Synergistic action of growth factors and dynamic loading for articular cartilage tissue engineering

It has previously been demonstrated that dynamic deformational loading of chondrocyte-seeded agarose hydrogels over the course of 1 month can increase construct mechanical and biochemical properties relative to free-swelling controls. The present study examines the manner in which two mediators of matrix biosynthesis, the growth factors TGF-beta1 and IGF-I, interact with applied dynamic deformational loading. Under free-swelling conditions in control medium (C), the [proteoglycan content][collagen content][equilibrium aggregate modulus] of cell-laden (10 x 10(6) cells/mL) 2% agarose constructs reached a peak of [0.54% wet weight (ww)][0.16% ww][13.4 kPa]c, whereas the addition of TGF-beta1 or IGF-I to the control medium led to significantly higher peaks of [1.18% ww][0.97% ww][23.6 kPa](C-TGF) and [1.00% ww][0.63% ww][19.3 kPa](C-IGF), respectively, by day 28 or 35 (p<0.01). Under dynamic loading in control medium (L), the measured parameters were [1.10% ww][0.52% ww][24.5 kPa]L, and with the addition of TGF-beta1 or IGF-I to the control medium these further increased to [1.49% ww][1.07% ww][50.5 kPa](L-TGF) and [1.48% ww][0.81% ww][46.2 kPa](L-IGF), respectively (p<0.05). Immunohistochemical staining revealed that type II collagen accumulated primarily in the pericellular area under free-swelling conditions, but spanned the entire tissue in dynamically loaded constructs. Applied in concert, dynamic deformational loading and TGF-beta1 or IGF-I increased the aggregate modulus of engineered constructs by 277 or 245%, respectively, an increase greater than the sum of either stimulus applied alone. These results support the hypothesis that the combination of chemical and mechanical promoters of matrix biosynthesis can optimize the growth of tissue-engineered cartilage constructs.     

The effect of devitalized trabecular bone on the formation of osteochondral tissue-engineered constructs

In the current study, evidence is presented demonstrating that devitalized trabecular bone has an inhibitory effect on in vitro chondral tissue development when used as a base material for the tissue-engineering of osteochondral constructs for cartilage repair. Chondrocyte-seeded agarose hydrogel constructs were cultured alone or attached to an underlying bony base in a chemically defined medium formulation that has been shown to yield engineered cartilaginous tissue with native Young's modulus (E(Y)) and glycosaminoglycan (GAG) content. By day 42 in culture the incorporation of a bony base significantly reduced these properties (E(Y)=87+/-12kPa, GAG=1.9+/-0.8%ww) compared to the gel-alone group (E(Y)=642+/-97kPa, GAG=4.6+/-1.4%ww). Similarly, the mechanical and biochemical properties of chondrocyte-seeded agarose constructs were inhibited when co-cultured adjacent to bone (unattached), suggesting that soluble factors rather than direct cell-bone interactions mediate the chondro-inhibitory bone effects. Altering the method of bone preparation, including demineralization, or the timing of bone introduction in co-culture did not ameliorate the effects. In contrast, osteochondral constructs with native cartilage properties (E(Y)=730+/-65kPa, GAG=5.2+/-0.9%ww) were achieved when a porous tantalum metal base material was adopted instead of bone. This work suggests that devitalized bone may not be a suitable substrate for long-term cultivation of osteochondral grafts.     

Influence of seeding density and dynamic deformational loading on the developing structure/function relationships of chondrocyte-seeded agarose hydrogels

Chondrocytes cultured in agarose hydrogels develop a functional extracellular matrix. Application of dynamic strain at physiologic levels to these constructs over time can increase their mechanical properties. In this study, the effect of seeding density (20 and 60×10⁶ cells/ml) on tissue elaboration was investigated. Higher seeding densities increased tissue properties in free-swelling culture, with constructs seeded at 20 and 60×10⁶ cells/ml reaching maximum values over the 63 day culture period of aggregate modulus H _A: 43±15 kPa, Youngs modulus E _Y: 39±3 kPa, and glycosaminglycan content [GAG]: 0.96%±0.13% wet weight; and H _A: 58±12 kPa, E _Y: 60±5 kPa, and [GAG]: 1.49% ± 0.26% wet weight, respectively. It was further observed that the application of daily dynamic deformational loading to constructs seeded at 20×10⁶ cells/ml enhanced biochemical content (150%) and mechanical properties (threefold) compared to free-swelling controls by day 28. However, at a concentration of 60×10⁶ cells/ml, no difference in mechanical properties was found in loaded samples versus their free-swelling controls. Multiple regression analysis showed that the mechanical properties of the tissue constructs depend more strongly on collagen content than GAG content; a finding that is more pronounced with the application of daily dynamic deformational loading. Our findings provide evidence for initial cell seeding density and nutrient accessibility as important parameters in modulating tissue development of engineered constructs, and their ability to respond to a defined mechanical stimulus. © 2002 Biomedical Engineering Society. PAC2002: 8717-d, 8719Rr     

Mechanical shear properties of cell-polymer cartilage constructs.

Cartilaginous constructs were created by using bovine chondrocytes on synthetic, biodegradable scaffolds made of fibrous polyglycolic acid (PGA). The constructs have previously been shown to resemble natural articular cartilage biochemically and histologically. The mechanical properties of articular cartilage mainly depend on the swollen extracellular matrix (ECM), which is a gel consisting of collagen fibers and proteoglycans in a fluid phase of water and electrolytes. The biomechanical properties of the constructs and the build-up of the ECM were studied using dynamic, nondestructive measurements in shear. A small, harmonic strain, lambda < or = 5 x 10(-4), was applied to the sample, and the resulting stress was recorded and used for calculating the complex shear modulus G*. The applied strain was much smaller than that used in confined compression. The shear modulus G* correlated well with both the collagen and glycosaminoglycan content of the constructs but did not reach the same level as in natural cartilage. Collagen is the dominant component contributing to the shear strength of cartilage, and G* was shown to depend approximately quadratically on the collagen content of the constructs. The difference in G* between the constructs and natural cartilage was shown to depend on both the biochemical composition and the microstructure of the constructs. () ()     

Transient exposure to transforming growth factor beta 3 under serum-free conditions enhances the biomechanical and biochemical maturation of tissue-engineered cartilage

A goal of cartilage tissue engineering is the production of cell-laden constructs possessing sufficient mechanical and biochemical features to enable native tissue function. This study details a systematic characterization of a serum-free (SF) culture methodology employing transient growth factor supplementation to promote robust maturation of tissue-engineered cartilage. Bovine chondrocyte agarose hydrogel constructs were cultured under free-swelling conditions in serum-containing or SF medium supplemented continuously or transiently with varying doses of transforming growth factor beta 3 (TGF-beta3). Constructs were harvested weekly or bi-weekly and assessed for mechanical and biochemical properties. Transient exposure (2 weeks) to low concentrations (2.5-5 ng/mL) of TGF-beta3 in chemically defined medium facilitated robust and highly reproducible construct maturation. Constructs receiving transient TGF-beta3 exposure achieved native tissue levels of compressive modulus (0.8 MPa) and proteoglycan content (6-7% of wet weight) after less than 2 months of in vitro culture. This maturation response was far superior to that observed after continuous growth factor supplementation or transient TGF-beta3 treatment in the presence of serum. These findings represent a significant advance in developing an ex vivo culture methodology to promote production of clinically relevant and mechanically competent tissue-engineered cartilage constructs for implantation to repair damaged articular surfaces.     

Design and validation of a compressive tissue stimulator with high-throughput capacity and real-time modulus measurement capability

Mechanical stimulation has been shown to impact the properties of engineered hyaline cartilage constructs and is relevant for engineering of cartilage and osteochondral tissues. Most mechanical stimulators developed to date emphasize precision over adaptability to standard tissue culture equipment and protocols. The realization of mechanical characteristics in engineered constructs approaching native cartilage requires the optimization of complex variables (type of stimulus, regimen, and bimolecular signals). We have proposed and validated a stimulator design that focuses on high construct capacity, compatibility with tissue culture plastic ware, and regimen adaptability to maximize throughput. This design utilizes thin force sensors in lieu of a load cell and a linear encoder to verify position. The implementation of an individual force sensor for each sample enables the measurement of Young's modulus while stimulating the sample. Removable and interchangeable Teflon plungers mounted using neodymium magnets contact each sample. Variations in plunger height and design can vary the strain and force type on individual samples. This allows for the evaluation of a myriad of culture conditions and regimens simultaneously. The system was validated using contact accuracy, and Young's modulus measurements range as key parameters. Contact accuracy for the system was excellent within 1.16% error of the construct height in comparison to measurements made with a micrometer. Biomaterials ranging from bioceramics (cancellous bone, 123?MPa) to soft gels (1% agarose, 20?KPa) can be measured without any modification to the device. The accuracy of measurements in conjunction with the wide range of moduli tested demonstrate the unique characteristics of the device and the feasibility of using this device in mapping real-time changes to Young's modulus of tissue constructs (cartilage, bone) through the developmental phases in ex vivo culture conditions.     

High mesenchymal stem cell seeding densities in hyaluronic acid hydrogels produce engineered cartilage with native tissue properties

Engineered cartilage based on adult mesenchymal stem cells (MSCs) is an alluring goal for the repair of articular defects. However, efforts to date have failed to generate constructs with sufficient mechanical properties to function in the demanding environment of the joint. Our findings with a novel photocrosslinked hyaluronic acid (HA) hydrogel suggest that stiff gels (high HA concentration, 5% w/v) foster chondrogenic differentiation and matrix production, but limit overall functional maturation due to the inability of the formed matrix to diffuse away from the point of production and form a contiguous network. In the current study, we hypothesized that increasing the MSC seeding density would decrease the required diffusional distance, and so expedite the development of functional properties. To test this hypothesis bovine MSCs were encapsulated at seeding densities of either 20,000,000 or 60,000,000 cells ml(-1) in 1%, 3%, and 5% (w/v) HA hydrogels. Counter to our hypothesis the higher concentration HA gels (3% and 5%) did not develop more rapidly with increased MSC seeding density. However, the biomechanical properties of the low concentration (1%) HA constructs increased markedly (nearly 3-fold with a 3-fold increase in seeding density). To ensure that optimal nutrient access was delivered, we next cultured these constructs under dynamic culture conditions (with orbital shaking) for 9 weeks. Under these conditions 1% HA seeded at 60,000,000 MSCs ml(-1) reached a compressive modulus in excess of 1 MPa (compared with 0.3-0.4 MPa for free swelling constructs). This is the highest level we have reported to date in this HA hydrogel system, and represents a significant advance towards functional stem cell-based tissue engineered cartilage.     

Chondrogenesis and Integration of Mesenchymal Stem Cells Within an <Emphasis Type="Italic">In</Emphasis> <Emphasis Type="Italic">Vitro</Emphasis> Cartilage Defect Repair Model

Integration of repair tissue is a key indicator of the long-term success of cell-based therapies for cartilage repair. The objective of this study was to compare the in vitro chondrogenic differentiation and integration of agarose hydrogels seeded with either chondrocytes or bone marrow-derived mesenchymal stem cells (MSCs) in defects created in cartilage explants. Chondrocytes and MSCs were isolated from porcine donors, suspended in 2% agarose and then injected into cylindrical defects within the explants. These constructs were maintained in a chemically defined medium supplemented with 10 ng/mL of TGF-β3. Cartilage integration was assessed by histology and mechanical push-out tests. After 6 weeks in culture, chondrocyte-seeded constructs demonstrated a higher integration strength (64.4 ± 8.3 kPa) compared to MSC-seeded constructs (22.7 ± 5.9 kPa). Glycosaminoglycan (GAG) (1.27 ± 0.3 vs. 0.19 ± 0.03 kPa) and collagen (0.31 ± 0.08 vs. 0.09 ± 0.01 kPa) accumulation in chondrocyte-seeded constructs was greater than that measured in the MSC-seeded group. The GAG, collagen, and DNA content of both chondrocyte- and MSC-seeded hydrogels cultured in cartilage explants was significantly lower than control constructs cultured in free swelling conditions. The results of this study suggest that the explant model may constitute a more rigorous in vitro test to assess MSC therapies for cartilage defect repair.     

The use of polyacrylamide gels for mechanical calibration of cartilage--a combined nanoindentation and unconfined compression study

This study investigates polyacrylamide (PA) gel as a calibration material to measure the nanomechanical compressive modulus of cartilage using nanoindentation. Both nanoindentation and unconfined compression testing were performed on PA gel and porcine rib cartilage. The equilibrium moduli measured by the two methods were discernable. Nanoindentation has the advantage of distinguishing between spatially dependent constituent properties that affect tissue mechanical function in heterogeneous and hierarchically structured tissues such as cartilage. Both sets of measurements exhibited similar positive correlation with increasing gel crosslinker concentration. The compressive modulus measurements from compression in the PA gels ranged from 300 kPa–1.4 MPa, whereas those from nanoindentation ranged from 100 kPa–1.1MPa. Using this data, a method for relating nanoindentation measurements to conventional mechanical property measurements is presented for porcine rib cartilage. It is shown that based on this relationship, the local tissue modulus as measured from nanoindentation (1.1–1.4 MPa) was able to predict the overall global modulus of the same sample of rib cartilage (2.2 MPa), as confirmed by experimental measurements from unconfined compression. This study supports the use of nanoindentation for the local characterization of cartilage tissues and may be applied to other soft tissues and constructs.     

Fractal network dimension and viscoelastic powerlaw behavior: I. A modeling approach based on a coarse-graining procedure combined with shear oscillatory rheometry

Recent advances in dynamic elastography and biorheology have revealed that the complex shear modulus, G*, of various biological soft tissues obeys a frequency-dependent powerlaw. This viscoelastic powerlaw behavior implies that mechanical properties are communicated in tissue across the continuum of scales from microscopic to macroscopic. For deriving constitutive constants from the dispersion of G* in a biological tissue, a hierarchical fractal model is introduced that accounts for multiscale networks. Effective-media powerlaw constants are derived by a constitutive law based on cross-linked viscoelastic clusters embedded in a rigid environment. The spatial variation of G* is considered at each level of hierarchy by an iterative coarse-graining procedure. The establishment of cross-links in this model network is associated with an increasing fractal dimension and an increasing viscoelastic powerlaw exponent. This fundamental relationship between shear modulus dynamics and fractal dimension of the mechanical network in tissue is experimentally reproduced in phantoms by applying shear oscillatory rheometry to layers of tangled paper strips embedded in agarose gel. Both model and experiments demonstrate the sensitivity of G* to the density of the mechanical network in tissue, corroborating disease-related alterations of the viscoelastic powerlaw exponent in human parenchyma demonstrated by in vivo elastography.     

Assessment of a bovine co-culture, scaffold-free method for growing meniscus-shaped constructs

Using a self-assembly (SA), scaffoldless method, five high-density co-cultures with varied ratios of meniscal fibrochondrocytes (MFCs) and articular chondrocytes (ACs) were seeded into novel meniscus-specific, ring-shaped agarose wells. The following ratios of MFCs to ACs were used: 0% MFC, 25% MFC, 50% MFC, 75% MFC, and 100% MFC. Over 4 weeks, all ratios of cells self-assembled into three-dimensional constructs with varying mechanobiological and morphological properties. All groups stained for collagen II (Col II), and all groups except the 0% MFC group stained for collagen I (Col I). It was found that the tensile modulus was proportional to the percentage of MFCs employed. The 100% MFC group yielded the greatest mechanical stiffness with 432.2 +/- 47 kPa tensile modulus and an ultimate tensile strength of 23.7 +/- 2.4 kPa. On gross inspection, the 50% MFC constructs were the most similar to our idealized meniscus shape, our primary criterion. A second experiment was performed to examine the anisotropy of constructs as well as to directly compare the scaffoldless, SA method with a poly-glycolic acid (PGA) scaffold-based construct. When compared to PGA constructs, the SA groups were 2-4 times stiffer and stronger in tension. Further, at 8 weeks, SA groups exhibited circumferential fiber bundles similar to native tissue. When pulled in the circumferential direction, the SA group had significantly higher tensile modulus (226 +/- 76 kPa) than when pulled in the radial direction (67 +/- 32 kPa). The PGA constructs had neither a directional collagen fiber orientation nor differences in mechanical properties in the radial or circumferential direction. It is suggested that the geometric constraint imposed by the ring-shaped, nonadhesive mold guides collagen fibril directionality and, thus, alters mechanical properties. Co-culturing ACs and MFCs in this manner appears to be a promising new method for tissue engineering fibrocartilaginous tissues exhibiting a spectrum of mechanical and biomechanical properties.     

High amplitude direct compressive strain enhances mechanical properties of scaffold-free tissue-engineered cartilage

Adult cartilage has a limited healing capacity. Damages resulting from disease or injury increase over time and cause severe pain. One approach to reinstate the cartilage function is tissue engineering (TE). However, the generation of TE cartilage is time consuming and expensive and its properties are so far suboptimal. As in vivo cartilage is subject to loading, it is assumed that mechanical stimulation may enhance the quality of TE cartilage. In this study the short-term influence of variable compressive strain amplitudes on mechanical and biochemical properties of scaffold-free TE cartilage was investigated. Primary porcine chondrocytes were isolated, proliferated, redifferentiated, and transferred onto hydroxyapatite carriers, resulting in scaffold-free cartilage-carrier constructs. These constructs were placed in a custom-made bioreactor. Compression amplitudes of 5%, 10%, and 20% were applied. In each experiment four constructs were loaded with dynamic compression (3000 cycles/day, 1?Hz) for 14 days and four constructs served as unloaded control. The cartilage was evaluated biochemically, histological, and mechanically. No difference in glycosaminoglycan or collagen content between the loaded and the control groups was found. However, a positive correlation between compression amplitude and normalized Young's modulus was detected (R(2)=0.59, p<0.001). The highest compression amplitude of 20% had the strongest positive effect on the mechanical properties of the TE cartilage (Young's modulus increase of 241±28% compared to unloaded control). The data presented suggest that preconditioning with higher load amplitudes might be an attractive way of generating stiffer tissue and may help accelerating the cultivation of mechanically competent TE cartilage.     

The mechanical and material properties of elderly human articular cartilage subject to impact and slow loading

The mechanical properties of articular cartilage vary enormously with loading rate, and how these properties derive from the composition and structure of the tissue is still unclear. This study investigates the mechanical properties of human articular cartilage at rapid rates of loading, compares these with measurements at slow rates of loading and explores how they relate to the gross composition of the tissue. Full-depth femoral head cartilage biopsies were subjected to a slow, unconfined compression test followed by an impact at an energy of 78.5 mJ and velocity 1.25 m s⁻¹. The modulus was calculated from the slope of the loading curve and the coefficient of restitution from the areas under the loading and unloading curves.Tissue composition was measured as water, collagen and glycosaminoglycan contents. The maximum dynamic modulus ranged from 25 to 150 MPa. These values compared with 1–3 MPa measured during quasi-static loading. The coefficient of restitution was 0.502 (0.066) (mean (standard deviation)) and showed no site variation. Water loss was not detectable. Composition was not strongly associated with modulus; water and collagen contents together predicted about 25% of the variance in modulus.     

Effect of a degraded core on the mechanical behaviour of tissueengineered cartilage constructs: A poro-elastic finite element analysis

The structure and functionality of tissue-engineered cartilage is determined by the tissue culture conditions and mechanical conditioning during growth. The quality of tissue-engineered cartilage can be evaluated using tests such as the confined compression test. Tissue-engineered cartilage constructs usually consist of an outer layer of cartilage and an inner core of either undeveloped cartilage or degrading scaffold material. A biphasic poro-elastic finite element model was used to demonstrate how such a core influences the reaction force-time curve obtained from a confined compression test. The finite element model predicted that higher volumes of degraded scaffold in the inner core would reduce the aggregate modulus calculated from the confined compression test and raised the estimate of tissue permeability. The predicted aggregate modulus reduced from 0.135 MPa, for a homogenous construct, to 0.068 MPa, for a construct that was only 70% cartilaginous. It was found that biphasic poro-elastic finite modelling should be used in preference to a one-dimensional model that assumed homogeneity in estimating the properties of tissue-engineered cartilage.     

Pulsatile dynamic stiffness of cartilage-like materials and use of agarose gels to validate mechanical methods and models

Stiffness is a fundamental indicator of the functional state of articular cartilage. Reported test modes include compressive incremental strain to determine the equilibrium modulus, and sinusoidal strain to determine the dynamic modulus and stress/strain loss angle. Here, initial development is described for a method recognizing that gait is pulsatile. Agarose gels have been used by others for validation or comparison of mechanical test methods and models for cartilage and proteoglycan aggregate. Accordingly, gels ranging from 0.5 to 20% agarose were prepared. Pulsatile stiffness in both indentation and unconfined compression were closely reproducible. Stiffness as a function of agarose concentration rose exponentially, as found using other methods. Indentation stiffness was higher than for unconfined compression and ranged from approximately 2.0 kPa for 0.5% gel to approximately 3,800 kPa for 20% gel. Pulsatile dynamic stiffness appears to be a useful method, although further development is needed. Agarose gel stiffness values obtained by other methods were reviewed for comparison. Unfortunately, reported values for a given agarose concentration ranged widely (e.g. fourfold) even when test methods were similar. Causes appear to include differences in molecular weight and gel preparation time-temperature regimens. Also, agarose is hygroscopic, leading to unintended variations in gel composition. Agarose gels are problematic materials for validation or comparison of cartilage mechanical test methods and models.     

A novel bioreactor for the dynamic stimulation and mechanical evaluation of multiple tissue-engineered constructs

Systematic advancements in the field of musculoskeletal tissue engineering require clear communication about the mechanical environments that promote functional tissue growth. To support the rapid discovery of effective mechanostimulation protocols, this study developed and validated a mechanoactive transduction and evaluation bioreactor (MATE). The MATE provides independent and consistent mechanical loading of six specimens with minimal hardware. The six individual chambers accurately applied static and dynamic loads (1 and 10?Hz) in unconfined compression from 0.1 to 10?N. The material properties of poly(ethylene glycol) diacrylate hydrogels and bovine cartilage were measured by the bioreactor, and these values were within 10% of the values obtained from a standard single-chamber material testing system. The bioreactor was able to detect a 1-day 12% reduction (2?kPa) in equilibrium modulus after collagenase was added to six collagenase sensitive poly(ethylene glycol) diacrylate hydrogels (p?=?0.03). By integrating dynamic stimulation and mechanical evaluation into a single batch-testing research platform, the MATE can efficiently map the biomechanical development of tissue-engineered constructs during long-term culture.     

Growth factor priming of synovium-derived stem cells for cartilage tissue engineering

This study investigated the potential use of synovium-derived stem cells (SDSCs) as a cell source for cartilage tissue engineering. Harvested SDSCs from juvenile bovine synovium were expanded in culture in the presence (primed) or absence (unprimed) of growth factors (1?ng/mL transforming growth factor-β(1), 10?ng/mL platelet-derived growth factor-ββ, and 5?ng/mL basic fibroblast growth factor-2) and subsequently seeded into clinically relevant agarose hydrogel scaffolds. Constructs seeded with growth factor-primed SDSCs that received an additional transient application of transforming growth factor-β(3) for the first 21 days (release) exhibited significantly better mechanical and biochemical properties compared to constructs that received sustained growth factor stimulation over the entire culture period (continuous). In particular, the release group exhibited a Young's modulus (267±96?kPa) approaching native immature bovine cartilage levels, with corresponding glycosaminoglycan content (5.19±1.45%ww) similar to native values, within 7 weeks of culture. These findings suggest that SDSCs may serve as a cell source for cartilage tissue engineering applications.     

Analysis of bending behavior of native and engineered auricular and costal cartilage

A large-deflection elasticity model was used to describe the mechanical behavior of cartilaginous tissues during three-point bending tests. Force-deflection curves were measured for 20-mm long x 4-mm wide x approximately 1-mm thick strips of porcine auricular and costal cartilage. Using a least-squares method with elastic modulus in bending as the only adjustable parameter, data were fit to a model based on the von Karman theory for large deflection of plates. This model described the data well, with an average RMS error of 14.8% and an average R(2) value of 0.98. Using this method, the bending modulus of auricular cartilage (4.6 MPa) was found to be statistically lower (p < 0.05) than that of costal cartilage (7.1 MPa). Material features of the cartilage samples influenced the mechanical behavior, including the orientation of the perichondrium in auricular cartilage. These methods also were used to determine the elastic moduli of engineered cartilage samples produced by seeding chondrocytes into fibrin glue. The modulus of tissue-engineered constructs increased statistically with time (p < 0.05), but still were statistically lower than the moduli of the native tissue samples (p > 0.05), reaching only about a third of the values of native samples.     

Low-Serum Media and Dynamic Deformational Loading in Tissue Engineering of Articular Cartilage

High-serum media have been shown to produce significant improvement in the properties of tissue-engineered articular cartilage when applied in combination with dynamic deformational loading. To mitigate concerns regarding the culture variability introduced by serum, we examined the interplay between low-serum/ITS-supplemented media and dynamic deformational loading. Our results show that low serum/ITS supplementation does not support the same level of tissue formation as compared to high serum controls. In free-swelling culture, using a combination of ITS with concentrations of FBS above 2% negated the beneficial effects of ITS. Although there were beneficial effects with loading and 0.2%FBS + ITS, these constructs significantly underperformed relative to 20%FBS constructs. At 2%FBS + ITS, the free-swelling construct stiffness and composition approached or exceeded that of 20%FBS constructs. With dynamic loading, the properties of 2%FBS + ITS constructs were significantly lower than free-swelling controls and 20%FBS constructs by day 42. By priming the chondrocytes in 20%FBS prior to exposure to low-serum/ITS media, we observed that low-serum/ITS media produced significant enhancement in tissue properties compared to constructs grown continuously in 20%FBS.     

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.