Dynamic compressive loading enhances cartilage matrix synthesis and distribution and suppresses hypertrophy in hMSC-laden hyaluronic acid hydrogels

Mesenchymal stem cells (MSCs) are being recognized as a viable cell source for cartilage repair, and there is growing evidence that mechanical signals play a critical role in the regulation of stem cell chondrogenesis and in cartilage development. In this study we investigated the effect of dynamic compressive loading on chondrogenesis, the production and distribution of cartilage specific matrix, and the hypertrophic differentiation of human MSCs encapsulated in hyaluronic acid (HA) hydrogels during long term culture. After 70 days of culture, dynamic compressive loading increased the mechanical properties, as well as the glycosaminoglycan (GAG) and collagen contents of HA hydrogel constructs in a seeding density dependent manner. The impact of loading on HA hydrogel construct properties was delayed when applied to lower density (20 million MSCs/ml) compared to higher seeding density (60 million MSCs/ml) constructs. Furthermore, loading promoted a more uniform spatial distribution of cartilage matrix in HA hydrogels with both seeding densities, leading to significantly improved mechanical properties as compared to free swelling constructs. Using a previously developed in vitro hypertrophy model, dynamic compressive loading was also shown to significantly reduce the expression of hypertrophic markers by human MSCs and to suppress the degree of calcification in MSC-seeded HA hydrogels. Findings from this study highlight the importance of mechanical loading in stem cell based therapy for cartilage repair in improving neocartilage properties and in potentially maintaining the cartilage phenotype.     

组织工程化软骨的应力—应变研究

为研究组织工程化软骨的应力－应变特性,采用生物力学方法对体外培养离心管软骨和裸鼠皮下培养胶原海绵构建的软骨在5％变形时的压缩模量进行了测试,结果表明,体外离心管培养软骨在4,8,12,16周时的压缩模量分别为0．3518,0．653,0．233,0．262MPa,其中在8周时达最高,低于天然人胎关节软骨压缩模量2个数量级,体内皮下培养的胶原海绵软骨16周时的压缩模量的动态变化提示它与软骨细胞分泌GAG的活性变化一致,都在8周时达高峰,离心管软骨压缩模量总体偏低的现象提示可能是由于培养液与人工软骨大分子之间缺乏屏障保护,细胞分泌的PGS分子易于流失到培养液中的结果,而体内皮下培养的软骨模量低于天然软骨的原因可能是软骨发育过程中缺乏负荷刺激,因此,从生物力学角度看,组织工程化软骨的体外培养方法尚需改进。     

Effects of auricular chondrocyte expansion on neocartilage formation in photocrosslinked hyaluronic acid networks

The overall objective of this study was to examine the effects of in vitro expansion on neocartilage formation by auricular chondrocytes photoencapsulated in a hyaluronic acid (HA) hydrogel as a next step toward the clinical application of tissue engineering therapies for treatment of damaged cartilage. Swine auricular chondrocytes were encapsulated either directly after isolation (p = 0), or after further in vitro expansion ( p = 1 and p = 2) in a 2 wt%, 50-kDa HA hydrogel and implanted subcutaneously in the dorsum of nude mice. After 12 weeks, constructs were explanted for mechanical testing and biochemical and immunohistochemical analysis and compared to controls of HA gels alone and native cartilage. The compressive equilibrium moduli of the p = 0 and p = 1 constructs (51.2 +/- 8.0 and 72.5 +/- 35.2 kPa, respectively) were greater than the p = 2 constructs (26.8 +/- 14.9 kPa) and the control HA gel alone (12.3 +/- 1.3 kPa) and comparable to auricular cartilage (35.1 +/- 12.2 kPa). Biochemical analysis showed a general decrease in glycosaminoglycan (GAG), collagen, and elastin content with chondrocyte passage, though no significant differences were found between the p = 0 and p = 1 constructs for any of the analyses. Histological staining showed intense and uniform staining for aggrecan, as well as greater type II collagen versus type I collagen staining in all constructs. Overall, this study illustrates that constructs with the p = 0 and p = 1 auricular chondrocytes produced neocartilage tissue that resembled native auricular cartilage after 12 weeks in vivo. However, these results indicate that further expansion of the chondrocytes (p = 2) can lead to compromised tissue properties.     

Effects of hypertonic (NaCl) two-dimensional and three-dimensional culture conditions on the properties of cartilage tissue engineered from an expanded mature bovine chondrocyte source

Clinically relevant mature cartilage cells (chondrocytes) present challenges for use in cartilage tissue engineering applications, given their low capacity for cell division and tissue production. Since the in situ environment of chondrocytes is hypertonic relative to standard culture medium conditions, in this study we tested the hypothesis that using culture medium of a hypertonic, more physiologic osmolarity during both two-dimensional (2D) expansion of mature bovine chondrocytes (MBCs) and their subsequent encapsulation culture in three-dimensional (3D) agarose hydrogel constructs produces improved engineered tissue construct mechanical and biochemical properties. Results demonstrate that 2D expansion of MBCs in hypertonic (NaCl) medium before encapsulation yielded improved construct mechanical properties. However, 3D encapsulation culture of cells in hypertonic (NaCl) medium yielded poorer construct mechanical properties. Osmolarity-related differences in construct biochemical content and organization may have contributed to differences in mechanical properties, as construct glycosaminoglycan content correlated moderately with construct mechanical properties, and construct collagen distribution varied between 3D osmotic culture groups. Results of this study suggest that application of hypertonic (NaCl) medium during 2D mature chondrocyte expansion, but not 3D encapsulated chondrocyte culture, may serve as a convenient and inexpensive method for improving mechanical properties of expanded cell-seeded constructs.     

The effect of the duration of mechanical stimulation and post-stimulation culture on the structure and properties of dynamically compressed tissue-engineered menisci

This study investigated the hypothesis that timing and duration of dynamic compression are integral to regulating extracellular matrix (ECM) assembly of tissue-engineered (TE) menisci. The goal of this study was to examine the effects of varying load and static culture duration on structure, composition, and mechanical properties of TE menisci. We accomplished this by varying the duration of dynamic loading over 4 weeks of culture, and by examining increasing periods of static culture after 2 weeks of dynamic loading. Bovine meniscal fibrochondrocytes were seeded into 2% w/v alginate, crosslinked with CaSO(4), injected into anatomical micro-computed tomography-based molds, and post-crosslinked with CaCl(2). Meniscal constructs were dynamically compressed three times a week via a custom bioreactor for a total of 2?h, with an hour of rest between loading cycles, for 1, 2, or 4 weeks. They were then placed in static culture. After 4 weeks of culture, increased load duration was found to be beneficial to matrix formation and mechanical properties, with superior mechanical and biochemical properties in samples loaded for 2 or 4 weeks. Further, the mechanical properties of these constructs were similar, suggesting that the additional 2 weeks of loading may not be necessary. Samples loaded for 2 weeks followed by a 4-week static culture period yielded the most mature matrix with significant improvements in collagen bundle formation, 2.8-fold increase in the glycosaminoglycan content, 2-fold increase in the collagen content, and 4.3-fold increase in the compressive equilibrium modulus. Overall, this study demonstrated the importance of timing and duration of loading. By switching to prolonged static culture after 2 weeks of loading, we decreased the amount of ECM lost to the media, while significantly increasing biochemical and mechanical properties of TE menisci.     

The effects of intermittent hydrostatic pressure on self-assembled articular cartilage constructs

To date, static culture for the tissue engineering of articular cartilage has shown to be inadequate in conferring functionality to constructs. Various forms of mechanical stimuli accompany articular cartilage development in vivo, and one of these is hydrostatic pressure. This study used histology, biochemistry, and biomechanics to examine the effects of intermittent hydrostatic pressure, applied at 10 MPa and 1 Hz for 4 h per day for 5 days per week for up to 8 weeks on self-assembled chondrocyte constructs. The self-assembling process is a novel approach that allows engineering of articular cartilage constructs without the use of exogenous scaffolds. The self-assembled constructs were found to be capable of enduring this loading regimen. Significant increases in collagen production were only observed in pressurized samples. Intermittent hydrostatic pressure prevented a significant decrease in total GAG, which was significant in controls. Aside from the beneficial effects intermittent hydrostatic pressure may have on ECM synthesis, its effects on mechanical properties may require longer culture periods to manifest. This study demonstrates the successful use of the self-assembling process to produce articular cartilage constructs. It also shows for the first time that long-term culture of tissue-engineered articular cartilage construct benefits from intermittent hydrostatic pressure.     

The influence of ascorbic acid,TGF-β1, and cell-mediated remodeling on the bulk mechanical properties of 3-D PEG–fibrinogen constructs

Two-dimensional cell culture studies have shown that matrix rigidity can influence cell function, but little is known about how matrix physical properties, and their changes with time, influence cell function in 3-D. Biosynthetic hydrogels based on PEGylated fibrinogen permit the initial decoupling of matrix chemical and mechanical properties, and are thus potentially attractive for addressing this question. However, the mechanical stability of these gels due to passive hydrolysis and cell-mediated remodeling has not previously been addressed. Here, we show that the bulk mechanical properties of acellular PEG–fibrinogen hydrogels significantly decrease over time in PBS regardless of matrix cross-linking density in 7 days. To compensate, smooth muscle cells (SMCs) were encapsulated and stimulated to produce their own matrix using ascorbic acid or TGF-β1. Ascorbic acid treatment improved the mechanical properties of the constructs after 14 days in less cross-linked matrices, but TGF-β1 did not. The increase in matrix modulus of the constructs was not due to an increase in type I collagen deposition, which remained low and pericellular regardless of cross-link density or the soluble factor applied. Instead, ascorbic acid, but not TGF-β1, preferentially enhanced the contractile SMC phenotype in the less cross-linked gels. Inhibition of contractility reduced cell spreading and the expression of contractile markers, and eliminated any beneficial increase in matrix modulus induced by cell-generated contraction of the gels. Together, these data show that PEG–fibrinogen hydrogels are susceptible to both hydrolysis and proteolysis, and suggest that some soluble factors may stimulate matrix remodeling by modulating SMC phenotype instead of inducing ECM synthesis in a 3-D matrix.     

A Paradigm for Functional Tissue Engineering of Articular Cartilage via Applied Physiologic Deformational Loading

Deformational loading represents a primary component of the chondrocyte physical environment in vivo. This review summarizes our experience with physiologic deformational loading of chondrocyte-seeded agarose hydrogels to promote development of cartilage constructs having mechanical properties matching that of the parent calf tissue, which has a Young's modulus E(Y) = 277 kPa and unconfined dynamic modulus at 1 Hz G* = 7 MPa. Over an 8-week culture period, cartilage-like properties have been achieved for 60 x 10(6) cells/ml seeding density agarose constructs, with E(Y) = 186 kPa, G* = 1.64 MPa. For these constructs, the GAG content reached 1.74% ww and collagen content 2.64% ww compared to 2.4% ww and 21.5% ww for the parent tissue, respectively. Issues regarding the deformational loading protocol, cell-seeding density, nutrient supply, growth factor addition, and construct mechanical characterization are discussed. In anticipation of cartilage repair studies, we also describe early efforts to engineer cylindrical and anatomically shaped bilayered constructs of agarose hydrogel and bone (i.e., osteochondral constructs). The presence of a bony substrate may facilitate integration upon implantation. These efforts will provide an underlying framework from which a functional tissue-engineering approach, as described by Butler and coworkers (2000), may be applied to general cell-scaffold systems adopted for cartilage tissue engineering.     

The influence of biological motifs and dynamic mechanical stimulation in hydrogel scaffold systems on the phenotype of chondrocytes

Primary bovine chondrocytes and PEG-based hydrogels were used to investigate the effects of scaffold composition and architecture on the cellular response to large dynamic compressive strain stimulation. Proteins and proteoglycans were conjugated to functionalized poly(ethylene glycol) (PEG) and immobilized in PEG hydrogels to create bio-synthetic scaffolds. Second passage articular chondrocytes were encapsulated into four different scaffold compositions: PEG-Proteoglycan (PP), PEG-Fibrinogen (PF), PEG-Albumin (PA), and PEG only and subjected to 15% dynamic compressive strain at 1-Hz frequency. Cellular response was evaluated in terms of cell number, glycosaminoglycans (GAGs), collagen type II and collagen type I accumulation in the constructs following 24h and 28 days of stimulated and static culture. Stimulation of the constructs resulted in an increase in the cell number in all scaffolds, with no statistical difference measured among them. Dynamic stimulation of PP, PF, PA and PEG constructs resulted in a respective increase in the GAGs by 33%, 53.4%, 240.5%, and 284.5%, compared to their static controls. The permissive PEG and PA scaffolds showed a significantly larger relative increase in the GAGs in comparison to the other scaffolds tested. Collagen type II content in the PF, PA and PEG constructs increased by 78%, 1266% and 896% respectively, compared to their static controls. Permissive constructs showed a significantly larger relative increase and final absolute values of GAGs and type II collagen, compared to the PF constructs. Immunostaining for collagen type I, an indicator for chondrocyte de-differentiation, indicated that stimulation inhibited its production. Correlation maps between scaffold properties highlighted the major differences between permissive and instructive scaffolds. These results support the hypothesis that both compressive strain and scaffold bioactivity have an important effect on the chondrocyte metabolic response to mechanical stimulation, and that the 3-D environment surrounding chondrocytes can actively participate in translating mechanical stimulation to the resident cells.     

Mechanical properties and remodeling of hybrid cardiac constructs made from heart cells, fibrin, and biodegradable, elastomeric knitted fabric

Hybrid cardiac constructs with mechanical properties suitable for in vitro loading studies and in vivo implantation were constructed from neonatal rat heart cells, fibrin (Fn), and biodegradable knitted fabric (Knit). Initial (2-h) constructs were compared with native heart tissue, studied in vitro with respect to mechanical function (stiffness, ultimate tensile strength [UTS], failure strain epsilon(f), strain energy density E) and compositional remodeling (collagen, DNA), and implanted in vivo. For 2-h constructs, stiffness was determined mainly by the Fn and was half as high as that of native heart, whereas UTS, epsilon(f), and E were determined by the Knit and were, respectively, 8-, 7-, and 30-fold higher than native heart. Over 1 week of static in vitro culture, cell-mediated, serum-dependent remodeling was demonstrated by a 5-fold increase in construct collagen content and maintenance of stiffness not observed in cell-free constructs. Cyclic stretch further increased construct collagen content in a manner dependent on loading regimen. The presence of cardiac cells in cultured constructs was demonstrated by immunohistochemistry (troponin I) and Western blot (connexin 43). However, in vitro culture reduced Knit mechanical properties, decreasing UTS, epsilon(f), and E of both constructs and cell-free constructs and motivating in vivo study of the 2-h constructs. Constructs implanted subcutaneously in nude rats for 3 weeks exhibited the continued presence of cardiomyocytes and blood vessel ingrowth by immunostaining for troponin I, connexin 43, and CD-31. Together, the data showed that hybrid cardiac constructs initially exhibited supraphysiologic UTS, epsilon(f), and E, and remodeled in response to serum and stretch in vitro and in an ectopic in vivo model.     

Quantitative Analysis of Temporal and Spatial Variations of Chondrocyte Behavior in Engineered Cartilage during Long-Term Culture

In this work, we present the fact that chondrocyte activity differs in relation to their position in an engineered cartilage construct. Chondrocytes from porcine articular cartilage were cultured in a monolayer. Then the cell/extracellular matrix (ECM) membrane was peeled off and centrifuged into a three-dimensional (3D) pellet-type construct. Cultivated in a static condition, the constructs were harvested at specific time intervals (1, 2, 3, and 5 weeks) and manually cored using a biopsy punch to separate the core from the remaining construct. The resultant parts, core and peripheral remnant were thus obtained and subjected to analysis individually. Cell density (10⁶ cells/cm³) of the core was significantly higher at 1 week than that of the periphery but this trend was reversed at later time points. Cell viability was remarkably better in the peripheral tissue. Alcian blue staining of glycosaminoglycan (GAG) revealed an intense blue staining from the periphery, exhibiting a steep gradient in distribution of GAG concentration. The gene expression ratio of collagen type II to I appeared to be more altered in the periphery, possibly suggesting cell dedifferentiation, especially at later time points (>2 weeks). The mRNA levels of matrix metalloproteinase-1 (MMP-1) and MMP-13 remained in the normal range, whereas collagen type X expression was more significantly upregulated at the periphery. This study showed that chondrocyte behavior could be highly variable in the extent of their proliferation, differentiation and dedifferentiation, depending on their physical location within 3D engineered cartilage construct.     

Iterative design of peptide-based hydrogels and the effect of network electrostatics on primary chondrocyte behavior

Iterative peptide design was used to generate two peptide-based hydrogels to study the effect of network electrostatics on primary chondrocyte behavior. MAX8 and HLT2 peptides have formal charge states of?+7 and?+5 per monomer, respectively. These peptides undergo triggered folding and self-assembly to afford hydrogel networks having similar rheological behavior and local network morphologies, yet different electrostatic character. Each gel can be used to directly encapsulate and syringe-deliver cells. The influence of network electrostatics on cell viability after encapsulation and delivery, extracellular matrix deposition, gene expression, and the bulk mechanical properties of the gel-cell constructs as a function of culture time was assessed. The less electropositive HLT2 gel provides a microenvironment more conducive to chondrocyte encapsulation, delivery, and phenotype maintenance. Cell viability was higher for this gel and although a moderate number of cells dedifferentiated to a fibroblast-like phenotype, many retained their chondrocytic behavior. As a result, gel-cell constructs prepared with HLT2, cultured under static in?vitro conditions, contained more GAG and type II collagen resulting in mechanically superior constructs. Chondrocytes delivered in the more electropositive MAX8 gel experienced a greater degree of cell death during encapsulation and delivery and the remaining viable cells were less prone to maintain their phenotype. As a result, MAX8 gel-cell constructs had fewer cells, of which a limited number were capable of laying down cartilage-specific ECM.     

Effect of construct properties on encapsulated chondrocyte expression of insulin-like growth factor-1

Hydrogels are a promising type of biomaterial for articular cartilage constructs since they have been shown to enable encapsulated chondrocytes to express their predominant phenotypic marker, type II collagen. Endogenously expressed signaling molecules, such as insulin-like growth factor-1 (IGF-1), are also known to facilitate the retention of this chondrocytic phenotype. Recent investigations have attempted to enhance the ability of encapsulated chondrocytes to regenerate cartilage through delivery of exogenous signaling molecules. However, we hypothesize that by altering construct properties, such as cell density and polymer concentration, we can augment the expression of endogenous IGF-1 in chondrocytes. To this end, bovine articular chondrocytes were encapsulated within alginate hydrogels at two different cell densities (25,000 and 100,000 cells/bead) and various alginate concentrations (0.8%, 1.2%, and 2.0% w/v). These parameters were chosen to simultaneously investigate cell-to-cell distance on paracrine signaling and water content on IGF-1 diffusion by chondrocytes. At 1, 4, and 8d, chondrocytes were analyzed for protein and mRNA expression of IGF-1 as well as type II collagen. Results suggest that cell density and alginate concentration at high cell density can significantly affect the endogenous IGF-1 expression by chondrocytes. Therefore, these results indicate that construct properties can impact chondrocyte gene expression and should be considered in order to create a proper engineered articular cartilage construct.     

The impact of substrate stiffness and mechanical loading on fibroblast-induced scaffold remodeling

Fibroblasts as many other cells are known to form, contract, and remodel the extracellular matrix (ECM). The presented study aims to gain an insight into how mechanical boundary conditions affect the production of ECM components, their remodeling, and the feedback of the altered mechanical cell environment on these processes. The influence of cyclic mechanical loading (f=1?Hz, 10% axial compression) and scaffold stiffness (E=1.2 and 8.5?kPa) on the mechanical properties of fibroblast-seeded scaffold constructs were investigated in an in vitro approach over 14 days of culture. To do so, a newly developed bioreactor system was employed. While mechanical loading resulted in a clear upregulation of procollagen-I and fibronectin production, scaffold stiffness showed to primarily influence matrix metalloproteinase-1 (MMP-1) secretion and cell-induced scaffold contraction. Higher stiffness of the collagen scaffolds resulted in an up to twofold higher production of collagen-degrading MMP-1. The changes of mechanical parameters like Young's modulus, maximum compression force, and elastic portion of compression force over time suggest that from initially distinct mechanical starting conditions (scaffold stiffness), the construct's mechanical properties converge over time. As a consequence of mechanical loading a shift toward higher construct stiffness was observed. The results suggest that scaffold stiffness has only a temporary effect on cell behavior, while the impact of mechanical loading is preserved over time. Thus, it is concluded that the mechanical environment of the cell after remodeling is depending on mechanical loading rather than on initial scaffold stiffness.     

Cell density alters matrix accumulation in two distinct fractions and the mechanical integrity of alginate-chondrocyte constructs

Chondrocyte density in articular cartilage is known to change with the development and growth of the tissue and may play an important role in the formation of a functional extracellular matrix (ECM). The objective of this study was to determine how initial chondrocyte density in an alginate hydrogel affects the matrix composition, its distribution between the cell-associated (CM) and further removed matrix (FRM) fractions, and the tensile mechanical properties of the developing engineered cartilage. Alginate constructs containing primary bovine chondrocytes at densities of 0, 4, 16, and 64 million cells/ml were fabricated and cultured for 1 or 2 weeks, at which time structural, biochemical, and mechanical properties were analyzed. Both matrix content and distribution varied with the initial cell density. Increasing cell density resulted in an increasing content of collagen and sulfated-glycosaminoglycan (GAG) and an increasing proportion of these molecules localized in the CM. While the equilibrium tensile modulus of cell-free alginate did not change with time in culture, the constructs with highest cell density were 116% stiffer than cell-free controls after 2 weeks of culture. The equilibrium tensile modulus was positively correlated with total collagen (r² = 0.47, p < 0.001) and GAG content (r² = 0.68, p < 0.001), and these relationships were enhanced when analyzing only those matrix molecules in the CM fraction (r² = 0.60 and 0.72 for collagen and GAG, respectively, each p < 0.001). Overall, the results of this study indicate that initial cell density has a considerable effect on the developing composition, structure, and function of alginate–chondrocyte constructs.     

Incorporation of intact elastin scaffolds in tissue-engineered collagen-based vascular grafts

Although collagen-based tissue-engineered blood vessels (TEBVs) have many interesting properties and have been utilized to study aspects of vascular biology, these constructs are too weak to be implanted as bypass grafts for in vivo investigations. This study presents a method to incorporate organized, intact elastin into collagen-based TEBVs to form hybrid constructs that better mimic arterial physiology and exhibit improved mechanical properties. Porcine carotids were digested with a series of autoclave and chemical treatments to elicit isolated elastin scaffolds. Elastin purity was verified via immunohistochemistry and amino acid analysis. Isolated scaffolds were combined with type I collagen and either human dermal fibroblasts (HDFs) or rat smooth muscle cells (RASMs) to form an elastin hybrid TEBV. Hybrid constructs exhibited increased tensile strengths (11-fold in HDFs; 7.5-fold in RASMs) and linear stiffness moduli (4-fold in HDFs; 1.8-fold in RASMs) compared with collagen control constructs with no exogenous elastin scaffold. Viscoelastic properties of the TEBVs also improved with the addition of an ancillary elastin scaffold as determined through stepwise stress relaxation analysis. Whereas the majority of resistance to deformation in collagen control constructs stemmed from viscous fluidlike effects, elastin hybrid constructs exhibited more ideal elastic solid mechanical behavior. Thus, elastin scaffolds can help recreate the elastic properties of native arteries. Future challenges include stimulating appropriate reorganization or synthesis of the collagen matrix to provide the necessary strength and viscoelastic properties for implantation.     

The effect of nanofiber alignment on the maturation of engineered meniscus constructs

The fibrocartilaginous menisci are load-bearing tissues vital to the normal functioning of the knee. Removal of damaged regions of the meniscus subsequent to injury impairs knee function and predisposes patients to osteoarthritis. In this study, we employed biodegradable nanofibrous scaffolds for the tissue engineering of the meniscus. Non-aligned (NA) or fiber-aligned (AL) nanofibrous scaffolds were seeded with meniscal fibrochondrocytes (MFCs) or mesenchymal stem cells (MSCs) to test the hypothesis that fiber-alignment would augment matrix content and organization, resulting in improved mechanical properties. Additionally, we proposed that MSCs could serve as an alternative to MFCs. With time in culture, MSC- and MFC-seeded NA and AL constructs increased in cellularity and extracellular matrix (ECM) content. Counter our initial hypothesis, NA and AL constructs contained comparable amounts of ECM, although a significantly larger increase in mechanical properties was observed for AL compared to NA constructs seeded with either cell type. Cell-seeded NA constructs increased in modulus by approximately 1MPa over 10 weeks while cell-seeded AL construct increased by >7MPa. Additionally, MSC-constructs yielded greater amounts of ECM and demonstrated comparable increases in mechanical properties, thereby confirming the utility of MSCs for meniscus tissue engineering. These results demonstrate that cell-seeded fiber-aligned nanofibrous scaffolds may serve as an instructive micro-pattern for directed tissue growth, reconstituting both the form and function of the native tissue.     

Enhancing mechanical properties of tissue-engineered constructs via lysyl oxidase crosslinking activity

A number of strategies have been investigated to enhance the mechanical stability of engineered tissues. In this study, we utilized lysyl oxidase (LO) to enzymatically crosslink extracellular matrix (ECM) proteins, particularly collagen and elastin, to enhance the mechanical integrity of the ECM and thereby impart mechanical strength to the engineered tissue. Vascular smooth muscle cells (VSMCs) were liposomally transfected with the LO gene. Both Northern and Western analyses confirmed increased LO expression. Increased LO activity was demonstrated using a fluorescent enzyme substrate assay and by observation of the presence of increased levels of desmosine, a product of LO crosslinking, in the ECM. The mechanical effects of altered crosslink densities within tissue-engineered constructs were demonstrated in a VSMC-populated collagen gel model. When smooth muscle cells transfected with lysyl oxidase were seeded in collagen gels, the tensile strength and elastic modulus in these constructs increased by approximately two-fold compared to constructs seeded with mock-transfected VSMCs. Also, desmosine levels in the LO-populated collagen gels were higher than they were in mock-seeded gels, as demonstrated via immunohistochemical staining. Compositional analysis of the ECM deposited by the transformed cells showed similar collagen and elastin levels, and cell proliferation rates were similar as well, thus attributing increased mechanical properties to ECM crosslinking.