Abnormal motion of the interventricular septum (ASM), seen post cardiac operation, with left bundle branch block or right ventricular pacing, may affect septal mitral annular motion and correlation of the ratio between the velocity of early diastolic mitral inflow and the early diastolic mitral annular velocity (E/Ea) with pulmonary capillary wedge pressure (PCWP). We examined the effect of ASM on the relationship between E/Ea and E/Vp (propagation velocity of mitral inflow) ratios and PCWP in adult patients in the intensive care unit (14 with normal septal motion [NSM], 36 with ASM) undergoing echocardiography and pulmonary artery catheterization. E/Ea correlated well with PCWP during NSM ( r = 0.86 lateral annulus, r = 0.75 septal annulus), but poorly during ASM ( r = 0.36 lateral annulus, r = 0.39 septal annulus). E/Vp correlated poorly with PCWP ( r = 0.05 NSM, r = 0.17 ASM). For patients who are critically ill, E/Vp ratios poorly estimate PCWP. During NSM, E/Ea ratios measured at the lateral or septal annulus correlate well with PCWP. ASM affects E/Ea ratios at both the septal and lateral annulus, making E/Ea ratios unreliable for estimating PCWP in this group.
Degradability is often a critical property of materials utilized in tissue engineering. Although alginate, a naturally derived polysaccharide, is an attractive material due to its biocompatibility and ability to form hydrogels, its slow and uncontrollable degradation can be an undesirable feature. In this study, we characterized gels formed using a combination of partial oxidation of polymer chains and a bimodal molecular weight distribution of polymer. Specifically, alginates were partially oxidized to a theoretical extent of 1% with sodium periodate, which created acetal groups susceptible to hydrolysis. The ratio of low MW to high MW alginates used to form gels was also varied, while maintaining the gel forming ability of the polymer. The rate of degradation was found to be controlled by both the oxidation and the ratio of high to low MW alginates, as monitored by the reduction of mechanical properties and corresponding number of crosslinks, dry weight loss, and molecular weight decrease. It was subsequently examined whether these modifications would lead to reduced biocompatibility by culturing C2C12 myoblast on these gels. Myoblasts adhered, proliferated, and differentiated on the modified gels at a comparable rate as those cultured on the unmodified gels. Altogether, this data indicates these hydrogels exhibit tunable degradation rates and provide a powerful material system for tissue engineering.
Multipotent cell types are rapidly becoming key components in a variety of tissue engineering schemes, and mesenchymal stem cells (MSCs) are emerging as an important tool in bone tissue regeneration. Although several soluble signals influencing osteogenic differentiation of MSCs in vitro are well-characterized, relatively little is known about the influence of substrate signals. This study was aimed at elucidating the effects of a bone-like mineral (BLM), which is vital in the process of bone bonding to orthopedic implant materials, on the osteogenic differentiation of human MSCs in vitro. Growth of a BLM film (carbonate apatite, Ca/P = 1.55) on poly(lactide-co-glycolide) (PLG) substrates was achieved via surface hydrolysis and subsequent incubation in a modified simulated body fluid. The BLM film demonstrated significantly increased adsorption of fibronectin, and supported enhanced proliferation of human mesenchymal stem cells (hMSCs) relative to PLG substrates. In the absence of osteogenic supplements hMSCs did not display a high expression of osteogenic markers on BLM or PLG. In the presence of osteogenic supplements hMSCs exhibited greater expression of osteogenic markers on PLG substrates than on BLM substrates, as measured by alkaline phosphatase activity and osteocalcin production. Taken together, these data support the concept that substrate signals significantly influence MSC growth and differentiation, highlighting the importance of carrier material composition in stem cell-based tissue engineering schemes.
OBJECTIVES: Describe current and future approaches to tissue engineering, specifically in the area of bone regeneration. These approaches will allow one to actively regulate the cellular populations participating in this process.
DESIGN: Many approaches to actively regulate cellular phenotype are under exploration, and these typically exploit known signal transduction pathways via presentation of specific receptor-binding ligands, and may also deliver mechanical information via the physical bridge formed by the receptor-ligand interactions. Cellular gene expression may also be directly modulated utilizing gene therapy approaches to control tissue regeneration.
CONCLUSIONS: Significant progress has been made to date in bone regeneration using inductive molecules and transplanted cells, and FDA approved therapies have resulted. While approaches to date have focused on delivery of single stimuli (e.g. one growth factor), future efforts will likely attempt to more closely mimic developmental processes by the delivery of multiple inputs to the cells in spatially and temporally regulated fashions.
Gene therapy approaches to bone tissue engineering have been widely explored. While localized delivery of plasmid DNA encoding for osteogenic factors is attractive for promoting bone regeneration, the low transfection efficiency inherent with plasmid delivery may limit this approach. We hypothesized that this limitation could be overcome by condensing plasmid DNA with nonviral vectors such as poly(ethylenimine) (PEI), and delivering the plasmid DNA in a sustained and localized manner from poly(lactic-co-glycolic acid) (PLGA) scaffolds. To address this possibility, scaffolds delivering plasmid DNA encoding for bone morphogenetic protein-4 (BMP-4) were implanted into a cranial critical-sized defect for time periods up to 15 weeks. The control conditions included no scaffold (defect left empty), blank scaffolds (no delivered DNA), and scaffolds encapsulating plasmid DNA (non-condensed). Histological and microcomputed tomography analysis of the defect sites over time demonstrated that bone regeneration was significant at the defect edges and within the defect site when scaffolds encapsulating condensed DNA were placed in the defect. In contrast, bone formation was mainly confined to the defect edges within scaffolds encapsulating plasmid DNA, and when blank scaffolds were used to fill the defect. Histomorphometric analysis revealed a significant increase in total bone formation (at least 4.5-fold) within scaffolds incorporating condensed DNA, relative to blank scaffolds and scaffolds incorporating uncondensed DNA at each time point. In addition, there was a significant increase both in osteoid and mineralized tissue density within scaffolds incorporating condensed DNA, when compared with blank scaffolds and scaffolds incorporating uncondensed DNA, suggesting that delivery of condensed DNA led to more complete mineralized tissue regeneration within the defect area. This study demonstrated that the scaffold delivery system encapsulating PEI-condensed DNA encoding for BMP-4 was capable of enhancing bone formation and may find applications in other tissue types.
Peptide modification of hydrogel-forming materials is being widely explored as a means to regulate the phenotype of cells immobilized within the gels. Alternatively, we hypothesized that the adhesive interactions between cells and peptides coupled to the gel-forming materials would also enhance the overall mechanical properties of the gels. To test this hypothesis, alginate polymers were modified with RGDSP-containing peptides and the resultant polymer was used to encapsulate C2C12 myoblasts. The mechanical properties of these gels were then assessed as a function of both peptide and cell density using compression and tensile tests. Overall, it was found that above a critical peptide and cell density, encapsulated myoblasts were able to provide additional mechanical integrity to hydrogels composed of peptide-modified alginate. This occurred presumably by means of cell-peptide cross-linking of the alginate polymers, in addition to the usual Ca++ cross-linking. These results are potentially applicable to other polymer systems and important for a range of tissue engineering applications.
UNLABELLED: Bone formation is a coordinated process involving various biological factors. We have developed a scaffold system capable of sustained and localized presentation of osteogenic (BMP-4) and angiogenic (VEGF) growth factors and human bone marrow stromal cells to promote bone formation at an ectopic site. Combined delivery of these factors significantly enhanced bone formation compared with other conditions.
INTRODUCTION: Tissue regeneration entails complex interactions between multiple signals and materials platforms. Orchestrating the presentation of these signals may greatly enhance the regeneration of lost tissue mass. Bone formation, for example, is dependent on the signaling of BMPs, molecules initiating vascularization (e.g., vascular endothelial growth factor [VEGF]), and osteogenic precursor cells capable of responding to these cues and forming bone tissue. It was hypothesized that combined and concerted delivery of these factors from biodegradable scaffolds would lead to enhanced bone formation.
MATERIALS AND METHODS: Poly(lactic-co-glycolic acid) scaffolds containing combinations of condensed plasmid DNA encoding for BMP-4, VEGF, and human bone marrow stromal cells (hBMSCs) were implanted into the subcutaneous tissue of SCID mice. Implants (n = 6) were retrieved at 3, 8, and 15 weeks after implantation. Bone and blood vessel formation was determined qualitatively and quantitatively by methods including histology, immmunostaining, and muCT.
RESULTS: Scaffolds delivering VEGF resulted in a prominent increase in blood vessel formation relative to the conditions without VEGF. BMP-4 expression in scaffolds encapsulating condensed DNA was also confirmed at the 15-week time-point, showing the characteristic of long-term delivery in this system. Combined delivery of all three types of factors resulted in a significant increase in the quantity of regenerated bone compared with any factor alone or any two factors combined, as measured with DXA, X-ray, and histomorphometric analysis. Furthermore, bone formed with all three factors had elastic moduli significantly higher than any other condition.
CONCLUSIONS: Concerted delivery of BMP-4, VEGF, and hBMSCs promoted greater bone formation relative to any single factor or combination of two factors. Materials systems that allows multifactorial presentation more closely mimic natural developmental processes, and these results may have important implications for bone regeneration therapeutics.
In the context of bone development and regeneration, the intimate association of the vascular endothelium with osteogenic cells suggests that endothelial cells (ECs) may directly regulate the differentiation of osteoprogenitor cells. To investigate this question, bone marrow stromal cells (BMSCs) were cultured: in the presence of EC-conditioned medium, on EC extracellular matrix, and in EC cocultures with and without cell contact. RNA and protein were isolated from ECs and analyzed by reverse transcriptase-polymerase chain reaction and Western blotting, respectively, for expression of bone morphogenetic protein 2 (BMP-2). In animal studies, BMSCs and ECs were cotransplanted into severe combined immunodeficient mice on biodegradable polymer matrices, and histomorphometric analysis was performed to determine the extent of new bone and blood vessel formation. ECs significantly increased BMSC osteogenic differentiation in vitro only when cultured in direct contact. ECs expressed BMP-2, and experiments employing interfering RNA inhibition confirmed its production as contributing to the increased BMSC osteogenic differentiation. In vivo, cotransplantation of ECs with BMSCs resulted in greater bone formation than did transplantation of BMSCs alone. These data suggest that ECs function not only to form the microvasculature that delivers nutrients to developing bone but also to modulate the differentiation of osteoprogenitor cells in vitro and in vivo.
The mechanical properties of cell adhesion substrates regulate cell phenotype, but the mechanism of this relation is currently unclear. It may involve the magnitude of traction force applied by the cell, and/or the ability of the cells to rearrange the cell adhesion molecules presented from the material. In this study, we describe a FRET technique that can be used to evaluate the mechanics of cell-material interactions at the molecular level and simultaneously quantify the cell-based nanoscale rearrangement of the material itself. We found that these events depended on the mechanical rigidity of the adhesion substrate. Furthermore, both the proliferation and differentiation of preosteoblasts (MC3T3-E1) correlated to the magnitude of force that cells generate to cluster the cell adhesion ligands, but not the extent of ligand clustering. Together, these data demonstrate the utility of FRET in analyzing cell-material interactions, and suggest that regulation of phenotype with substrate stiffness is related to alterations in cellular traction forces.
Nonviral delivery vectors are attractive for gene therapy approaches in tissue engineering, but suffer from low transfection efficiency and short-term gene expression. We hypothesized that the sustained delivery of poly(ethylenimine) (PEI)-condensed DNA from three-dimensional biodegradable scaffolds that encourage cell infiltration could greatly enhance gene expression. To test this hypothesis, a PEI-condensed plasmid encoding beta-galactosidase was incorporated into porous poly(lactide-co-glycolide) (PLG) scaffolds, using a gas foaming process. Four conditions were examined: condensed DNA and uncondensed DNA encapsulated into PLG scaffolds, blank scaffolds, and bolus delivery of condensed DNA in combination with implantation of PLG scaffolds. Implantation of scaffolds incorporating condensed beta-galactosidase plasmid into the subcutaneous tissue of rats resulted in a high level of gene expression for the entire 15-week duration of the experiment, as exemplified by extensive positive staining for beta-galactosidase gene expression observed on the exterior surface and throughout the cross-sections of the explanted scaffolds. No positive staining could be observed for the control conditions either on the exterior surface or in the cross-section at 8- and 15-week time points. In addition, a high percentage (55-60%) of cells within scaffolds incorporating condensed DNA at 15 weeks demonstrated expression of the DNA, confirming the sustained uptake and expression of the encapsulated plasmid DNA. Quantitative analysis of beta-galactosidase gene expression revealed that expression levels in scaffolds incorporating condensed DNA were one order of magnitude higher than those of other conditions at the 2- week time point and nearly two orders of magnitude higher than those of the control conditions at the 8- and 15-week time points. This study demonstrated that the sustained delivery of PEI-condensed plasmid DNA from PLG scaffolds led to an in vivo long-term and high level of gene expression, and this system may find application in areas such as bone tissue engineering.
Non-viral gene vectors are commonly used for gene therapy owing to safety concerns with viral vectors. However, non-viral vectors are plagued by low levels of gene transfection and cellular expression. Current efforts to improve the efficiency of non-viral gene delivery are focused on manipulations of the delivery vector, whereas the influence of the cellular environment in DNA uptake is often ignored. The mechanical properties (for example, rigidity) of the substrate to which a cell adheres have been found to mediate many aspects of cell function including proliferation, migration and differentiation, and this suggests that the mechanics of the adhesion substrate may regulate a cell's ability to uptake exogeneous signalling molecules. In this report, we present a critical role for the rigidity of the cell adhesion substrate on the level of gene transfer and expression. The mechanism relates to material control over cell proliferation, and was investigated using a fluorescent resonance energy transfer (FRET) technique. This study provides a new material-based control point for non-viral gene therapy.
PURPOSE: We hypothesized that sustained delivery of vascular endothelial growth factor (VEGF) using a polymer [85:15 poly(lactide-co-glycolide) (PLG)] would enhance angiogenesis and improve perfusion of ischemic tissue.
METHODS: C57BL/6J mice (n = 20/group) underwent unilateral hind limb ischemia surgery and were randomized to groups of no scaffold implantation (0-Implant), unloaded scaffold implantation (Empty-PLG), or implantation of scaffolds incorporating 3 microg of VEGF165 (PLG-VEGF). Endpoints included laser Doppler perfusion imaging (LDPI, ischemic/nonischemic limb, %), local vessel counts, immunohistochemistry for CD31, and alpha-smooth muscle actin. In vitro release kinetics of VEGF from PLG was also measured.
RESULTS: PLG-VEGF resulted in improved lower extremity perfusion vs. controls as measured by LDPI% at 7, 14, 21, and 28 days (p < 0.05). PLG-VEGF was associated with significantly greater percentage of vessels staining for CD31 and alpha-smooth muscle actin compared to the Empty-PLG or 0-Implant (p < 0.05 for both).
CONCLUSIONS: The PLG-VEGF scaffolds resulted in sustained VEGF delivery, improved tissue perfusion, greater capillary density, and more mature vasculature compared to the controls. The sustained-release PLG polymer vehicle is a promising delivery system for therapeutic neovascularization applications.
This study characterized the attachment of chondrocytes to RGD-functionalized alginate by examining the effect of substrate stiffness on cell attachment and morphology. Bovine chondrocytes were added to wells coated with 2% alginate or RGD-alginate. The alginate was crosslinked with divalent cations ranging from 1.25 to 62.5 mmol/g alginate. Attachment to RGD-alginate was 10-20 times higher than attachment to unmodified alginate and was significantly inhibited by antibodies to integrin subunits alpha3l and beta1, cytochalasin-D, and soluble RGD peptide. The equilibrium level and rate of attachment increased with crosslink density and substrate stiffness. Substrate stiffness also regulated chondrocyte morphology, which changed from a rounded shape with nebulous actin on weaker substrates to a predominantly flat morphology with actin stress fibers on stiffer substrates. The dependence of attachment on integrins and substrate stiffness suggests that chondrocyte integrins may play a role in sensing the mechanical properties of the matrices to which they are attached.
Maintenance of liver-specific function of hepatocytes in culture is still difficult. Improved culture conditions may enhance the cell growth and function of cultured cells. We investigated the effect of three-dimensional culture under flow conditions, and the influence of surface modifications in hepatocyte cultures. Hepatocytes were harvested from Lewis rats. Cells were cultured on three-dimensional polymeric poly-lactic-co-glycolic acid (PLGA) matrices in static culture, or in a pulsatile flow-bioreactor system. Different surface modifications of matrices were investigated: coating with collagen I, collagen IV, laminin, or fibronectin; or uncoated matrix. Hepatocyte numbers, DNA content, and albumin secretion rate were assessed over the observation period. Culture under flow condition significantly enhanced cell numbers. An additional improvement of this effect was observed, when matrix coating was used. Cellular function also showed a significant increase (4- to 5-fold) under flow conditions when compared with static culture. Our data showed that culture under flow conditions improves cell number, and strongly enhances cellular function. Matrix modification by coating with extracellular matrix showed overall an additive stimulatory effect. Our conclusion is that combining three-dimensional culture under flow conditions and using matrix modification significantly improves culture conditions and is therefore attractive for the development of successful culture systems for hepatocytes.
Angiogenesis and biomineral substrates play major roles in bone development and regeneration. We hypothesized that macroporous scaffolds of biomineralized 85:15 poly(lactide-co-glycolide), which locally release vascular endothelial growth factor-165 (VEGF), would direct simultaneous regeneration of bone and vascular tissue. The presence of a bone-like biomineral substrate significantly increased regeneration of osteoid matrix (32 +/- 7% of total tissue area; mean +/- SD; p < 0.05) and mineralized tissue (14 +/- 2%; P < 0.05) within a rat cranium critical defect compared with a non-mineralized polymer scaffold (19 +/- 8% osteoid and 10 +/- 2% mineralized tissue). Further, the addition of VEGF to a mineralized substrate significantly increased the generation of mineralized tissue (19 +/- 4%; P < 0.05) compared with mineralized substrate alone. This appeared to be due to a significant increase in vascularization throughout VEGF-releasing scaffolds (52 +/- 9 vessels/mm(2); P < 0.05) compared with mineralized scaffolds without VEGF (34 +/- 4 vessels/mm(2)). Surprisingly, there was no significant difference in total osteoid between the two samples, suggesting that increased vascularization enhances mineralized tissue generation, but not necessarily osteoid formation. These results indicate that induced angiogenesis can enhance tissue regeneration, supporting the concept of therapeutic angiogenesis in tissue-engineering strategies.
Hydrogels, chemically cross-linked or physically entangled, have found a number of applications as novel delivery vehicles of drugs and cells. However, the narrow ranges of degradation rates and mechanical strength currently available from many hydrogels limits their applications. We have hypothesized that utilization of multi-functional cross-linking molecules to form hydrogels could provide a wider range and tighter control over the degradation rates and mechanical stiffness of gels than bi-functional cross-linking molecules. To address the possibility, we isolated alpha-L-guluronate residues of sodium alginate, and oxidized them to prepare poly(aldehyde guluronate) (PAG). Hydrogels were formed with either poly(acrylamide-co-hydrazide) (PAH) as a multi-functional cross-linking molecule or adipic acid dihydrazide (AAD) as a bi-functional cross-linking molecule. The initial properties and degradation behavior of both PAG gel types were monitored. PAG/PAH hydrogels showed higher mechanical stiffness before degradation and degraded more slowly than PAG/AAD gels, at the same concentration of cross-linking functional groups. The enhanced mechanical stiffness and prolonged degradation behavior could be attributed to the multiple attachment points of PAH in the gel at the same concentration of functional groups. This approach to regulating gel properties with multifunctional cross-linking molecules could be broadly used in hydrogels.
A developing therapy for complete or partial loss of function in various tissues and organs involves transplanting an appropriate cell population, capable of compensating for the existing deficiencies. Clinical application of this type of strategy is currently limited by the death or dedifferentiation of the transplanted cells after delivery to the recipient. A delay in thorough vascularization of the implant area creates an environment low in oxygen and other nutrients, and likely contributes to the initial death of transplanted cells. We have addressed this problem by sustained delivery of vascular endothelial growth factor (VEGF), an initiator of angiogenesis, from a porous polymer matrix utilized simultaneously for cell delivery. As expected from previous studies, VEGF delivered from these constructs elicited an enhanced angiogenic response over a 2-week period when implanted subcutaneously in SCID mice. Hepatocytes implanted using VEGF-containing matrices demonstrated significantly greater survival after 1 week in vivo as compared with cells implanted on matrices without growth factor. The results of this study therefore indicate that enhancing vascularization in the location of transplanted cells promotes their survival. In addition, this delivery system may be used in future studies to directly promote cell survival and function by also providing growth factors specific to the transplanted cells.
Alginate hydrogels are currently being employed and explored for a broad range of medical applications including cell encapsulation, drug delivery, and tissue engineering. In these capacities, knowledge of the mechanical and material properties of the hydrogels and the properties that govern and influence them is necessary to adequately design and effectively use these systems. Although much is known about the mechanical properties of alginate in compression and shear, little is known about the tensile characteristics. Thus, an extensive tensile assessment of alginate hydrogels was completed as a function of alginate type, formulation, gelling conditions, incubation, and strain rate. In general, the initial tensile behavior and properties of alginate hydrogels were highly dependent on the choice of the alginate polymer and how it was processed. Specifically, high guluronic acid containing alginate polymers yielded stronger, more ductile hydrogels than high mannuronic acid containing alginates. The ultimate stress, ultimate strain, and tensile modulus were decreased by increased phosphate concentrations, solution reconstitution with phosphate buffered saline instead of culture media, and peptide modification. Incubation of hydrogels for at least 7 days diminished many of the initial tensile property differences associated with formulation and gelling conditions. Overall, by controlling the specific alginate polymer and processing methods, a wide range of tensile properties are available from these hydrogels.
Bone regeneration can be enhanced or accelerated by the delivery of osteogenic signalling factors or bone forming cells. These factors have commonly provided benefit when retained at the defect site with a delivery vehicle formed from natural or synthetic materials. Growth factors can be directly delivered as recombinant proteins or expressed by genetically modified cells to induce bone formation. Furthermore, bone regeneration has been achieved with the transplantation of various cell types that can participate in bone healing. Carriers utilised for the delivery of osteoinductive material allow for a prolonged presentation at the repair site and the timing of presentation can be readily adjusted to correspond to the extent necessary for bone regeneration. This review examines some of the recent developments in delivery systems used to manage the presentation of these factors at the desired site. Moreover, the authors provide suggestions for continued progress in bone regeneration.