IU Indianapolis ScholarWorks :: Browsing by Author "Moldovan, Leni"

Browsing by Author "Moldovan, Leni"

Now showing 1 - 10 of 12

3D Printing of Human Ossicle Models for the Biofabrication of Personalized Middle Ear Prostheses
(MDPI, 2022-10-31) Dairaghi, Jacob; Rogozea, Dan; Cadle, Rachel; Bustamante, Joseph; Moldovan, Leni; Petrache, Horia I.; Moldovan, Nicanor I.; Physics, School of Science
The middle ear bones (‘ossicles’) may become severely damaged due to accidents or to diseases. In these situations, the most common current treatments include replacing them with cadaver-derived ossicles, using a metal (usually titanium) prosthesis, or introducing bridges made of biocompatible ceramics. Neither of these solutions is ideal, due to the difficulty in finding or producing shape-matching replacements. However, the advent of additive manufacturing applications to biomedical problems has created the possibility of 3D-printing anatomically correct, shape- and size-personalized ossicle prostheses. To demonstrate this concept, we generated and printed several models of ossicles, as solid, porous, or soft material structures. These models were first printed with a plottable calcium phosphate/hydroxyapatite paste by extrusion on a solid support or embedded in a Carbopol hydrogel bath, followed by temperature-induced hardening. We then also printed an ossicle model with this ceramic in a porous format, followed by loading and crosslinking an alginate hydrogel within the pores, which was validated by microCT imaging. Finally, ossicle models were printed using alginate as well as a cell-containing nanocellulose-based bioink, within the supporting hydrogel bath. In selected cases, the devised workflow and the printouts were tested for repeatability. In conclusion, we demonstrate that moving beyond simplistic geometric bridges to anatomically realistic constructs is possible by 3D printing with various biocompatible materials and hydrogels, thus opening the way towards the in vitro generation of personalized middle ear prostheses for implantation.
Adeno-Associated Virus Overexpression of Angiotensin-Converting Enzyme-2 Reverses Diabetic Retinopathy in Type 1 Diabetes in Mice
(Elsevier, 2016) Dominguez, James M., II; Hu, Ping; Caballero, Sergio; Moldovan, Leni; Verma, Amrisha; Oudit, Gavin Y.; Li, Qiuhong; Grant, Maria B.; Department of Ophthalmology, School of Medicine
Angiotensin-converting enzyme (ACE)-2 is the primary enzyme of the vasoprotective axis of the renin angiotensin system that regulates the classic renin angiotensin system axis. We aimed to determine whether local retinal overexpression of adenoassociated virus (AAV)-ACE2 prevents or reverses diabetic retinopathy. Green fluorescent protein (GFP)-chimeric mice were generated to distinguish resident (retinal) from infiltrating bone marrow-derived inflammatory cells and were made diabetic using streptozotocin injections. Retinal digestion using trypsin was performed and acellular capillaries enumerated. Capillary occlusion by GFP(+) cells was used to measure leukostasis. Overexpression of ACE2 prevented (prevention cohort: untreated diabetic, 11.3 ± 1.4; ACE2 diabetic, 6.4 ± 0.9 per mm(2)) and partially reversed (reversal cohort: untreated diabetic, 15.7 ± 1.9; ACE2 diabetic, 6.5 ± 1.2 per mm(2)) the diabetes-associated increase of acellular capillaries and the increase of infiltrating inflammatory cells into the retina (F4/80(+)) (prevention cohort: untreated diabetic, 24.2 ± 6.7; ACE2 diabetic, 2.5 ± 1.6 per mm(2); reversal cohort: untreated diabetic, 56.8 ± 5.2; ACE2 diabetic, 5.6 ± 2.3 per mm(2)). In both study cohorts, intracapillary bone marrow-derived cells, indicative of leukostasis, were only observed in diabetic animals receiving control AAV injections. These results indicate that diabetic retinopathy, and possibly other diabetic microvascular complications, can be prevented and reversed by locally restoring the balance between the classic and vasoprotective renin angiotensin system.
Bone Marrow–Derived Cell Recruitment to the Neurosensory Retina and Retinal Pigment Epithelial Cell Layer Following Subthreshold Retinal Phototherapy
(ARVO, 2017-10) Caballero, Sergio; Kent, David L.; Sengupta, Nilanjana; Li Calzi, Sergio; Shaw, Lynn; Beli, Eleni; Moldovan, Leni; Dominguez, James M.; Moorthy, Ramana S.; Grant, Maria B.; Medicine, School of Medicine
Purpose We investigated whether subthreshold retinal phototherapy (SRPT) was associated with recruitment of bone marrow (BM)–derived cells to the neurosensory retina (NSR) and RPE layer. Methods GFP chimeric mice and wild-type (WT) mice were subjected to SRPT using a slit-lamp infrared laser. Duty cycles of 5%, 10%, 15%, and 20% (0.1 seconds, 250 mW, spot size 50 μm) with 30 applications were placed 50 to 100 μm from the optic disc. In adoptive transfer studies, GFP+ cells were given intravenously immediately after WT mice received SRPT. Immunohistochemistry was done for ionized calcium-binding adapter molecule-1 (IBA-1+), CD45, Griffonia simplicifolia lectin isolectin B4, GFP or cytokeratin). Expression of Ccl2, Il1b, Il6, Hspa1a, Hsp90aa1, Cryab, Hif1a, Cxcl12, and Cxcr4 mRNA and flow cytometry of the NSR and RPE-choroid were performed. Results Within 12 to 24 hours of SRPT, monocytes were detected in the NSR and RPE-choroid. Detection of reparative progenitors in the RPE occurred at 2 weeks using flow cytometry. Recruitment of GFP+ cells to the RPE layer occurred in a duty cycle–dependent manner in chimeric mice and in mice undergoing adoptive transfer. Hspa1a, Hsp90aa1, and Cryab mRNAs increased in the NSR at 2 hours post laser; Hif1a, Cxcl12, Hspa1a increased at 4 hours in the RPE-choroid; and Ccl2, Il1b, Ifng, and Il6 increased at 12 to 24 hours in the RPE-choroid. Conclusions SRPT induces monocyte recruitment to the RPE followed by hematopoietic progenitor cell homing at 2 weeks. Recruitment occurs in a duty cycle–dependent manner and potentially could contribute to the therapeutic efficacy of SRPT.
Comparison of Biomaterial-Dependent and -Independent Bioprinting Methods for Cardiovascular Medicine
(Elsevier, 2017) Moldovan, Leni; Babbey, Clifford; Murphy, Michael; Moldovan, Nicanor I.; Department of Biomedical Engineering, School of Engineering and Technology
There is an increasing need of human organs for transplantation, of alternatives to animal experimentation, and of better in vitro tissue models for drug testing. All these needs create unique opportunities for the development of novel and powerful tissue engineering methods, among which the 3D bioprinting is one of the most promising. However, after decades of incubation, ingenuous efforts, early success and much anticipation, biomaterial-dependent 3D bioprinting, although shows steady progress, is slow to deliver the expected clinical results. For this reason, alternative ‘scaffold-free’ 3D bioprinting methods are developing in parallel at an accelerated pace. In this opinion paper we discuss comparatively the two approaches, with specific examples drawn from the cardiovascular field. Moving the emphasis away from competition, we show that the two platforms have similar goals but evolve in complementary technological niches. We conclude that the biomaterial-dependent bioprinting is better suited for tasks requiring faster, larger, anatomically-true, cell-homogenous and matrix-rich constructs, while the scaffold-free biofabrication is more adequate for cell-heterogeneous, matrix-poor, complex and smaller constructs, but requiring longer preparation time.
Design and Implementation of Anatomically Inspired Mesenteric and Intestinal Vascular Patterns for Personalized 3D Bioprinting
(MDPI, 2022-04-27) Cadle, Rachel; Rogozea, Dan; Moldovan, Leni; Moldovan, Nicanor I.; Surgery, School of Medicine
Recent progress in bioprinting has made possible the creation of complex 3D intestinal constructs, including vascularized villi. However, for their integration into functional units useful for experimentation or implantation, the next challenge is to endow them with a larger-scale, anatomically realistic vasculature. In general, the perfusion of bioprinted constructs has remained difficult, and the current solution is to provide them with mostly linear and simply branched channels. To address this limitation, here we demonstrated an image analysis-based workflow leading through computer-assisted design from anatomic images of rodent mesentery and colon to the actual printing of such patterns with paste and hydrogel bioinks. Moreover, we reverse-engineered the 2D intestinal image-derived designs into cylindrical objects, and 3D-printed them in a support hydrogel. These results open the path towards generation of more realistically vascularized tissue constructs for a variety of personalized medicine applications.
Electroacupuncture Promotes Central Nervous System-Dependent Release of Mesenchymal Stem Cells
(Wiley, 2017-05) Salazar, Tatiana E.; Richardson, Matthew R.; Beli, Eleni; Ripsch, Matthew S.; George, John; Kim, Youngsook; Duan, Yaqian; Moldovan, Leni; Yan, Yuanqing; Bhatwadekar, Ashay; Jadhav, Vaishnavi; Smith, Jared A.; McGorray, Susan; Bertone, Alicia L.; Traktuev, Dmitri O.; March, Keith L.; Colon-Perez, Luis M.; Avin, Keith; Sims, Emily; Mund, Julie A.; Case, Jamie; Deng, Shaolin; Kim, Min Su; McDavitt, Bruce; Boulton, Michael E.; Thinschmidt, Jeffrey; Calzi, Sergio Li; Fitz, Stephanie D.; Fuchs, Robyn K.; Warden, Stuart J.; McKinley, Todd; Shekhar, Anantha; Febo, Marcelo; Johnson, Phillip L.; Chang, Lung Ji; Gao, Zhanguo; Kolonin, Mikhail G.; Lai, Song; Ma, Jinfeng; Dong, Xinzhong; White, Fletcher A.; Xie, Huisheng; Yoder, Mervin C.; Grant, Maria B.; Ophthalmology, School of Medicine
Electroacupuncture (EA) performed in rats and humans using limb acupuncture sites, LI-4 and LI-11, and GV-14 and GV-20 (humans) and Bai-hui (rats) increased functional connectivity between the anterior hypothalamus and the amygdala and mobilized mesenchymal stem cells (MSCs) into the systemic circulation. In human subjects, the source of the MSC was found to be primarily adipose tissue, whereas in rodents the tissue sources were considered more heterogeneous. Pharmacological disinhibition of rat hypothalamus enhanced sympathetic nervous system (SNS) activation and similarly resulted in a release of MSC into the circulation. EA-mediated SNS activation was further supported by browning of white adipose tissue in rats. EA treatment of rats undergoing partial rupture of the Achilles tendon resulted in reduced mechanical hyperalgesia, increased serum interleukin-10 levels and tendon remodeling, effects blocked in propranolol-treated rodents. To distinguish the afferent role of the peripheral nervous system, phosphoinositide-interacting regulator of transient receptor potential channels (Pirt)-GCaMP3 (genetically encoded calcium sensor) mice were treated with EA acupuncture points, ST-36 and LIV-3, and GV-14 and Bai-hui and resulted in a rapid activation of primary sensory neurons. EA activated sensory ganglia and SNS centers to mediate the release of MSC that can enhance tissue repair, increase anti-inflammatory cytokine production and provide pronounced analgesic relief.
iPSC-Derived Vascular Cell Spheroids as Building Blocks for Scaffold-Free Biofabrication
(Wiley, 2017) Moldovan, Leni; Barnard, April; Gil, Chang-Hyun; Lin, Y.; Grant, Maria B.; Yoder, Mervin C.; Prasain, Nutan; Moldovan, Nicanor I.; Biomedical Engineering, School of Engineering and Technology
Recently a protocol is established to obtain large quantities of human induced pluripotent stem cells (iPSC)-derived endothelial progenitors, called endothelial colony forming cells (ECFC), and of candidate smooth-muscle forming cells (SMFC). Here, the suitability for assembling in spheroids, and in larger 3D cell constructs is tested. iPSC-derived ECFC and SMFC are labeled with tdTomato and eGFP, respectively. Spheroids are formed in ultra-low adhesive wells, and their dynamic proprieties are studied by time-lapse microscopy, or by confocal microscopy. Spheroids are also tested for fusion ability either in the wells, or assembled on the Regenova 3D bioprinter which laces them in stainless steel micro-needles (the “Kenzan” method). It is found that both ECFC and SMFC formed spheroids in about 24 h. Fluorescence monitoring indicated a continuous compaction of ECFC spheroids, but stabilization in those prepared from SMFC. In mixed spheroids, the cell distribution changed continuously, with ECFC relocating to the core, and showing pre-vascular organization. All spheroids have the ability of in-well fusion, but only those containing SMFC are robust enough to sustain assembling in tubular structures. In these constructs a layered distribution of alpha smooth muscle actin-positive cells and extracellular matrix deposition is found. In conclusion, iPSC-derived vascular cell spheroids represent a promising new cellular material for scaffold-free biofabrication.
Labeling of endothelial cells with magnetic microbeads by angiophagy
(Springer, 2018-08) Thomas, Jessica; Jones, Desiree; Moldovan, Leni; Anghelina, Mirela; Gooch, Keith J.; Moldovan, Nicanor I.; Ophthalmology, School of Medicine
Objectives Attachment of magnetic particles to cells is needed for a variety of applications but is not always possible or efficient. Simpler and more convenient methods are thus desirable. In this study, we tested the hypothesis that endothelial cells (EC) can be loaded with micron-size magnetic beads by the phagocytosis-like mechanism ‘angiophagy’. To this end, human umbilical vein EC (HUVEC) were incubated with magnetic beads conjugated or not (control) with an anti-VEGF receptor 2 antibody, either in suspension, or in culture followed by re-suspension using trypsinization. Results In all conditions tested, HUVEC incubation with beads induced their uptake by angiophagy, which was confirmed by (i) increased cell granularity assessed by flow cytometry, and (ii) the presence of an F-actin rich layer around many of the intracellular beads, visualized by confocal microscopy. For confluent cultures, the average number of beads per cell was 4.4 and 4.2, with and without the presence of the anti-VEGFR2 antibody, respectively. However, while the actively dividing cells took up 2.9 unconjugated beads on average, this number increased to 5.2 if binding was mediated by the antibody. Magnetic pulldown increased the cell density of beads-loaded cells in porous electrospun poly-capro-lactone scaffolds by a factor of 4.5 after 5 min, as compared to gravitational settling (p < 0.0001). Conclusion We demonstrated that EC can be readily loaded by angiophagy with micron-sized beads while attached in monolayer culture, then dispersed in single-cell suspensions for pulldown in porous scaffolds and for other applications.
Loss of Angiotensin-Converting Enzyme 2 Exacerbates Diabetic Retinopathy by Promoting Bone Marrow Dysfunction
(Wiley, 2018-09) Duan, Yaqian; Beli, Eleni; Calzi, Sergio Li; Quigley, Judith L.; Miller, Rehae C.; Moldovan, Leni; Feng, Dongni; Salazar, Tatiana E.; Hazra, Sugata; Al-Sabah, Jude; Chalam, Kakarla V.; Trinh, Thao Le Phuong; Meroueh, Marya; Markel, Troy A.; Murray, Matthew C.; Vyas, Ruchi J.; Boulton, Michael E.; Parsons-Wingerter, Patricia; Oudit, Gavin Y.; Obukhov, Alexander G.; Grant, Maria B.; Cellular and Integrative Physiology, School of Medicine
Angiotensin-converting enzyme 2 (ACE2) is the primary enzyme of the vasoprotective axis of the renin angiotensin system (RAS). We tested the hypothesis that loss of ACE2 would exacerbate diabetic retinopathy by promoting bone marrow dysfunction. ACE2-/y were crossed with Akita mice, a model of type 1 diabetes. When comparing the bone marrow of the ACE2-/y-Akita mice to that of Akita mice, we observed a reduction of both short-term and long-term repopulating hematopoietic stem cells, a shift of hematopoiesis towards myelopoiesis, and an impairment of lineage-c-kit+ hematopoietic stem/progenitor cell (HS/PC) migration and proliferation. Migratory and proliferative dysfunction of these cells was corrected by exposure to angiotensin-1–7 (Ang-1–7), the protective peptide generated by ACE2. Over the duration of diabetes examined, ACE2 deficiency led to progressive reduction in electrical responses assessed by electroretinography and to increases in neural infarcts observed by fundus photography. Compared to Akita mice, ACE2-/y-Akita at 9-months of diabetes showed an increased number of acellular capillaries indicative of more severe diabetic retinopathy. In diabetic and control human subjects, CD34+ cells, a key bone marrow HS/PC population, were assessed for changes in mRNA levels for MAS, the receptor for Ang-1–7. Levels were highest in CD34+ cells from diabetics without retinopathy. Higher serum Ang-1–7 levels predicted protection from development of retinopathy in diabetics. Treatment with Ang-1–7 or alamandine restored the impaired migration function of CD34+ cells from subjects with retinopathy. These data support that activation of the protective RAS within HS/PCs may represent a therapeutic strategy for prevention of diabetic retinopathy.
Of balls, inks and cages: Hybrid biofabrication of 3D tissue analogs
(2019) Moldovan, Nicanor I.; Moldovan, Leni; Raghunath, Michael; Biomedical Engineering, School of Engineering and Technology
The overarching principle of three-dimensional (3D) bioprinting is the placing of cells or cell clusters in the 3D space to generate a cohesive tissue microarchitecture that comes close to in vivo characteristics. To achieve this goal, several technical solutions are available, generating considerable combinatorial bandwidth: (i) Support structures are generated first, and cells are seeded subsequently; (ii) alternatively, cells are delivered in a printing medium, so-called “bioink,” that contains them during the printing process and ensures shape fidelity of the generated structure; and (iii) a “scaffold-free” version of bioprinting, where only cells are used and the extracellular matrix is produced by the cells themselves, also recently entered a phase of accelerated development and successful applications. However, the scaffold-free approaches may still benefit from secondary incorporation of scaffolding materials, thus expanding their versatility. Reversibly, the bioink-based bioprinting could also be improved by adopting some of the principles and practices of scaffold-free biofabrication. Collectively, we anticipate that combinations of these complementary methods in a “hybrid” approach, rather than their development in separate technological niches, will largely increase their efficiency and applicability in tissue engineering.

Browsing by Author "Moldovan, Leni"

Results Per Page

Sort Options