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Biomedical subjects

Gordana Vunjak-Novakovic

Publications and source records attributed to Gordana Vunjak-Novakovic.

At least 19 recordsLinked to original sources
PubMed2007

Microfluidic patterning for fabrication of contractile cardiac organoids.

The development of in vitro methods of engineering three-dimensional cardiac tissues can be useful for tissue replacement, diagnostics and drug discovery. Here, we introduce the use of patterned hyaluronic acid (HA) substrates generated using microfluidic patterning as a method of fabricating 3D cardiac organoids. HA micropatterns served as inductive templates for organoid assembly. Upon seeding, cardiomyocytes elongated and aligned along the pattern direction attaching preferentially to the glass substrate and the interface between HA patterns and glass substrate. After 3 days in culture, the linearly aligned myocytes detached from the surface and formed contractile cardiac organoids. The procedure can be utilized to simply, rapidly and inexpensively create in vitro cardiac tissue models.

Animals↗
PubMed2006

Control of in vitro tissue-engineered bone-like structures using human mesenchymal stem cells and porous silk scaffolds.

Natural bone consists of cortical and trabecular morphologies, the latter having variable pore sizes. This study aims at engineering different bone-like structures using scaffolds with small pores (112-224 microm) in diameter on one side and large pores (400-500 microm) on the other, while keeping scaffold porosities constant among groups. We hypothesized that tissue engineered bone-like structure resulting from silk fibroin (SF) implants is pre-determined by the scaffolds' geometry. To test this hypothesis, SF scaffolds with different pore diameters were prepared and seeded with human mesenchymal stem cells (hMSC). As compared to static seeding, dynamic cell seeding in spinner flasks resulted in equal cell viability and proliferation, and better cell distribution throughout the scaffold as visualized by histology and confocal microscopy, and was, therefore, selected for subsequent differentiation studies. Differentiation of hMSC in osteogenic cell culture medium in spinner flasks for 3 and 5 weeks resulted in increased alkaline phosphatase activity and calcium deposition when compared to control medium. Micro-computed tomography (microCT) detailed the pore structures of the newly formed tissue and suggested that the structure of tissue-engineered bone was controlled by the underlying scaffold geometry.

Biocompatible Materials↗
PubMed2006

Bone and cartilage tissue constructs grown using human bone marrow stromal cells, silk scaffolds and rotating bioreactors.

Human bone marrow contains a population of bone marrow stromal cells (hBMSCs) capable of forming several types of mesenchymal tissues, including bone and cartilage. The present study was designed to test whether large cartilaginous and bone-like tissue constructs can be selectively engineered using the same cell population (hBMSCs), the same scaffold type (porous silk) and same hydrodynamic environment (construct settling in rotating bioreactors), by varying the medium composition (chondrogenic vs. osteogenic differentiation factors). The hBMSCs were harvested, expanded and characterized with respect to their differentiation potential and population distribution. Passage two cells were seeded on scaffolds and cultured for 5 weeks in bioreactors using osteogenic, chondrogenic or control medium. The three media yielded constructs with comparable wet weights and compressive moduli ( approximately 25 kPa). Chondrogenic medium yielded constructs with higher amounts of DNA (1.5-fold) and glycosaminoglycans (GAG, 4-fold) per unit wet weight (ww) than control medium. In contrast, osteogenic medium yielded constructs with higher dry weight (1.6-fold), alkaline phosphatase (AP) activity (8-fold) and calcium content (100-fold) per unit ww than control medium. Chondrogenic medium yielded constructs that were weakly positive for GAG by contrast-enhanced MRI and alcian blue stain, whereas osteogenic medium yielded constructs that were highly mineralized by microCT and von Kossa stain. Engineered bone constructs were large (8mm diameter x 2mm thick disks) and resembled trabecular bone with respect to structure and mineralized tissue volume fraction (12%).

Biocompatible Materials↗
PubMed2006

Stem cell-based tissue engineering with silk biomaterials.

Silks are naturally occurring polymers that have been used clinically as sutures for centuries. When naturally extruded from insects or worms, silk is composed of a filament core protein, termed fibroin, and a glue-like coating consisting of sericin proteins. In recent years, silk fibroin has been increasingly studied for new biomedical applications due to the biocompatibility, slow degradability and remarkable mechanical properties of the material. In addition, the ability to now control molecular structure and morphology through versatile processability and surface modification options have expanded the utility for this protein in a range of biomaterial and tissue-engineering applications. Silk fibroin in various formats (films, fibers, nets, meshes, membranes, yarns, and sponges) has been shown to support stem cell adhesion, proliferation, and differentiation in vitro and promote tissue repair in vivo. In particular, stem cell-based tissue engineering using 3D silk fibroin scaffolds has expanded the use of silk-based biomaterials as promising scaffolds for engineering a range of skeletal tissues like bone, ligament, and cartilage, as well as connective tissues like skin. To date fibroin from Bombyx mori silkworm has been the dominant source for silk-based biomaterials studied. However, silk fibroins from spiders and those formed via genetic engineering or the modification of native silk fibroin sequence chemistries are beginning to provide new options to further expand the utility of silk fibroin-based materials for medical applications.

Animals↗
PubMed2006

Osteogenesis by human mesenchymal stem cells cultured on silk biomaterials: comparison of adenovirus mediated gene transfer and protein delivery of BMP-2.

Bone tissue engineering, gene therapy based on human mesenchymal stem cells (MSCs) and silk fibroin biomaterials were combined to study the impact of viral transfection on MSC osteogenic performance in vitro. MSCs were transduced with adenovirus containing a human BMP-2 (Ad-BMP-2) gene at clinically reasonable viral concentrations and cultured for 4 weeks. Controls with nontransfected MSCs, but exposed to exogenous BMP-2 concentrations on an analogous time profile as that secreted by the Ad-BMP-2 group, were compared. Both the Ad-BMP-2 MSC group and the exogenous protein BMP-2 group strongly expressed osteopontin and bone sialoprotein. Cells secreted a matrix that underwent mineralization on the silk fibroin scaffolds, forming clusters of osseous material, as determined by micro-computed tomography. The expression of osteogenic marker proteins and alkaline phosphatase was significantly higher in the Ad-BMP-2 MSC group than in the exogenous protein BMP-2 group, and no significant differences in mineralization were observed in two of the three MSC sources tested. The results demonstrate that transfection resulted in higher levels of expression of osteogenic marker genes, no change in proliferation rate and did not impact the capacity of the cells to calcify tissues on these protein scaffolds. These findings suggest additional options to control differentiation where exogenous additions of growth factors or morphogens can be replaced with transfected MSCs.

Adenoviridae↗
PubMed2006

A photolithographic method to create cellular micropatterns.

Here we describe a simple and rapid system for creation of patterned cell culture substrates. This technique is based on (1) printing a mask on a standard overhead transparency, (2) coating a thin layer of a photocrosslinkable chitosan on a slide, (3) exposing the slide and mask to ultraviolet (UV) light, and (4) rinsing the uncrosslinked polymer to expose the underlying cell-repellent patterns. Photocross-linkable chitosan does not require photoinitiators, it is non-toxic and forms flexible, biocompatible hydrogel upon short ( approximately min) UV exposure. Patterns of various shapes (lanes, squares, triangles, circles) were created on two surfaces commonly used for cell culture: glass and tissue culture polystyrene. The pattern size could be varied with a mum resolution using a single mask and varying UV exposure time. Cardiac fibroblasts formed stable patterns for up to 18 days in culture. Cardiomyocytes, patterned in lanes 68-99 microm wide, exhibited expression of cardiac Troponin I, well developed contractile apparatus and they contracted synchronously in response to electrical field stimulation. Osteoblasts (SAOS-2) localized in the exposed glass regions (squares, triangles, or circles; 0.063-0.5mm(2)). They proliferated to confluence in 5 days, expressed alkaline phosphatase and produced a mineralized matrix.

Animals↗
PubMed2006

Oxygen gradients correlate with cell density and cell viability in engineered cardiac tissue.

For clinical utility, cardiac grafts should be thick and compact, and contain physiologic density of metabolically active, differentiated cells. This involves the need to control the levels of nutrients, and most critically oxygen, throughout the construct volume. Most culture systems involve diffusional transport within the constructs, a situation associated with gradients of oxygen concentration, cell density, cell viability, and function. The goal of our study was to measure diffusional gradients of oxygen in statically cultured cardiac constructs, and to correlate oxygen gradients to the spatial distributions of cell number and cell viability. Using microelectrodes, we measured oxygen distribution in a disc-shaped constructs (3.6 mm diameter, 1.8 mm thickness) based on neonatal rat cardiomyocytes cultured on collagen scaffolds for 16 days in static dishes. To rationalize experimental data, a mathematical model of oxygen distribution was derived as a function of cell density, viability, and spatial position within the construct. Oxygen concentration and cell viability decreased linearly and the live cell density decreased exponentially with the distance from the construct surface. Physiological density of live cells was present only within the first 128 microm of the construct thickness. Medium flow significantly increased oxygen concentration within the construct, correlating with the improved tissue properties observed for constructs cultured in convectively mixed bioreactors.

Animals↗
PubMed2006

Porous silk fibroin 3-D scaffolds for delivery of bone morphogenetic protein-2 in vitro and in vivo.

Bone morphogenetic protein-2 (BMP-2) plays a key role in osteogenesis. Biomaterials used for the sustained delivery of BMP-2 in vivo have shown therapeutic benefits. In the present study, BMP-2 was loaded in porous silk fibroin scaffolds derived from silkworm cocoons (2.4 +/- 0.14 microg per scaffold). The release profile of BMP-2 under dynamic culture conditions (spinner flasks) showed that after 1 week in culture 25% of the initial BMP-2 was retained adsorbed to the scaffold; up to 4 weeks no additional BMP-2 was released. BMP-2 induced human bone marrow stromal cells (hMSCs) to undergo osteogenic differentiation when the seeded scaffolds were cultured in medium supplemented with osteogenic stimulants for 4 weeks, based on elevated alkaline phosphatase activity, calcium deposition, and transcript levels for bone sialoprotein, osteopontin, osteocalcin, BMP-2, and cbfa-1. Micro-computed tomography revealed densely deposited mineral at the center of the scaffolds. In contrast, hMSCs cultured in control scaffolds (no BMP-2) exhibited limited osteogenesis. When implanted in critical sized cranial defects in mice, scaffolds loaded with BMP-2 and seeded with hMSCs resulted in significant bone ingrowth. These results were qualitatively similar to scaffolds loaded with BMP-2 but no hMSCs or with BMP-2 and hMSCs but not pregrown into bone-like tissue. Bone-related outcomes were improved when compared with the scaffold controls implanted without BMP-2. These studies illustrate the potential use of slow degrading silk fibroin 3-D scaffolds loaded with BMP-2, in combination with hMSCs, in osteogenesis studies in vitro and in vivo, and provide a new range of material properties for these applications.

Adsorption↗
PubMed2006

Size-based microfluidic enrichment of neonatal rat cardiac cell populations.

Native heart consists of myocytes and non-myocytes. We demonstrate here the feasibility of a size-based microfluidic separation of myocytes and non-myocytes from the neonatal rat myocardium. The device consists of a middle channel (50 microm wide, 200 microm tall, and 4 cm long) connected to adjacent side channels by microsieves (80 microm wide, 5 microm tall and 40 microm in length). The side channels increase in width in a flared shape along the length of the device to ensure constant pressure gradient across all sieves. In the first step, non-myocytes were removed from the myocytes by a conventional pre-plating method for 75 min. Subsequently, the non-myocytes were further enriched in a microfluidic device at 20 microl/min. We demonstrated that the cells in the middle and side channels maintained viability during sorting and the ability to attach and grow in culture. Upon culture for 48 h cardiomyocytes from the reservoir (control) and middle channel stained positive for cardiac Troponin I, exhibited a well developed contractile apparatus and contracted spontaneously and in response to electrical field stimulation. Most of the cells in the side channel expressed a non-myocyte marker vimetin. Fluorescent activated cell sorting indicated significant enrichment in the side channel (p < 0.001) for non-myocytes. Original cell suspension had a bimodal cell size distribution with the peaks in the range from 7-9 microm and 15-17 microm. Upon cell sorting the distribution was Gaussian in both side channel and middle channel with the peaks in the range 7-9 microm and 9-11 microm respectively, indicating that the separation by size occurred.

Animals↗
PubMed2006

Biomimetic approach to cardiac tissue engineering: oxygen carriers and channeled scaffolds.

We report that the functional assembly of engineered cardiac muscle can be enhanced by oxygen supply provided by mechanisms resembling those in normal vascularized tissues. To mimic the capillary network, cardiomyocytes and fibroblasts isolated from the neonatal rat hearts were cultured on a highly porous elastomer with a parallel array of channels that were perfused with culture medium. To mimic oxygen supply by hemoglobin, culture medium was supplemented with a perfluorocarbon (PFC) emulsion; constructs perfused with unsupplemented culture medium served as controls. In PFC-supplemented medium, the decrease in the partial pressure of oxygen in the aqueous phase was only 50% of that in control medium (28 mmHg vs. 45 mmHg between the construct inlet and outlet at a flow rate of 0.1 mL/min). Consistently, constructs cultivated in the presence of PFC contained higher amounts of DNA and cardiac markers (troponin I, connexin-43) and had significantly better contractile properties as compared to control constructs. In both groups, electron microscopy revealed open channels and the presence of cells at the channel surfaces as well as within constructs. Improved properties of cardiac constructs could be correlated with the enhanced supply of oxygen to the cells, by a combined use of channeled scaffolds and PFC.

Animals↗
PubMed2006

Biophysical regulation during cardiac development and application to tissue engineering.

Tissue engineering combines the principles of biology, engineering and medicine to create biological substitutes of native tissues, with an overall objective to restore normal tissue function. It is thought that the factors regulating tissue development in vivo (genetic, molecular and physical) can also direct cell fate and tissue assembly in vitro. In light of this paradigm, tissue engineering can be viewed as an effort of "imitating nature". We first discuss biophysical regulation during cardiac development and the factors of interest for application in tissue engineering of the myocardium. Then we focus on the biomimetic approach to cardiac tissue engineering which involves the use of culture systems designed to recapitulate some aspects of the actual in vivo environment. To mimic cell signaling in native myocardium, subpopulations of neonatal rat heart cells were cultured at a physiologically high cell density in three-dimensional polymer scaffolds. To mimic the capillary network, highly porous elastomer scaffolds with arrays of parallel channels were perfused with culture medium. To mimic oxygen supply by hemoglobin, culture medium was supplemented with an oxygen carrier. To enhance electromechanical coupling, tissue constructs were induced to contract by applying electrical signals mimicking those in native heart. Over only eight days of cultivation, the biomimetic approach resulted in tissue constructs which contained electromechanically coupled cells expressing cardiac differentiation markers and cardiac-like ultrastructure and contracting synchronously in response to electrical stimulation. Ongoing studies are aimed at extending this approach to tissue engineering of functional cardiac grafts based on human cells.

Animals↗
PubMed2005

Silk implants for the healing of critical size bone defects.

Bone (re)-generation and bone fixation strategies utilize biomaterial implants, which are gradually replaced by autologous tissues. Ideally, these biomaterials should be biodegradable, osteoconductive, and provide mechanical strength and integrity until newly formed host tissues can maintain function. Some protein-based biomaterials such as collagens are promising because of their biological similarities to natural proteins on bone surfaces. However, their use as bone implant materials is largely hampered by poor mechanical properties. In contrast, silks offer distinguishing mechanical properties that are tailorable, along with slow degradability to permit adequate time for remodeling. To assess the suitability of silk-based biomaterials as implants for bone healing, we explored the use of novel porous silk fibroin scaffolds as templates for the engineering of bone tissues starting from human bone marrow derived stem cells cultured under osteogenic conditions for up to 5 weeks. The slowly degrading protein matrix permitted adequate temporal control of hydroxyapatite deposition and resulted in the formation of a trabecular-like bone matrix in bioreactor studies. The organic and inorganic components of the engineered bone tissues resembled those of bone, as shown by gene expression analysis, biochemical assays, and X-ray diffractometry. Implantation of the tissue-engineered bone implants (grown in bioreactors for 5 weeks prior to implantation) into calvarial critical size defects in mice demonstrated the capacity of these systems to induce advanced bone formation within 5 weeks, whereas the implantation of stem cell loaded silk scaffolds, and scaffolds alone resulted in less bone formation. These results demonstrate the feasibility of silk-based implants with engineered bone for the (re-)generation of bone tissues and expand the class of protein-based bone-implant materials with a mechanically stable and durable option.

Animals↗
PubMed2005

Development and remodeling of engineered cartilage-explant composites in vitro and in vivo.

OBJECTIVE: Development and remodeling of engineered cartilage-explant composites were studied in vitro and in vivo. DESIGN: Individual and interactive effects of cell chondrogenic potential (primary or fifth passage bovine calf chondrocytes), scaffold degradation rate (hyaluronan benzyl ester or polyglycolic acid), and adjacent tissue cell activity and architecture (vital trabecular bone (VB), articular cartilage (AC), devitalized bone (DB) or digested cartilage (DC)) were evaluated over 8 weeks in vitro (bioreactor cultures) and in vivo (ectopic implants). RESULTS: In vitro, significant effects of cell type on construct adhesive strength (P<0.001) and scaffold type on adhesive strength (P<0.001), modulus (P=0.014), glycosaminoglycans (GAG) (P<0.001), and collagen (P=0.039) were observed. Chondrogenesis was best when the scaffold degradation rate matched the extracellular matrix deposition rate. In vivo, adjacent tissue type affected adhesive strength (P<0.001), modulus (P<0.001), and GAG (P<0.001) such that 8-week values obtained for bone (VB and DB) were higher than for cartilage (AC). In the AC/construct group, chondrogenesis appeared attenuated in the region of the construct close to the AC. In contrast, in the VB/construct group, a 500 microm thick zone of mature hyaline-like cartilage formed at the interface, and signs of active remodeling were present in the bone that included osteoclastic and osteoblastic activity and trabecular rebuttressing; these features were not present in the DB group or in vitro. CONCLUSIONS: Development and remodeling of composites based on engineered cartilage were mediated in vitro by cell chondrogenic potential and scaffold degradation rate, and in vivo by type of adjacent tissue and time.

Animals↗
PubMed2005

The inflammatory responses to silk films in vitro and in vivo.

Silks have a long history of biomedical use as sutures. Silk can be purified, chemically modified to attach RGD sequences and processed into highly porous scaffolds for tissue engineering. We report biocompatibility studies of silk films (with or without covalently bound RGD) that were seeded with bone-marrow derived mesenchymal stem cells (MSC) and (a) cultured in vitro with human MSC or (b) seeded with autologous rat MSC and implanted in vivo. Controls for in vitro studies included tissue culture plastic (TCP; negative control), TCP with lipopolysaccharide (LPS) in the cell culture medium (positive control), and collagen films; controls for in vivo studies included collagen, PLA and TCP. After 9 h of culture, the expression of the pro-inflammatory Interleukin 1 beta (IL-1beta) and inflammatory cyclooxygenase 2 (COX-2) in human MSC were comparable for silk, collagen and TCP. After 30 and 96 h, gene expression of IL-1beta and COX-2 in MSC returned to the baseline (pre-seeding) levels. These data were corroborated by measuring IL-1beta and prostaglandin E2 levels in culture medium. The rate of cell proliferation was higher on silk films than either on collagen or TCP. In vivo, films made of silk, collagen or PLA were seeded with rat MSCs, implanted intramuscularly in rats and harvested after 6 weeks. Histological and immunohistochemical evaluation of silk explants revealed the presence of circumferentially oriented fibroblasts, few blood vessels, macrophages at the implant-host interface, and the absence of giant cells. Inflammatory tissue reaction was more conspicuous around collagen films and even more around PLA films when compared to silk. These data suggest that (a) purified degradable silk is biocompatible and (b) the in vitro cell culture model (hMSC seeded and cultured on biomaterial films) gave inflammatory responses that were comparable to those observed in vivo.

Animals↗
PubMed2005

Influence of macroporous protein scaffolds on bone tissue engineering from bone marrow stem cells.

The aim of this study was to investigate the effect of three-dimensional silk fibroin scaffold preparation methods (aqueous and solvent) on osteogenic responses by human bone marrow stem cells (hMSCs). Macroporous 3D protein scaffolds with similar sized pores of 900+/-50 microm were prepared either by an organic solvent process (hexafluoro-2-propanol, HFIP) or an aqueous process. hMSCs were expanded, seeded on the scaffolds, and cultured up to 28 days under static conditions in osteogenic media. hMSCs seeded onto the water-based silk scaffolds showed a significant increase in cell numbers (p<0.01) vs. the HFIP-prepared silk scaffolds. Significantly higher (p<0.01) alkaline phosphatase (ALPase) activity and calcium deposition were apparent after 28 days of culture in the water-based silk scaffolds when compared to the HFIP-derived silk scaffolds. Transcript levels for collagen type I (Col I), ALP, and osteopontin (OP) increased (p<0.05) in the water-based silk scaffolds in comparison to the HFIP-derived materials. At early stages of culture, increased expression of OP and collagen type II (Col II) were also observed in both scaffolds. Expression of Col II, MMP 13, Col I, and OP proteins increased in the water-based silk scaffolds in comparison to the HFIP-derived scaffolds while bone sialoprotein (BSP) proteins increased in the HFIP-derived silk scaffolds in comparison to the water-based scaffolds after 28 days of culture. Histological analysis showed the development of bone-like trabeculae with cuboid cells in an extracellular matrix (ECM) in the water-based silk scaffolds with more organization than in the HFIP-derived material after 28 days of culture. Alcian blue staining demonstrated the presence of proteoglycan in the ECM formed in the water-based scaffolds but not in the HFIP-prepared silk scaffolds. The results suggest that macroporous 3D aqueous-derived silk fibroin scaffolds provide improved bone-related outcomes in comparison to the HFIP-derived systems. These data illustrate the importance of materials processing on biological outcomes, as the same protein, silk fibroin, was used in both preparations.

Bone Substitutes↗
PubMed2005

Growth factor induced fibroblast differentiation from human bone marrow stromal cells in vitro.

Utilizing a two-dimensional tissue culture plastic screening system and a fractional factorial design, specific media formulations and growth factor combinations were determined that support human bone marrow stromal cell (BMSC) differentiation toward fibroblast characteristics for utilization in tissue engineering, specifically cell morphology and alignment, metabolic activity, abundant expression of collagen types I and III, and negligible expression of other tissue-specific markers. BMSCs were cultured for up to 14 days on tissue culture plastic, supplemented with Dulbecco's Minimal Essential Medium (DMEM)/10% FBS or Advanced DMEM(ADMEM)/5% FBS. Each medium base was supplemented with one of nine possible growth factor combinations and ascorbate-2-phosphate (Asc-2-P) for the duration of culture. ADMEM supported comparable cell viability with half the serum content of the DMEM formulation. Asc-2-P was potent in promoting BMSC proliferation, in the absence of a mitogen, supporting significant increases in cell activity over 14 days of culture. DMEM promoted significant increases in cell viability for 7 of 9 growth factor groups when compared to their ADMEM counterparts. ADMEM, however, promoted increased cell transcript and protein expression, as 5 of 9 growth factor combinations induced a 200% increase in collagen type I versus equivalent DMEM cultures. Cell morphology and collagen type I immunostaining, when assessed in context of MTT and RNA results, identified 3 growth factor and medium combinations that supported fibroblast differentiation for future development of ligament tissue in vitro.

Bone Marrow Cells↗
PubMed2004

Functional assembly of engineered myocardium by electrical stimulation of cardiac myocytes cultured on scaffolds.

The major challenge of tissue engineering is directing the cells to establish the physiological structure and function of the tissue being replaced across different hierarchical scales. To engineer myocardium, biophysical regulation of the cells needs to recapitulate multiple signals present in the native heart. We hypothesized that excitation-contraction coupling, critical for the development and function of a normal heart, determines the development and function of engineered myocardium. To induce synchronous contractions of cultured cardiac constructs, we applied electrical signals designed to mimic those in the native heart. Over only 8 days in vitro, electrical field stimulation induced cell alignment and coupling, increased the amplitude of synchronous construct contractions by a factor of 7, and resulted in a remarkable level of ultrastructural organization. Development of conductive and contractile properties of cardiac constructs was concurrent, with strong dependence on the initiation and duration of electrical stimulation.

Animals↗