Chandrasekhar R. Kothapalli

Microfluidic devices for studying heterotypic cell-cell interactions and tissue specimen cultures under controlled microenvironments.

Microfluidic devices allow for precise control of the cellular and noncellular microenvironment at physiologically relevant length- and time-scales. These devices have been shown to mimic the complex in vivo microenvironment better than conventional in vitro assays, and allow real-time monitoring of homotypic or heterotypic cellular interactions. Microfluidic culture platforms enable new assay designs for culturing multiple different cell populations and∕or tissue specimens under controlled user-defined conditions. Applications include fundamental studies of cell population behaviors, high-throughput drug screening, and tissue engineering. In this review, we summarize recent developments in this field along with studies of heterotypic cell-cell interactions and tissue specimen culture in microfluidic devices from our own laboratory.

3D matrix microenvironment for targeted differentiation of embryonic stem cells into neural and glial lineages

The onset of neurodegenerative disorders is characterized by the progressive dysfunction and loss of subpopulations of specialized cells within specific regions of the central nervous system (CNS). Since CNS has a limited ability for self-repair and regeneration under such conditions, clinical transplantation of stem cells has been explored as an alternative. Although embryonic stem cells (ESCs) offer a promising therapeutic platform to treat a variety of neurodegenerative disorders, the niche microenvironment, which could regulate their differentiation into specialized lineages on demand, needs to be optimized for successful clinical transplantation. Here, we evaluated the synergistic role of matrix microenvironment (type, architecture, composition, stiffness) and signaling molecules (type, dosage) on murine ESC differentiation into specific neural and glial lineages. ESCs were cultured as embryoid bodies on either 2D substrates or within 3D scaffolds, in the presence or absence of retinoic acid (RA) and sonic hedgehog (Shh). Results showed that ESCs maintained their stemness even after 4 days in the absence of exogenous signaling molecules, as evidenced by Oct-4 staining. RA at 1 μM dosage was deemed optimal for neural differentiation and neurite outgrowth on collagen-1 coated substrates. Significant neural differentiation with robust neurite outgrowth and branching was evident only on collagen-1 coated 2D substrates and within 3D matrigel scaffolds, in the presence of 1 μM RA. Blocking α6 or β1 integrin subunits on differentiating cells inhibited matrigel-induced effects on neural differentiation and neurite outgrowth. Hydrogel concentration strongly regulated formation of neural and astrocyte lineages in 1 μM RA additive cultures. When RA and Shh were provided, either alone or together, 3D collagen-1 scaffolds enhanced significant motor neuron formation, while 3D matrigel stimulated dopaminergic neuron differentiation. These results suggest a synergistic role of microenvironmental cues for ESC differentiation and maturation, with potential applications in cell transplantation therapy.

Transforming Growth Factor Beta 1 and Hyaluronan Oligomers Synergistically Enhance Elastin Matrix Regeneration by Vascular Smooth Muscle Cells

Elastin is a vital structural and regulatory matrix protein that plays an important role in conferring elasticity to blood vessel wall. Previous tissue engineering approaches to regenerate elastin in situ or within tissue engineering constructs are curtailed by innate poor elastin synthesis potential by adult vascular smooth muscle cells (SMCs). Currently, we seek to develop cellular cues to enhance tropoelastin synthesis and improve elastin matrix yield, stability, and ultrastructure. Our earlier studies attest to the elastogenic utility of hyaluronan (HA)-based cellular cues, though their effects are fragment size dependent and dose dependent, with HA oligomers deemed most elastogenic. We presently show transforming growth factor beta 1 (TGF-beta1) and HA oligomers, when provided concurrently, to synergistically and dramatically improve elastin matrix regeneration by adult vascular SMCs. Together, these cues suppress SMC proliferation, enhance synthesis of tropoelastin (8-fold) and matrix elastin protein (5.5-fold), and also improve matrix elastin yield (45% of total elastin vs. 10% for nonadditive controls), possibly by more efficient recruitment of tropoelastin for crosslinking. The density of desmosine crosslinks within the elastin matrix was itself attenuated, although the cues together modestly increased production and activity of the elastin crosslinking enzyme, lysyl oxidase. TGF-beta1 and HA oligomers together induced much greater assembly of mature elastin fibers than they did separately, and did not induce matrix calcification. The present outcomes might be great utility to therapeutic regeneration of elastin matrix networks in situ within elastin-compromised vessels, and within tissue-engineered vascular graft replacements.

Copper nanoparticle cues for biomimetic cellular assembly of crosslinked elastin fibers

Elastin, a structural protein distributed in the extracellular matrix of vascular tissues, is critical to maintaining the elastic stability and mechanical properties of blood vessels, as well as regulating cell-signaling pathways involved in vascular injury response and morphogenesis. Pathological degradation of vascular elastin or its malformation within native vessels and the poor ability to tissue-engineer elastin-rich vascular replacements due to innately poor elastin synthesis by adult vascular cells can compromise vascular homeostasis, and must thus be addressed. Our recent studies attest to the utility of hyaluronan (HA) oligomers for elastin synthesis and organization by adult vascular smooth muscle cells (SMCs), though the elastin matrix yields in these cases were quite low relative to total elastin produced. Thus, in this study, we investigated the utility of copper (Cu(2+)) ions to enhance cellular elastin deposition, crosslinking and maturation into structural fibers. Copper nanoparticles (CuNPs; 80-100 nm) in the dose range of 1-100 ng ml(-1) were tested for Cu(2+) ion release, and based on mathematical modeling of their release profiles, CuNPs (1, 10, and 400 ng ml(-1)) were chosen for supplementation to adult SMC cultures. The 400 ng ml(-1) dose of CuNPs cumulatively delivered Cu(2+) doses in the range of 0.1 M, over the 21 day culture period. It was observed that while exogenous CuNP supplements do not up-regulate tropoelastin production by vascular SMCs, they promoted formation of crosslinked elastin matrices. The deposition of crosslinked matrix elastin was further improved by the additional presence of HA oligomers in these cultures. Immunofluorescence imaging and structural analysis of the isolated elastin matrices indicate that amorphous elastin clumps were formed within non-additive control cultures, while aggregating elastin fibrils were observed within SMC cultures treated with CuNPs (1-10 ng ml(-1)) alone or together with HA oligomers. The presence of 400 ng ml(-1) of CuNPs concurrent with HA oligomers furthered aggregation of these elastin fibrils into mature fibers with diameters ranging from 200 to 500 nm. Ultrastructural analysis of elastin matrix within cultures treated with HA oligomers and 400 ng ml(-1) of CuNPs suggest that elastin matrix deposition as stimulated by Cu(2+) ions proceeds via a fibrillin-mediated assembly process, with enhanced crosslinking occurring via stimulation of lysyl oxidase. Overall, the data suggest that CuNPs and HA oligomers are highly useful for regenerating crosslinked, fibrillar elastin matrices by adult vascular SMCs. These results have immense utility in tissue-engineering vascular replacements.

Benefits of concurrent delivery of hyaluronan and IGF-1 cues to regeneration of crosslinked elastin matrices by adult rat vascular cells.

Elastin, a major component of vascular matrices, critically determines vascular mechanics and maintains the quiescence of smooth muscle cells (SMCs). Attempts to regenerate elastin in elastin‐compromised blood vessels using tissue‐engineering approaches is limited by the unavailability of elastogenic cues to upregulate poor elastin output and matrix assembly by adult vascular cells. We previously showed that hyaluronan (HA) elastogenically stimulates aortic SMCs, although these effects are highly specific to HA fragment size. The elastogenic response of SMCs can also be modulated with growth factors such as insulin‐like growth factor (IGF‐1). Here, we evaluate the benefits of concurrent delivery of HA fragments (0.76–2000 kDa) and IGF‐1 (500 ng/ml) to elastin synthesis, organization and crosslinking. The study outcomes show that, relative to supplement‐free cultures, IGF‐1 and long‐chain HA/large HA fragments, but not HA oligomers, together induce multifold increases in the synthesis of elastin precursors, structural elastin matrix yields and crosslink densities within cell layers, and encourage elastic fibre formation. These outcomes are not all obtained when either of the cues is provided separately. IGF‐1 and large HA fragments (>20 kDa) also together inhibit cell proliferation, a concern in elastin‐compromised vessels, where SMC hyperproliferation is common. The results will benefit efforts to provide exogenous or scaffold‐based elastogenic cues (IGF‐1 + HMW HA/large HA fragments) to enable robust and faithful regeneration of elastin matrix structures in vivo or in vitro. The present outcomes may be used to restore elastin matrix homeostasis in de‐elasticized vessels and tissue‐engineered constructs that may be grafted as a substitute. Copyright

Lysyl Oxidase Enhances Elastin Synthesis and Matrix Formation by Vascular Smooth Muscle Cells

Lysyl oxidase (LOX) is a copper‐dependent enzyme that initiates covalent crosslinking of elastin precursors by oxidizing peptidyl lysine to aminoadipic semi‐aldehydes. Previous studies have shown LOX deficiency to affect crosslinking of elastin and collagen in vivo, resulting in disorganized connective tissue formation. In this study, we investigated the utility of exogenously supplemented LOX peptides (50–100 µl/well) to elastin synthesis, crosslinking efficiency and matrix deposition in adult rat aortic smooth muscle cell (RASMC) cultures. Additionally, we also examined the role of LOX peptides on SMC proliferation and matrix metalloproteinase (MMP) synthesis in these cultures. Highly purified bovine aorta LOX peptide was found to increase matrix elastin synthesis by 40–80% to that in control cultures in a dose‐dependent manner, while the crosslinking efficiency significantly (as measured by the ratio of matrix elastin protein to the total elastin protein synthesized) improved to 45–55% of total elastin synthesized under these conditions. However, LOX peptides affected neither SMC proliferation relative to controls, nor elastin precursor (tropoelastin) synthesis, nor the total elastin synthesis on a per‐cell basis. In general, LOX peptides also did not affect MMP‐2 and MMP‐9 activities relative to control cultures, except for MMP‐9 activity suppression at a higher LOX dose, suggesting that these LOX peptide cues could be safely used to enhance tropoelastin crosslinking into matrix structures and elastin matrix yield, within tissue‐engineered constructs, a major challenge in the field. Copyright

Characterization of glycidyl methacrylate - crosslinked hyaluronan hydrogel scaffolds incorporating elastogenic hyaluronan oligomers.

Prior studies on two-dimensional cell cultures suggest that hyaluronic acid (HA) stimulates cell-mediated regeneration of extracellular matrix structures, specifically those containing elastin, though such biologic effects are dependent on HA fragment size. Towards being able to regenerate three-dimensional (3-D) elastic tissue constructs, the present paper studies photo-crosslinked hydrogels containing glycidyl methacrylate (GM)-derivatized bio-inert high molecular weight (HMW) HA (1 × 10(6)Da) and a bioactive HA oligomer mixture (HA-o: MW ∼0.75 kDa). The mechanical (rheology, degradation) and physical (apparent crosslinking density, swelling ratio) properties of the gels varied as a function of incorporated HA oligomer content; however, overall, the mechanics of these hydrogels were too weak for vascular applications as stand-alone materials. Upon in vivo subcutaneous implantation, only a few inflammatory cells were evident around GM-HA gels, however their number increased as HA-o content within the gels increased, and the collagen I distribution was uniform. Smooth muscle cells (SMC) were encapsulated into GM hydrogels, and calcein acetoxymethyl detection revealed that the cells were able to endure twofold the level of UV exposure used to crosslink the gels. After 21 days of culture, SMC elastin production, measured by immunofluorescence quantification, showed HA-o to increase cellular deposition of elastic matrix twofold relative to HA-o-free GM-HA gels. These results demonstrate that cell response to HA/HA-o is not altered by their methacrylation and photo-crosslinking into a hydrogel, and that HA-o incorporation into cell-encapsulating hydrogel scaffolds can be useful for enhancing their production of elastic matrix structures in a 3-D space, important for regenerating elastic tissues.

Biomimetic Regeneration of Elastin Matrices Using Hyaluronan and Copper Ion Cues

Current efforts to tissue engineer elastin-rich vascular constructs and grafts are limited because of the poor elastogenesis of adult vascular smooth muscle cells (SMCs) and the unavailability of appropriate cues to upregulate and enhance cross-linking of elastin precursors (tropoelastin) into organized, mature elastin fibers. We earlier showed that hyaluronan (HA) fragments greatly enhance tropo- and matrix-elastin synthesis by SMCs, although the yield of matrix elastin is low. To improve matrix yields, here we investigate the benefits of adding copper (Cu(2+)) ions (0.01 M and 0.1 M), concurrent with HA (756-2000 kDa), to enhance lysyl oxidase (LOX)-mediated elastin cross-linking machinery. Although absolute elastin amounts in test groups were not different from those in controls, on a per-cell basis, 0.1 M of Cu(2+) ions slowed cell proliferation (5.6 +/- 2.3-fold increase over 21 days vs 22.9 +/- 4.2-fold for non-additive controls), stimulated synthesis of collagen (4.1 +/- 0.4-fold), tropoelastin (4.1 +/- 0.05-fold) and cross-linked matrix elastin (4.2 +/- 0.7-fold). LOX protein synthesis increased 2.5 times in the presence of 0.1 M of Cu(2+) ions, and these trends were maintained even in the presence of HA fragments, although LOX functional activity remained unchanged in all cases. The abundance of elastin and LOX in cell layers cultured with 0.1 M of Cu(2+) ions and HA fragments was qualitatively confirmed using immunoflourescence. Scanning electron microscopy images showed that SMC cultures supplemented with 0.1 M of Cu(2+) ions and HA oligomers and large fragments exhibited better deposition of mature elastic fibers ( approximately 1 mum diameter). However, 0.01 M of Cu(2+) ions did not have any beneficial effect on elastin regeneration. In conclusion, the results suggest that supplying 0.1 M of Cu(2+) ions to SMCs to concurrently (a) enhance per-cell yield of elastin matrix while allowing cells to remain viable and synthetic and not density-arrested in long-term culture because of their moderating effects on otherwise rapid cell proliferation and (b) provide additional benefits of enhanced elastin fiber formation and cross-linking within these tissue-engineered constructs.

Induced elastin regeneration by chronically activated smooth muscle cells for targeted aneurysm repair

Elastin breakdown in vascular aneurysms is mediated by cytokines such as tumor necrosis factor alpha (TNF-alpha, which induces vascular smooth muscle cell (SMC) activation and regulates their deposition of matrix. We previously demonstrated that exogenous supplementation with TGF-beta1 (1 ng ml(-1)) and hyaluronan oligomers (0.786 kDa, 0.2 microg ml(-1)) cues the upregulation of elastin matrix synthesis by healthy cultured SMCs. Here, we determine whether these cues likewise enhance elastin matrix synthesis and assembly by TNF-alpha-stimulated SMCs, while restoring their healthy phenotype. Adult rat aortic SMCs were treated with TNF-alpha alone or together with TGF-beta1/hyaluronan oligomeric cues and the release of inflammatory markers were monitored during over a 21 day culture. Biochemical analysis was used to quantify cell proliferation, matrix protein synthesis and cross-linking efficiency, while immunofluorescence and electron microscopy were used to analyze the elastin matrix quality. It was observed that SMC activation with TNF-alpha (10 ng ml(-1)) induced matrix calcification and promoted production of elastolytic MMP-2 and MMP-9. However, these effects were attenuated by the addition of TGF-beta1 and HA oligomer cues to TNF-alpha-stimulated cultures, which also enhanced tropoelastin and collagen production, improved elastin matrix yield and cross-linking, promoted elastin fiber formation and suppressed elastase activity, although the release of MMP-2 and MMP-9 was not affected. Overall, the results suggest that TGF-beta1 and HA oligomers are potentially useful in suppressing SMC activation and inducing regenerative elastin repair within aneurysms.

Tuning composition and architecture of biomimetic scaffolds for enhanced matrix synthesis by murine cardiomyocytes

A major onset of heart failure is myocardial infarction, which causes the myocardium to lose cardiomyocytes and transform into a scar tissue. Since mammalian infarcted cardiac tissue has a limited ability to regenerate, alternative strategies including implantation of tissue-engineered scaffolds at the site of damaged myocardium have been explored. The goal is to enable in situ cardiac reconstruction at the injured myocardium site, replace the lost cardiomyocytes, deliver the required biomolecules, and remodel the extracellular matrix (ECM). ECM synthesis and deposition by cardiomyocytes within such scaffolds remains categorically unexplored. Here, we investigated the survival, ECM synthesis and deposition, and matrix metalloproteinases (MMPs) release by cardiomyocytes within three-dimensional (3D) substrates. Rat cardiomyocytes were cultured for three weeks within two structurally different substrates: 3D collagen hydrogels or polycaprolactone (PCL) nanofibrous scaffolds. The concentration and composition of the hydrogels was varied, while PCL nanofibers were surface-modified with various ECM proteins. Results showed that myocyte attachment and survival was higher within collagen hydrogels, while myocyte alignment and beating was noted only within PCL scaffolds. Total protein synthesis by myocytes within PCL scaffolds was significantly higher compared to that within collagen hydrogels, although more protein was deposited as matrix within hydrogels. Significant ECM synthesis and matrix deposition, TIMP-1, and MMP release were noted within modified collagen hydrogels and PCL nanofiber scaffolds. These results were qualitatively confirmed by imaging techniques. Results attest to the prominent role of scaffold composition and architecture in influencing cardiomyocyte phenotype, matrix synthesis and cytokines release, with significant applications in cardiac tissue remodeling strategies.