Cartilage Tissue Engineering Studies Research Articles

The Ogden model for hydrogels in tissue engineering: Modulus determination with compression to failure

Hydrogel mechanical properties for tissue engineering are often reported in terms of a compressive elastic modulus derived from a linear regression of a typically non-linear stress-strain plot. There is a need for an alternative model to fit the full strain range of tissue engineering hydrogels. Fortunately, the Ogden model provides a shear modulus, μ0, and a nonlinear parameter, α, for routine analysis of compression to failure. Three example hydrogels were tested: (1) pentenoate-modified hyaluronic acid (PHA), (2) dual-crosslinked PHA and polyethylene glycol diacrylate (PHA-PEGDA), and (3) composite PHA-PEGDA hydrogel with cryoground devitalized cartilage (DVC) at 5, 10, and 15%w/v concentration (DVC5, DVC10, and DVC15, respectively). Gene expression analyses suggested that the DVC hydrogels supported chondrogenesis of human bone marrow mesenchymal stem cells to some degree. Both linear regression (5 to 15% strain) and Ogden fits (to failure) were performed. The compressive elastic modulus, E, was over 4-fold higher in the DVC15 group relative to the PHA group (129 kPa). Similarly, the shear modulus, μ0, was over 3-fold higher in the DVC15 group relative to the PHA group (37 kPa). The PHA group exhibited a much higher degree of nonlinearity (α = 10) compared to the DVC15 group (α = 1.4). DVC hydrogels may provide baseline targets of μ0 and α for future cartilage tissue engineering studies. The Ogden model was demonstrated to fit the full strain range with high accuracy (R2 = 0.998 ± 0.001) and to quantify nonlinearity. The current study provides an Ogden model as an attractive alternative to the elastic modulus for tissue engineering constructs.

Research progress of self-assembling peptide hydrogels in repairing cartilage defects

Due to the lack of blood vessels, nerves and lymphatic vessels, the capacity of articular cartilage to heal is extremely limited. Once damaged, it is urgent for articular cartilage to repair the injury. In recent years, there has been an increase in cartilage tissue engineering studies. Self-assembling peptide hydrogel as a kind of hydrogels composed of peptides and water is widely used in cartilage tissue engineering. Under noncovalent interactions such as electrostatic interaction, hydrophobic interaction, hydrogen bonding and pi-pi stacking force, peptides self-assemble into three-dimensional (3D) structures that mimic the natural extracellular matrix and allow cells to grow, proliferate and differentiate. Because SAPHs have excellent biocompatibility and biodegradability, variable mechanical properties, low immunogenicity, injectability, and the ability to load cells and bioactive substances, many researchers utilized them to promote the repair and regeneration of articular cartilage after damage. Therefore, the purpose of this review is to sum up the composition, injury characteristics, and treatments of articular cartilage, as well as the action of SAPHs in repairing articular cartilage damage.

Deformation Thresholds for Chondrocyte Death and the Protective Effect of the Pericellular Matrix

In cartilage tissue engineering studies, the stimulatory effect of mechanical perturbation declines after the first 2 weeks of culture. Similarly, it is known that chondrocyte-agarose constructs should not be loaded within the first days after seeding, to prevent considerable cell death, suggesting a mechanical threshold. This study aims to establish a relationship between chondrocyte deformation and death, and to evaluate the protective effect of the pericellular matrix (PCM) that is formed in 3D cultures. Chondrocyte viability was monitored every hour for 24 h after applying a strain range of 0% to 25% to agarose constructs containing chondrocytes, cultured for 1, 3, 5, 7, or 10 days. At these culture time points, the PCM thickness and chondrocyte deformation were assessed by means of histology and assayed for biochemical contents. Inverse finite element (FE) simulations were used to evaluate the change of mechanical properties of the chondrocyte and PCM over the 10-day culture duration. Chondrocyte death was demonstrated to be dependent on both the magnitude and duration of straining. The highest cell death was observed at day 1 (43%), reducing over culture duration (15% at day 3 and 2.5% at day 10). Cell deformation at 25% compression decreased significantly over culture duration (aspect ratio of 2.24±0.67 at day 1 and 1.45±0.24 at day 3) and with increased matrix production. Inverse FE simulations showed an increasing PCM Young's modulus of 45 kPa at day 3 to 162 kPa at day 10. The current results provide evidence for a mechanical threshold for chondrocyte death and for the protective effect of the PCM. As such, these insights may help in establishing mechanical loading protocols for cartilage tissue engineering studies.

Hybrid hyaluronic acid hydrogel/poly(ɛ‐caprolactone) scaffold provides mechanically favorable platform for cartilage tissue engineering studies

Hybrid scaffolds for cartilage tissue engineering provide the potential for high stiffness properties in tension and compression while exhibiting the viscoelastic response found in hydrogels and native cartilage tissue. We investigate the impact of a hybrid scaffold fabricated from a hyaluronic acid (HA)-based hydrogel combined with porous poly(ε-caprolactone) (PCL) material formed by a particulate leaching method to study dedifferentiated chondrocyte response. The material properties of the hybrid scaffold showed mean Young's moduli in tension which were similar to human articular cartilage but not statistically different between the hybrid and porous PCL scaffolds at 2.02 and 2.07 MPa, respectively. For both the hybrid and porous PCL control scaffolds in compression at low loading frequencies (<1 Hz) and 10% strain peak amplitude the Young's moduli are not statistically distinct. However, at frequencies in the range of normal human gait from 1 to2 Hz, hybrid scaffolds exhibit significantly (p < 0.01) increased loss moduli indicating additional contribution of the viscous phase to stiffness. Dedifferentiated chondrocytes seeded onto the scaffolds exhibited a rounded morphology in hybrid scaffolds however ECM protein expression levels of collagen type I, collagen type II, and aggrecan are not different from the PCL control scaffolds. These results provide a model platform to investigate cell response to mechanical and chemical cues in a hybrid scaffold system with mechanical properties similar to human cartilage that does not contribute to differentiation in order to identify the appropriate design and development parameters to promote formation of extracellular matrix and investigate chondrocyte scaffold interactions.

Biochemical and Biomechanical Properties of the Rabbit Nasal Septum

Objectives: 1) Characterize biochemical and biomechanical properties of New Zealand White rabbit septal cartilage for future utilization in rabbit septal cartilage tissue engineering studies. 2) Determine whether tensile and bending properties vary when measured in different axes. Methods: Nasal septa were harvested from adult New Zealand White rabbits post-mortem. Confined compression testing was performed. Samples cut in the vertical and anterior-posterior (AP) plane were subjected to tensile testing and 3-point bending. Biochemical assays determined the Glycosaminoglycans (GAG), total collagen, and DNA concentrations in each sample. Results: Confined compression demonstrated an average compressive modulus (HAO) of 0.82 (±0.23 MPa). In tensile testing, peak stress of samples cut in the vertical plane (6.61±1.28 MPa) were significantly greater (p=0.02) than in the AP plane (5.22±1.31 MPa). Tensile stiffness at failure strain for vertical (22.90±5.51 MPa) and AP (18.67±9.56 MPa) samples did not differ significantly (p &gt; 0.05). The flexural moduli of samples cut in the vertical plane (39.31±16.6 MPa) and AP plane (39.32±20.24 MPa) were also not significantly different (p &gt; 0.05). Biochemical characterization demonstrated an average concentration of 43.81±8.34 µg GAG, 104.71±21.52 µg total collagen, and 0.23±0.05 µg DNA per mg wet weight. Conclusions: Rabbit septal cartilage is anisotropic; tensile strength was higher in the vertical plane than in the AP plane. Compared with published properties of human septal cartilage, rabbit septal cartilage is mechanically stronger, stiffer, and less flexible. Rabbit septal cartilage has a similar DNA content, with higher GAG and total collagen levels than values published for human septal cartilage.

217102 共焦点顕微鏡とImageJによる再生軟骨の階層性評価(OS07 細胞・生体組織1,オーガナイズド・セッション)

Many cartilage tissue engineering studies use mechanical forces to enhance the biomechanical properties of the engineered tissue. Then it is measured mainly as overall property. But, partial property is also important, because cartilage has configurationality. So, this paper describes a new estimation of regenerated cartilage configurationality by confocal microscope and ImageJ. First, three-dimensional image of cartilage is obtained by confocal microscope. Second, the image is analyzed by imageJ and it outputs volume, surface fraction, intensity and position of the chondrocytes. This study applies this method to native cartilage, cultured cartilage, static cultured cartilage and rotating cultured cartilage. The result shows that this method captures cartilage configurationality.

The effect of concentration, thermal history and cell seeding density on the initial mechanical properties of agarose hydrogels

Agarose hydrogels are commonly used for cartilage tissue engineering studies and to provide a three dimensional environment to investigate cellular mechanobiology. Interpreting the results of such studies requires accurate quantification of the mechanical properties of the hydrogel. There is significant variation in the reported mechanical properties of agarose hydrogels, and little is reported on the influence of factors associated with mixing these hydrogels with cell suspensions on their initial mechanical properties. The objective of this study was to determine the influence of agarose concentration, the cooling rate during gelation, the thermal history following gelation and the cell seeding density on the initial mechanical properties of agarose hydrogels. The average ramp modulus of 2% agarose gel in tension was 24.9 kPa (±1.7, n = 10 ), compared with 55.6 kPa (±0.5, n = 10 ) in compression. The average tensile equilibrium modulus was 39.7 kPa (±5.7, n = 6 ), significantly higher than the compressive equilibrium modulus of 14.2 kPa (±1.6, n = 10 ). The equilibrium and dynamic compressive modulus of agarose hydrogels were observed to reduce if maintained at 37 ∘C following gelation compared with samples maintained at room temperature. Depending on the methodology used to encapsulate chondrocytes within agarose hydrogels, the equilibrium compressive modulus was found to be significantly higher for acellular 2% agarose gels compared with 2% agarose gels seeded at approximately 40×10 6 cells/mL. These results have important implications for interpreting the results of chondrocyte mechanobiology studies in agarose hydrogels.

Comparison of Multipotent Differentiation Potentials of Murine Primary Bone Marrow Stromal Cells and Mesenchymal Stem Cell Line C3H10T1/2

Murine C3H10T1/2 cells have many features of mesenchymal stem cells (MSCs). Whether or not the multipotent differentiation capability of C3H10T1/2 cells is comparable to that of primary bone marrow-derived MSCs (BM-MSCs) was investigated in this study. For in vitro osteogenic differentiation, both BM-MSCs and C3H10T1/2 cells differentiated to osteoblastic cell lineage and showed positive staining for alkaline phosphatase (ALP) and increased mRNA expression of Runx2, Col1alphaI, and osteocalcin. C3H10T1/2 cells and BM-MSCs induced similar amounts of bone formation in the biomaterials. Under chondrogenic induction in the presence of TGF-beta1, cell pellets of both BM-MSCs and C3H10T1/2 cells formed cartilage-like tissues with cartilage matrix components including proteoglycan, type II collagen, and aggrecan. However, C3H10T1/2 cells presented lower adipogenic differentiation potential, with only about 10% C3H10T1/2 cells (but about 70% of BM-MSCs) being committed to adipogenesis. In this study we confirmed that C3H10T1/2 cells coimplanted with osteoconductive scaffolds can form bone spontaneously in vivo and that C3H10T1/2 cells have a basal level of osteocalcin expression, suggesting that they may be a good alternative source of primary BM-MSCs for investigating osteogenic and chondrogenic differentiation in bone or cartilage tissue engineering studies. Caution is needed when using C3H10T1/2 cells for adipogenic studies as they appear to have lower adipogenic potential than BM-MSCs.

Assessment of tissue repair in full thickness chondral defects in the rabbit using magnetic resonance imaging transverse relaxation measurements

The purpose of this study was to determine if the noninvasive and nondestructive technique of magnetic resonance imaging could be used to quantify the amount of repair tissue that fills surgically-induced chondral defects in the rabbit. Sixteen 4-mm diameter full-thickness chondral defects were created. A photopolymerizable hydrogel was used to seal the defects as a treatment modality. At 5 weeks, the animals were sacrificed and the distal femur was subjected to MRI analyses at high field (9.4 T). The transverse relaxation time (T(2)) in each defect was measured. Histology and histomorphometric analysis were used to quantify the amount of repair tissue that filled each defect. The relationship between T(2) and percent tissue fill was found to fit well to a negatively sloped, linear model. The linear (Pearson's product-moment) correlation coefficient was found to be r = -0.82 and the associated coefficient of determination was r(2) = 0.67. This correlation suggests that the MRI parameter T(2) can be used to track changes in the amount of repair tissue that fills cartilage defects. This would be especially useful in in vivo cartilage tissue engineering studies that attempt to determine optimal biomaterials for scaffold design.

Collagens of Articular Cartilage: Structure, Function, and Importance in Tissue Engineering

Collagen is a crucial matrix component of articular cartilage. Because articular cartilage is a load bearing tissue, developing mechanical integrity is a central goal of tissue engineering. The significant role of collagen in cartilage biomechanics necessitates creating a collagen network in tissue engineered constructs. An extensive network of collagen fibrils provides cartilage with mechanical integrity, but developing strategies to replicate this collagen network remains a challenge for articular cartilage tissue engineering efforts. To study the structure and biomechanics of the collagen network, many experimental and computational methodologies have been developed. However, despite extensive cartilage tissue engineering research, few studies have assessed collagen type, crosslinks, or fibril orientation. Further study of the collagen network, both within native tissue and engineered neotissue, will enable more robust constructs to be developed. This review focuses on the biology and biomechanics of the collagen network, relevant experimental methods for assessing the collagen network, and articular cartilage tissue engineering studies that have examined collagen.

Chondrogenic differentiation and functional maturation of bovine mesenchymal stem cells in long-term agarose culture

The developmental history of the chondrocyte results in a cell whose biosynthetic activities are optimized to maintain the concentration and organization of a mechanically functional cartilaginous extracellular matrix. While useful for cartilage tissue engineering studies, the limited supply of healthy autologous chondrocytes may preclude their clinical use. Consequently, multipotential mesenchymal stem cells (MSCs) have been proposed as an alternative cell source. While MSCs undergo chondrogenesis, few studies have assessed the mechanical integrity of their forming matrix. Furthermore, efficiency of matrix formation must be determined in comparison to healthy chondrocytes from the same donor. Given the scarcity of healthy human tissue, this study determined the feasibility of isolating bovine chondrocytes and MSCs, and examined their long-term maturation in three-dimensional agarose culture. Bovine MSCs were seeded in agarose and induced to undergo chondrogenesis. Mechanical and biochemical properties of MSC-laden constructs were monitored over a 10-week period and compared to those of chondrocytes derived from the same group of animals maintained similarly. Our results show that while chondrogenesis does occur in MSC-laden hydrogels, the amount of the forming matrix and measures of its mechanical properties are lower than that produced by chondrocytes under the same conditions. Furthermore, some important properties, particularly glycosaminoglycan content and equilibrium modulus, plateau with time in MSC-laden constructs, suggesting that diminished capacity is not the result of delayed differentiation. These findings suggest that while MSCs do generate constructs with substantial cartilaginous properties, further optimization must be done to achieve levels similar to those produced by chondrocytes.