Hai Yao

Regeneration of the articular surface of the rabbit synovial joint by cell homing: a proof of concept study

BACKGROUND A common approach for tissue regeneration is cell delivery, for example by direct transplantation of stem or progenitor cells. An alternative, by recruitment of endogenous cells, needs experimental evidence. We tested the hypothesis that the articular surface of the synovial joint can regenerate with a biological cue spatially embedded in an anatomically correct bioscaffold. METHODS In this proof of concept study, the surface morphology of a rabbit proximal humeral joint was captured with laser scanning and reconstructed by computer-aided design. We fabricated an anatomically correct bioscaffold using a composite of poly-epsilon-caprolactone and hydroxyapatite. The entire articular surface of unilateral proximal humeral condyles of skeletally mature rabbits was surgically excised and replaced with bioscaffolds spatially infused with transforming growth factor beta3 (TGFbeta3)-adsorbed or TGFbeta3-free collagen hydrogel. Locomotion and weightbearing were assessed 1-2, 3-4, and 5-8 weeks after surgery. At 4 months, regenerated cartilage samples were retrieved from in vivo and assessed for surface fissure, thickness, density, chondrocyte numbers, collagen type II and aggrecan, and mechanical properties. FINDINGS Ten rabbits received TGFbeta3-infused bioscaffolds, ten received TGFbeta3-free bioscaffolds, and three rabbits underwent humeral-head excision without bioscaffold replacement. All animals in the TGFbeta3-delivery group fully resumed weightbearing and locomotion 3-4 weeks after surgery, more consistently than those in the TGFbeta3-free group. Defect-only rabbits limped at all times. 4 months after surgery, TGFbeta3-infused bioscaffolds were fully covered with hyaline cartilage in the articular surface. TGFbeta3-free bioscaffolds had only isolated cartilage formation, and no cartilage formation occurred in defect-only rabbits. TGFbeta3 delivery yielded uniformly distributed chondrocytes in a matrix with collagen type II and aggrecan and had significantly greater thickness (p=0.044) and density (p<0.0001) than did cartilage formed without TGFbeta3. Compressive and shear properties of TGFbeta3-mediated articular cartilage did not differ from those of native articular cartilage, and were significantly greater than those of cartilage formed without TGFbeta3. Regenerated cartilage was avascular and integrated with regenerated subchondral bone that had well defined blood vessels. TGFbeta3 delivery recruited roughly 130% more cells in the regenerated articular cartilage than did spontaneous cell migration without TGFbeta3. INTERPRETATION Our findings suggest that the entire articular surface of the synovial joint can regenerate without cell transplantation. Regeneration of complex tissues is probable by homing of endogenous cells, as exemplified by stratified avascular cartilage and vascularised bone. Whether cell homing acts as an adjunctive or alternative approach of cell delivery for regeneration of tissues with different organisational complexity warrants further investigation. FUNDING New York State Stem Cell Science; US National Institutes of Health.

New insight into deformation-dependent hydraulic permeability of gels and cartilage, and dynamic behavior of agarose gels in confined compression

Equilibrium, creep, and dynamic behaviors of agarose gels (2.0-14.8%) in confined compression were investigated in this study. The hydraulic permeabilities of gels were determined by curve-fitting creep data to the biphasic model (J. Biomech. Eng. 102 (1980) 73) and found to be similar in value to those published in the literature (AIChE J. 42 (1996) 1220). A new relationship between intrinsic permeability and volume fraction of water was found for agarose gel, capable of predicting deformation-dependent permeabilities of bovine articular cartilage and 2% agarose gel published in literature. This relationship is accurate for gels and cartilage over a wide range of permeabilities (four orders of magnitude variation). The dynamic stiffness of the gels increases with gel concentration and loading frequency (0.01-1.0Hz). The increase in dynamic stiffness with loading frequency is less pronounced for gels with higher concentrations. The results of this study provide a new insight into deformation-dependent permeability behavior of agarose gel and cartilage, and are important for understanding biological responses of cells to interstitial fluid flow in gel or in cartilage under dynamic mechanical loading.

Engineering alginate as bioink for bioprinting.

Recent advances in three-dimensional (3-D) printing offer an excellent opportunity to address critical challenges faced by current tissue engineering approaches. Alginate hydrogels have been used extensively as bioinks for 3-D bioprinting. However, most previous research has focused on native alginates with limited degradation. The application of oxidized alginates with controlled degradation in bioprinting has not been explored. Here, a collection of 30 different alginate hydrogels with varied oxidation percentages and concentrations was prepared to develop a bioink platform that can be applied to a multitude of tissue engineering applications. The authors systematically investigated the effects of two key material properties (i.e. viscosity and density) of alginate solutions on their printabilities to identify a suitable range of material properties of alginates to be applied to bioprinting. Further, four alginate solutions with varied biodegradability were printed with human adipose-derived stem cells (hADSCs) into lattice-structured, cell-laden hydrogels with high accuracy. Notably, these alginate-based bioinks were shown to be capable of modulating proliferation and spreading of hADSCs without affecting the structure integrity of the lattice structures (except the highly degradable one) after 8days in culture. This research lays a foundation for the development of alginate-based bioink for tissue-specific tissue engineering applications.

Effect of micrometer-scale roughness of the surface of Ti6Al4V pedicle screws in vitro and in vivo.

BACKGROUND Titanium implants that have been grit-blasted and acid-etched to produce a rough microtopography support more bone integration than do smooth-surfaced implants. In vitro studies have suggested that this is due to a stimulatory effect on osteoblasts. It is not known if grit-blasted and acid-etched Ti6Al4V implants also stimulate osteoblasts and increase bone formation clinically. In this study, we examined the effects of micrometer-scale-structured Ti6Al4V surfaces on cell responses in vitro and on tissue responses in vivo. METHODS Ti6Al4V disks were either machined to produce smooth surfaces with an average roughness (Ra) of 0.2 microm or grit-blasted, resulting in an Ra of 2.0, 3.0, or 3.3 microm. Human osteoblast-like cells were cultured on the disks and on tissue culture polystyrene. The cell number, markers of osteoblast differentiation, and levels of local factors in the conditioned media were determined at confluence. In addition, Ti6Al4V pedicle screws with smooth or rough surfaces were implanted into the L4 and L5 vertebrae of fifteen two-year-old sheep. Osteointegration was evaluated at twelve weeks with histomorphometry and on the basis of removal torque. RESULTS The cell numbers on the Ti6Al4V surfaces were lower than those on the tissue culture polystyrene; the effect was greatest on the roughest surface. The alkaline-phosphatase-specific activity of cell lysates was decreased in a surface-dependent manner, whereas osteocalcin, prostaglandin E(2), transforming growth factor-beta1, and osteoprotegerin levels were higher on the rough surfaces. Bone-implant contact was greater around the rough-surfaced Ti6Al4V screws, and the torque needed to remove the rough screws from the bone was more than twice that required to remove the smooth screws. CONCLUSIONS Increased micrometer-scale surface roughness increases osteoblast differentiation and local factor production in vitro, which may contribute to increased bone formation and osteointegration in vivo. There was a correlation between in vitro and in vivo observations, indicating that the use of screws with rough surfaces will result in better bone-implant contact and implant stability.

Diffusivity of Ions in Agarose Gels and Intervertebral Disc: Effect of Porosity

The effect of tissue porosity on ion (sodium, potassium, and chloride) diffusivity in agarose gels and porcine intervertebral disc tissues was investigated using an electrical conductivity method. An empirical, constitutive model for diffusivity (D) of solutes in porous fibrous media was proposed: D/Do=exp [−α(rs/κ1/2)β] where rs is the Stokes radius of a solute, κ is the Darcy permeability of the porous medium, Do is the diffusivity in free solution, α and β are two positive parameters whose values depend on material structure. It is found that α=1.25±0.138, β=0.681±0.059 (95% confidence interval, R2=0.92, n=72) for agarose gels and α=1.29±0.171 and β=0.372±0.088 (95% confidence interval, R2=0.88, n=86) for porcine annulus fibrosus. The functional relationship between solute diffusivity and tissue deformation was derived. Comparisons of our model prediction with experimental data on diffusion coefficients of macromolecules (proteins, dextrans, polymer beads) in agarose gels in the literature were made. Our results were also compared to the data on ion diffusivity in charged gels and in cartilaginous tissues reported in the literature. There was a good agreement between our model prediction and the data in the literature. The present study provides additional information on solute diffusivity in uncharged gels and charged tissues, and is important for understanding nutritional transport in avascular cartilaginous tissues under different mechanical loading conditions.

Effects of Swelling Pressure and Hydraulic Permeability on Dynamic Compressive Behavior of Lumbar Annulus Fibrosus

AbstractThe objective of this study was to investigate the effects of swelling pressure and hydraulic permeability on the dynamic behavior of intervertebral disk tissue in confined compression. Normal (served as a control) and trypsin-treated, axial annulus fibrosus (AF) specimens from the porcine lumbar disks were tested and their swelling strain, swelling pressure, equilibrium compressive modulus (HA, dynamic modulus, and hydraulic permeability (k) were determined at 30% and 40% swelling strain levels. The proteoglycan depletion due to trypsin treatment resulted in significantly lower values of the free swelling strain, swelling pressure, equilibrium modulus, dynamic modulus, and higher value of hydraulic permeability for trypsin-treated group, comparing to those for the control group. At the 30% swelling strain level, the equilibrium moduli were 51.84±14.53 kPa (n=8) for the control group and 15.11±5.67 kPa (n=8) for the trypsin-treated group; and the hydraulic permeabilities were 4.50E-15±1.60E-15 m4/Ns and 8.43E-15±4.29E-15 m4/Ns for control and trypsin-treated groups, respectively. No statistically significant difference in wet tissue density or dry tissue density was found between control and trypsin-treated groups. There was a significant correlation between swelling pressure and compressive (aggregate) modulus (R2=0.824, m=22). The decrease in measured dynamic modulus for trypsin-treated group was attributed to the reduced swelling pressure (or modulus HA and increased hydraulic permeability (k) due to PG depletion.

Caveolin-1 knockout mice have increased bone size and stiffness

The skeletal phenotype of the cav‐1−/− mouse, which lacks caveolae, was examined. μCT and histology showed increased trabecular and cortical bone caused by the gene deletion. Structural changes were accompanied by increased mechanical properties. Cell studies showed that cav‐1 deficiency leads to increased osteoblast differentiation. These results suggest that cav‐1 helps to maintain osteoblast progenitors in a less differentiated state.

Effects of Hydration and Fixed Charge Density on Fluid Transport in Charged Hydrated Soft Tissues

AbstractThe effects of tissue hydration and fixed charge density on hydraulic permeability and creep behavior of cartilaginous tissues have been investigated using the triphasic theory and finite element methods. The empirical model for hydraulic permeability of uncharged gels and Mackie and Meares (1955) model for ion diffusivity were used in the numerical analysis. The hydraulic permeabilities of normal and trypsin-treated porcine annulus fibrosus tissues were measured indirectly. Analysis of the experimental data from this study and in literature indicates that the water content plays a more important role in regulating tissue permeability than fixed charge density for normal tissues. A change in glycosaminoglycan content will change both triphasic closed-circuit (or intrinsic) and biphasic open-circuit permeabilities of cartilaginous tissues. Analysis also shows that both fixed charge density and water content play an important role in tissue creep response. This study adds new knowledge to the permeability and creep behavior of cartilaginous tissues and is important for understanding the nutrition in intervertebral disk.

Electrical conductivity of lumbar anulus fibrosis: effects of porosity and fixed charge density.

Study Design. Experimental investigation of the electrical conductivity of normal and trypsin-treated lumbar anulus fibrosis specimens. Objectives. To measure the electrical conductivity of intervertebral disc tissues and to study the effects of tissue porosity (volume fraction of water) and fixed charge density on the electrical conductivity of anulus fibrosis in physiologic saline. Summary of Background Data. Specific electrical conductivity is one of the material properties of intervertebral discs. Their value depends on ion concentrations and ion diffusivities within the tissue, which in turn are functions of tissue composition and structure. To our knowledge, the electrical conductivity of intervertebral discs has not been studied. Investigation of the electrical conductivity of intervertebral discs and understanding of their relationship to tissue porosity and fixed charge density will provide insights into electromechanical phenomena (e.g., streaming potential) and ion transport in intervertebral discs. Methods. A total of 35 porcine lumbar anulus fibrosis specimens were divided into two groups: one control group (n = 10) and one trypsin-treated group (n = 25). The specimens in the control group were subjected to one-dimensional free swelling in phosphate-buffered saline (pH 7.4), and electrical conductivity and porosity (water content) were measured over a period of about 45 minutes. The specimens in the treated group were immersed in a trypsin solution (372 U/mL phosphate-buffered saline) for 45 minutes at room temperature, and the electrical conductivity and porosity were measured after treatment. The electrical conductivity was correlated to tissue porosity for the control and treated specimens. The influences of porosity and fixed charge density were studied. Results. The average value for control specimens was 5.60 ± 0.89 mS/cm (mean ± SD; n = 10) before swelling and 9.11 ± 0.90 mS/cm (mean ± SD; n = 10) after swelling. Tissue porosity increased from 0.74 ± 0.03 (mean ± SD; n = 10) before swelling to 0.83 ± 0.02 (mean ± SD; n = 10) after swelling. The trypsin treatment reduced anulus fibrosis porosity by 3.6% (P < 0.05) and conductivity by 13% (P < 0.05) compared to those for control specimens after swelling. No significant changes werefound in wet and dry tissue densities between control and treated groups. There was a significant, linear correlation between conductivity and porosity for control anulus fibrosis specimens (R2 = 0.87; 86 measurements). Conclusions. Measured electrical conductivity was sensitive to tissue porosity, but not to fixed charged density for anulus fibrosis specimens in phosphate-buffered saline.

The region-dependent biphasic viscoelastic properties of human temporomandibular joint discs under confined compression

The objective of this study was to determine the biphasic viscoelastic properties of human temporomandibular joint (TMJ) discs, correlate these properties with disc biochemical composition, and examine the relationship between these properties and disc dynamic behavior in confined compression. The equilibrium aggregate modulus (H(A)), hydraulic permeability (k), and dynamic modulus were examined between five disc regions. Biochemical assays were conducted to quantify the amount of water, collagen, and glycosaminoglycan (GAG) content in each region. The creep tests showed that the average equilibrium moduli of the intermediate, lateral, and medial regions were significantly higher than for the anterior and posterior regions (69.75+/-11.47kPa compared to 22.0+/-5.15kPa). Permeability showed the inverse trend with the largest values in the anterior and posterior regions (8.51+/-1.36x10(-15)m(4)/Ns compared with 3.75+/-0.72x10(-15)m(4)/Ns). Discs were 74.5% water by wet weight, 62% collagen, and 3.2% GAG by dry weight. Regional variations were only observed for water content which likely results in the regional variation in biphasic mechanical properties. The dynamic modulus of samples during confined compression is related to the aggregate modulus and hydraulic permeability of the tissue. The anterior and posterior regions displayed lower complex moduli over all frequencies (0.01-3Hz) with average moduli of 171.8-609.3kPa compared with 454.6-1613.0kPa for the 3 central regions. The region of the TMJ disc with higher aggregate modulus and lower permeability had higher dynamic modulus. Our results suggested that fluid pressurization plays a significant role in the load support of the TMJ disc under dynamic loading conditions.