Albert C. Chen

Compressive properties and function-composition relationships of developing bovine articular cartilage.

The composition of cartilage is known to change during fetal and postnatal development. The objectives of this study were to characterize the compressive biomechanical properties of the 1 mm thick articular layer of cartilage of the distal femur from third‐trimester bovine fetuses, from 1 to 3 week old bovine calf and from young adult bovine knees, and to correlate these properties with tissue components. The confined compression modulus increased 180% from the fetus to the calf and adult. The hydraulic permeability at 45% offset compression (relative to the free‐swelling thickness) decreased by 70% from fetus to adult. These development‐associated changes in biomechanical properties were primarily associated with a marked (∼2–3‐fold) increase during development in collagen content and no detectable change in glycosaminoglycan (GAG) content. A role for collagen in the compressive properties of cartilage and the gradual increase in collagen during development suggest that collagen metabolism is critical for cartilage tissue engineering and repair therapies.

Static and dynamic compression modulate matrix metabolism in tissue engineered cartilage

Static and dynamic compression are known to modulate the metabolism of articular cartilage. The present study focused on determining the effects of compressive loading on the metabolism of sulfated glycosaminoglycans (S‐GAG) and protein in tissue engineered cartilage constructs. Cartilage constructs were subjected to static or dynamic compression for 24 h and radiolabeled with 35SO4 and 3H‐proline to assess the total synthesis and percentage retention of S‐GAG and total protein, respectively. Static compression at an amplitude of 50% suppressed the synthesis of both S‐GAG and protein by 35% and 57%, respectively. Dynamic compression at an amplitude of 5% had stimulatory effects on synthesis that were dependent on the static offset compression amplitude (10% or 50%) and dynamic compression frequency (0.001 or 0.1 Hz). Thus, tissue engineered cartilage demonstrated the ability to respond to mechanical loading in a manner similar to that observed with articular cartilage. Mechanical loading may therefore potentially be used to modulate the growth of cartilaginous tissues in vitro, potentially facilitating the culture of functional cartilage tissues suitable for implantation.

Prolonged Storage Effects on the Articular Cartilage of Fresh Human Osteochondral Allografts

BACKGROUND Fresh osteochondral allograft transplantation is a well-established technique for the treatment of cartilage defects of the knee. It is believed that the basic paradigm of the technique is that the transplantation of viable chondrocytes maintains the articular cartilage matrix over time. Allograft tissue is typically transplanted up to forty-two days after the death of the donor, but it is unknown how the conditions and duration of storage affect the properties of fresh human osteochondral allografts. This study examined the quality of human allograft cartilage as a function of storage for a duration of one, seven, fourteen, and twenty-eight days. We hypothesized that chondrocyte viability, chondrocyte metabolic activity, and the biochemical and biomechanical properties of articular cartilage would remain unchanged after storage for twenty-eight days. METHODS Sixty osteochondral plugs were harvested from ten fresh human femoral condyles within forty-eight hours after the death of the donor and were stored in culture medium at 4 degrees C. At one, seven, fourteen, and twenty-eight days after harvest, the osteochondral plugs were analyzed for (1) viability and viable cell density by confocal microscopy, (2) proteoglycan synthesis by quantification of (35)SO(4) incorporation, (3) glycosaminoglycan content, (4) indentation stiffness, (5) compressive modulus and hydraulic permeability by static and dynamic compression testing, and (6) tensile modulus by equilibrium tensile testing. RESULTS Chondrocyte viability and viable cell density remained unchanged after storage for seven and fourteen days (p > 0.7) and then declined at twenty-eight days (p < 0.001). Proteoglycan synthesis remained unchanged at seven days (p > 0.1) and then declined at fourteen days (p < 0.01) and twenty-eight days (p < 0.001). No significant differences were detected in glycosaminoglycan content (p > 0.8), indentation stiffness (p > 0.4), compressive modulus (p > 0.05), permeability (p > 0.3), or equilibrium tensile modulus after storage for twenty-eight days (p > 0.9). CONCLUSIONS These data demonstrate that fresh human osteochondral allograft tissue stored for more than fourteen days undergoes significant decreases in chondrocyte viability, viable cell density, and metabolic activity, with preservation of glycosaminoglycan content and biomechanical properties. The cartilage matrix is preserved during storage for twenty-eight days, but the chondrocytes necessary to maintain the matrix after transplantation decreased over that time-period.

Depth- and strain-dependent mechanical and electromechanical properties of full-thickness bovine articular cartilage in confined compression

Compression tests have often been performed to assess the biomechanical properties of full-thickness articular cartilage. We tested whether the apparent homogeneous strain-dependent properties, deduced from such tests, reflect both strain- and depth-dependent material properties. Full-thickness bovine articular cartilage was tested by oscillatory confined compression superimposed on a static offset up to 45%. and the data fit to estimate modulus, permeability, and electrokinetic coefficient assuming homogeneity. Additional tests on partial-thickness cartilage were then performed to assess depth- and strain-dependent properties in an inhomogeneous model, assuming three discrete layers (i = 1 starting from the articular surface, to i = 3 up to the subchondral bone). Estimates of the zero-strain equilibrium confined compression modulus (H(A0)), the zero-strain permeability (kp0) and deformation dependence constant (M), and the deformation-dependent electrokinetic coefficient (ke) differed among individual layers of cartilage and full-thickness cartilage. HiA0 increased from layer 1 to 3 (0.27 to 0.71 MPa), and bracketed the apparent homogeneous value (0.47 MPa). ki(p0) decreased from layer 1 to 3 (4.6 x 10(-15) to 0.50 x 10(-15) m2/Pa s) and was less than the homogeneous value (7.3 x 10(-15) m2/Pa s), while Mi increased from layer 1 to 3 (5.5 to 7.4) and became similar to the homogeneous value (8.4). The amplitude of ki(e) increased markedly with compressive strain, as did the homogeneous value: at low strain, it was lowest near the articular surface and increased to a peak in the middle-deep region. These results help to interpret the biomechanical assessment of full-thickness articular cartilage.

Tensile mechanical properties of bovine articular cartilage: variations with growth and relationships to collagen network components

One approach to repairing articular defects is to regenerate cartilage by recapitulating the changes that occur during fetal and postnatal growth into adulthood, and to thereby restore functional biomechanical properties, especially those of the normally strong superficial region. The objectives of this study were (1) to characterize and compare tensile biomechanical properties of the superficial region of articular cartilage of the patellofemoral groove (PFG) and femoral condyle (FC) from bovine animals over a range of growth stages (third‐trimester fetal, 1–3 week‐old calf, and adult), and (2) to determine if these properties were correlated with collagen network components. With growth from the fetus to the adult, the equilibrium and dynamic tensile moduli and strength of cartilage samples increased by an average of 391‐1060%, while the strain at the failure decreased by 43%. The collagen concentration (per wet weight) increased by 98%, and the pyridinoline cross‐link concentration increased by 730%, while the glycosaminoglycan concentration remained unchanged or decreased slightly. Some growth‐associated changes were location‐specific, with tensile moduli and strength attaining higher values in the PFG than the FC. The growth‐associated variation in tensile moduli and strength were associated strongly with variation in the contents of collagen and pyridinoline cross‐link, but not sulfated glycosaminoglycan. The marked changes in the tensile properties and collagen network components of articular cartilage with growth suggest that such parameters may be used to evaluate the degrees to which regenerated cartilage recapitulates normal development and growth.

Matrix stiffness drives epithelial–mesenchymal transition and tumour metastasis through a TWIST1–G3BP2 mechanotransduction pathway

Matrix stiffness potently regulates cellular behaviour in various biological contexts. In breast tumours, the presence of dense clusters of collagen fibrils indicates increased matrix stiffness and correlates with poor survival. It is unclear how mechanical inputs are transduced into transcriptional outputs to drive tumour progression. Here we report that TWIST1 is an essential mechanomediator that promotes epithelial–mesenchymal transition (EMT) in response to increasing matrix stiffness. High matrix stiffness promotes nuclear translocation of TWIST1 by releasing TWIST1 from its cytoplasmic binding partner G3BP2. Loss of G3BP2 leads to constitutive TWIST1 nuclear localization and synergizes with increasing matrix stiffness to induce EMT and promote tumour invasion and metastasis. In human breast tumours, collagen fibre alignment, a marker of increasing matrix stiffness, and reduced expression of G3BP2 together predict poor survival. Our findings reveal a TWIST1–G3BP2 mechanotransduction pathway that responds to biomechanical signals from the tumour microenvironment to drive EMT, invasion and metastasis.

The effects of storage on fresh human osteochondral allografts.

Historically, fresh human osteochondral allografts have been stored in lactated Ringer’s solution at 4° C and then transplanted as quickly as possible, generally within 2 to 5 days, to ensure delivery of a high level of viable chondrocytes. Recently, allograft distribution companies have begun to provide fresh osteochondral allografts that are stored in a proprietary culture medium usually for at least 2 weeks before delivery to the surgeon for implantation. The effects of such storage on human cartilage have not been well-defined. In the current study the effects of storage in lactated Ringer’s solution and in culture media were assessed. After 7 days of storage in lactated Ringer’s solution, a significant decline in chondrocyte viability and metabolic activity was seen. Culture media provided significantly better preservation of the cartilage with viability and metabolic activity remaining essentially unchanged from baseline for as many as 14 days. The biochemical and biomechanical properties of the extracellular matrix remained stable with storage in both solutions with time. These data suggest that osteochondral allografts stored under traditional conditions in lactated Ringer’s solution should continue to be implanted as quickly as possible and certainly within 7 days of donor death. If kept in culture media, the storage duration may be extended to approximately 2 weeks.

Potential of 3-D tissue constructs engineered from bovine chondrocytes/silk fibroin-chitosan for in vitro cartilage tissue engineering.

The use of cell-scaffold constructs is a promising tissue engineering approach to repair cartilage defects and to study cartilaginous tissue formation. In this study, silk fibroin/chitosan blended scaffolds were fabricated and studied for cartilage tissue engineering. Silk fibroin served as a substrate for cell adhesion and proliferation while chitosan has a structure similar to that of glycosaminoglycans, and shows promise for cartilage repair. We compared the formation of cartilaginous tissue in silk fibroin/chitosan blended scaffolds seeded with bovine chondrocytes and cultured in vitro for 2 weeks. The constructs were analyzed for cell viability, histology, extracellular matrix components glycosaminoglycan and collagen types I and II, and biomechanical properties. Silk fibroin/chitosan scaffolds supported cell attachment and growth, and chondrogenic phenotype as indicated by Alcian Blue histochemistry and relative expression of type II versus type I collagen. Glycosaminoglycan and collagen accumulated in all the scaffolds and was highest in the silk fibroin/chitosan (1:1) blended scaffolds. Static and dynamic stiffness at high frequencies was higher in cell-seeded constructs than non-seeded controls. The results suggest that silk/chitosan scaffolds may be a useful alternative to synthetic cell scaffolds for cartilage tissue engineering.

Follow-up of Osteochondral Plug Transfers in a Goat Model A 6-Month Study

Background Osteochondral transfer procedures are increasingly used to resurface full-thickness articular cartilage defects. There has not been long-term assessment/description of autogenous donor and recipient sites. Hypothesis The healing process occurs at the donor/host cartilage and bone interfaces. Study Design Histologic, biochemical, and biomechanical changes were assessed 6 months after an osteochondral transfer in a goat model. Methods Eight adult goats were studied. In the 6 osteochondral transfer goats, 2 autogenous plugs were transferred from the femoral trochlea to defects in the weightbearing portion of the medial femoral condyle. The goats were allowed free range for 6 months. Randomly assigned plugs were assessed. Results Knees of the sacrificed animals had preservation of the joint space with mild chondromalacic changes in both transfer and contralateral control groups. Histologically, no evidence of cartilage (host/donor) healing was seen. Subchondral bone of the plug was contiguous with the surrounding recipient bone. Cellular viability in the autogenous osteochondral plug was seen, and 35SO4 uptake of the articular cartilage was not statistically different from the contralateral control condyle. The indentation stiffness of the transfer plug (mosaicplasty) and the contralateral donor site were similar—much stiffer than normal cartilage including surrounding condylar cartilage. Large structural stiffness of transferred cores and donor sites appeared to be related to their thinner cartilage layer. Conclusions At 6-month follow-up, a cleft between host and transferred articular regions remained, with no integration between the two. Clinical Relevance Autogenous transplantation of osteochondral plugs is possible with integration of subchondral bone and preservation of chondral viability.

A morphologic, biochemical, and biomechanical assessment of short-term effects of osteochondral autograft plug transfer in an animal model.

PURPOSE The objective of this study was to assess the short-term changes that occur after an osteochondral autograft plug transfer from the femoral trochlea to the medial femoral condyle in a goat model. TYPE OF STUDY Articular cartilage repair animal study. METHODS Six adult male goats were used in this study. Two 4.5-mm osteochondral plugs were transferred from the superolateral femoral trochlea to 2 recipient sites in the central portion of the medial femoral condyle for a survival period of 12 weeks. Postmortem, the global effects of the procedure were assessed by gross morphologic inspection and by analyzing the synovial DNA for inflammatory response. The recipient sites were also evaluated histologically and biomechanically. Metabolic activity was determined by (35)SO(4) uptake, and viability was assessed using a live/dead stain and by confocal laser microscopy. RESULTS There was no evidence of significant gross morphologic or histologic changes in the operative knee as a result of the osteochondral donor or recipient sites. The patella, tibial plateau, and medial meniscus did not show any increased degenerative changes as a result of articulating against the donor or recipient sites of the osteochondral autografts. Analysis of synovial DNA revealed no inflammatory response. Biomechanically, 6- to 7-fold greater stiffness was noted in the cartilage of the transferred plugs compared with the control medial femoral condyle. Furthermore, on histologic examination, the healing subchondral bone interface at the recipient site had increased density. Glycosaminoglycan synthesis as determined by (35)SO(4) uptake was upregulated in the transplanted cartilage plug relative to the contralateral control, showing a repair response at the site of implantation. And finally, confocal microscopy showed 95% viability of the transferred plugs in the medial femoral condyle region. CONCLUSIONS Our findings demonstrate the ability to successfully transfer an osteochondral autograft plug with maintenance of chondrocyte cellular viability. The transferred cartilage is stiffer than the control medial femoral condyle cartilage, and there is concern regarding the increased trabecular mass in the healing subchondral plate, but these do not result in increased degenerative changes of the opposing articular surfaces in the short term.