissue regeneration and the cellular and molecular mechanisms of tissue turnover (both cellular and extracellular components) at different periods of human life and in different diseases. Systematic in silico, in vitro and in vivo research is a foundation for further progress in regenerative medicine. Regenerative medicine is a rapidly advancing field that opens new and exciting opportunities for completely revolutionary therapeutic modalities and technologies. Regenerative medicine is, at its essence, an emergence of applied stem cell and developmental biology.

Recruitment of New Cells into the Postnatal Heart

Abstract:  Establishment of the circulatory system occurs very early in development to support the rapid growth of the embryo. Therefore, the heart is the first functional organ to be formed during both avian and mammalian development. Historically, cardiac development has been considered to occur only during embryogenesis from cell sources located within the primordial structures that generate the myocardium and associated coronary vascular endothelium and smooth muscle and cardiac fibroblasts. Recently, however, contribution to the cardiac structures has been demonstrated to occur during embryonic development from extracardiac sources, like the anterior heart field, raising questions as to whether cardiogenesis may be an ongoing process that extends into adult life. In this brief article, we describe the contribution of circulating adult bone marrow hematopoietic stem cells to the cardiac cell populations and the potential regulation of their differentiation by the extracellular matrix protein, periostin.

Codistribution analysis of elastin and related fibrillar proteins in early vertebrate development

Elastin is an extracellular matrix protein found in adult and neonatal vasculature, lung, skin and connective tissue. It is secreted as tropoelastin, a soluble protein that is cross-linked in the tissue space to form an insoluble elastin matrix. Cross-linked elastin can be found in association with several microfibril-associated proteins including fibrillin-1, fibrillin-2 and fibulin-1 suggesting that these proteins contribute to elastic fiber assembly, structure or function. To date, the earliest reported elastin expression was in the conotruncal region of the developing avian heart at 3.5 days of gestation. Here we report that elastin expression begins at significantly earlier developmental stages. Using a novel immunolabeling method, the deposition of elastin, fibrillin-1 and -2 and fibulin-1 was analyzed in avian embryos at several time points during the first 2 days of development. Elastin was found at the midline associated with axial structures such as the notochord and somites at 23 h of development. Fibrillin-1 and -2 and fibulin-1 were also expressed at the embryonic midline at this stage with fibrillin-1 and fibulin-1 showing a high degree of colocalization with elastin in fibers surrounding midline structures. The expression of these genes was confirmed by conventional immunoblotting and mRNA detection methods. Our results demonstrate that elastin polypeptide deposition occurs much earlier than was previously appreciated. Furthermore, the results suggest that elastin deposition at the early embryonic midline is accompanied by the deposition and organization of a number of extracellular matrix polypeptides. These filamentous extracellular matrix structures may act to transduce or otherwise stabilize dynamic forces generated during embryogenesis.