Effects of equine joint injury on boundary lubrication of articular cartilage by synovial fluid: role of hyaluronan.

OBJECTIVE: To compare equine synovial fluid (SF) from injured and control joints for cartilage boundary lubrication function; concentrations of the putative boundary lubricant molecules hyaluronan (HA), proteoglycan 4 (PRG4), and surface-active phospholipids (SAPLs); relationships between lubrication function and composition; and lubrication restoration by addition of HA. METHODS: Equine SF from normal joints, joints with acute injury, and joints with chronic injury were analyzed for boundary lubrication of normal articular cartilage (kinetic friction coefficient [µ(kinetic) ]). Equine SF samples were analyzed for HA, PRG4, and SAPL concentrations and HA molecular weight distribution. The effect of the addition of HA, of different concentrations and molecular weight, on the µ(kinetic) of equine SF samples from normal joints and joints with acute injury was determined. RESULTS: The µ(kinetic) of equine SF from joints with acute injury (0.036) was higher (+39%) than that of equine SF from normal joints (0.026). Compared to normal equine SF, SF from joints with acute injury had a lower HA concentration (-30%) of lower molecular weight forms, higher PRG4 concentration (+83%), and higher SAPL concentration (+144%). Equine SF from joints with chronic injury had µ(kinetic) , PRG4, and SAPL characteristics intermediate to those of equine SF from joints with acute injury and normal equine SF. Regression analysis revealed that the µ(kinetic) value decreased with increasing HA concentration in equine SF. The friction-reducing properties of HA alone improved with increasing concentration and molecular weight. The addition of high molecular weight HA (4,000 kd) to equine SF from joints with acute injury reduced the µ(kinetic) to a value near that of normal equine SF. CONCLUSION: In the acute postinjury stage, equine SF exhibits poor boundary lubrication properties, as indicated by a high µ(kinetic) . HA of diminished concentration and molecular weight may be the basis for this, and adding HA to deficient equine SF restored lubrication function.

Articular cartilage tensile integrity: modulation by matrix depletion is maturation-dependent.

Articular cartilage function depends on the molecular composition and structure of its extracellular matrix (ECM). The collagen network (CN) provides cartilage with tensile integrity, but must also remodel during growth. Such remodeling may depend on matrix molecules interacting with the CN to modulate the tensile behavior of cartilage. The objective of this study was to determine the effects of increasingly selective matrix depletion on tensile properties of immature and mature articular cartilage, and thereby establish a framework for identifying molecules involved in CN remodeling. Depletion of immature cartilage with guanidine, chondroitinase ABC, chondroitinase AC, and Streptomyces hyaluronidase markedly increased tensile integrity, while the integrity of mature cartilage remained unaltered after depletion with guanidine. The enhanced tensile integrity after matrix depletion suggests that certain ECM components of immature matrix serve to inhibit CN interactions and may act as modulators of physiological alterations of cartilage geometry and tensile properties during growth/maturation.

Shaped, stratified, scaffold-free grafts for articular cartilage defects.

One goal of treatment for large articular cartilage defects is to restore the anatomic contour of the joint with tissue having a structure similar to native cartilage. Shaped and stratified cartilaginous tissue may be fabricated into a suitable graft to achieve such restoration. We asked if scaffold-free cartilaginous constructs, anatomically shaped and targeting spherically-shaped hips, can be created using a molding technique and if biomimetic stratification of the shaped constructs can be achieved with appropriate superficial and middle/deep zone chondrocyte subpopulations. The shaped, scaffold-free constructs were formed from the alginate-released bovine calf chondrocytes with shaping on one (saucer), two (cup), or neither (disk) surfaces. The saucer and cup constructs had shapes distinguishable quantitatively (radius of curvature of 5.5 +/- 0.1 mm for saucer and 2.8 +/- 0.1 mm for cup) and had no adverse effects on the glycosaminoglycan and collagen contents and their distribution in the constructs as assessed by biochemical assays and histology, respectively. Biomimetic stratification of chondrocyte subpopulations in saucer- and cup-shaped constructs was confirmed and quantified using fluorescence microscopy and image analysis. This shaping method, combined with biomimetic stratification, has the potential to create anatomically contoured large cartilaginous constructs.

Three-dimensional (3-D) imaging of chondrocytes in articular cartilage: growth-associated changes in cell organization.

Three-dimensional (3-D) imaging and analysis techniques can be used to assess the organization of cells in biological tissues, providing key insights into the role of cell arrangement in growth, homeostasis, and degeneration. The objective of the present study was to use such methods to assess the growth-related changes in cell organization of articular cartilage from different sites in the bovine knee. Three-dimensional images of fetal, calf, and adult cartilage were obtained and processed to identify cell nuclei. The density of cells was lower with growth and with increasing depth from the articular surface. The cell organization, assessed by the angle to the nearest neighboring cell, also varied with growth, and reflected the classical organization of cells in adult tissue, with neighboring cells arranged horizontally in the superficial zone (average angle of 20 degrees) and vertically in the deep zone (60 degrees). In all other regions and growth stages of cartilage, the angle was approximately 32 degrees, indicative of an isotropic organization. On the contrary, the nearest neighbor distance did not vary significantly with growth or depth. Together, these results indicate that cartilage growth is associated with distinctive 3-D arrangements of groups of chondrocytes.

Short-term retention of labeled chondrocyte subpopulations in stratified tissue-engineered cartilaginous constructs implanted in vivo in mini-pigs.

It is likely that effective application of cell-laden implants for cartilage defects depends on retention of implanted cells and interaction between implanted and host cells. The objectives of this study were to characterize stratified cartilaginous constructs seeded sequentially with superficial (S) and middle (M) chondrocyte subpopulations labeled with fluorescent cell tracking dye PKH26 (*) and determine the degree to which these stratified cartilaginous constructs maintain their architecture in vivo after implantation in mini-pigs for 1 week. Alginate-recovered cells were seeded sequentially to form stratified S*/M (only S cells labeled) and S*/M* (both S and M cells labeled) constructs. Full-thickness defects (4 mm diameter) were created in the patellofemoral groove of adult Yucatan mini-pigs and filled with portions of constructs or left empty. Constructs were characterized biochemically, histologically, and biomechanically, and stratification visualized and quantified, before and after implant. After 1 week, animals were sacrificed and implants retrieved. After 1 week in vivo, glycosaminoglycan and collagen content of constructs remained similar to that at implant, whereas DNA content increased. Histological analyses revealed features of an early repair response, with defects filled with tissues containing little matrix and abundant cells. Some implanted (PKH26-labeled) cells persisted in the defects, although constructs did not maintain a stratified organization. Of the labeled cells, 126 +/- 38% and 32 +/- 8% in S*/M and S*/M* constructs, respectively, were recovered. Distribution of labeled cells indicated interactions between implanted and host cells. Longer-term in vivo studies will be useful in determining whether implanted cells are sufficient to have a positive effect in repair.

Microenvironment regulation of PRG4 phenotype of chondrocytes.

Articular cartilage is a heterogeneous tissue with superficial (S), middle (M), and deep (D) zones. Chondrocytes in the S zone secrete the lubricating PRG4 protein, while chondrocytes from the M and D zones are more specialized in producing large amounts of the glycosaminoglycan (GAG) component of the extracellular matrix. Soluble and insoluble chemicals and mechanical stimuli regulate cartilage development, growth, and homeostasis; however, the mechanisms of regulation responsible for the distinct PRG4-positive and negative phenotypes of chondrocytes are unknown. The objective of this study was to determine if interaction between S and M chondrocytes regulates chondrocyte phenotype, as determined by coculture in monolayer at different ratios of S:M (100:0, 75:25, 50:50, 25:75, 0:100) and at different densities (240,000, 120,000, 60,000, and 30,000 cells/cm(2)), and by measurement of PRG4 secretion and expression, and GAG accumulation. Coculture of S and M cells resulted in significant up-regulation in PRG4 secretion and the percentage of cells expressing PRG4, with simultaneous down-regulation of GAG accumulation. Tracking M cells with PKH67 dye in coculture revealed that they maintained a PRG4-negative phenotype, and proliferated less than S cells. Taken together, these results indicate that the up-regulated PRG4 expression in coculture is a result of preferential proliferation of PRG4-expressing S cells. This finding may have practical implications for generating a large number of phenotypically normal S cells, which can be limited in source, for tissue engineering applications.

Effect of bone morphogenetic proteins 2 and 7 on septal chondrocytes in alginate.

OBJECTIVE: To determine the effects of bone morphogenetic proteins (BMP)-2 and -7, and serum, on extracellular matrix production by human septal chondrocytes in alginate. STUDY DESIGN: Human nasal septal chondrocytes were expanded, suspended in alginate, and cultured in BMP-2 or 7, with and without serum. The optimal concentration of each growth factor was determined based on matrix production. Next, the synergistic effects of BMP-2 and -7 at optimal concentrations were determined on separate beads, based on matrix quantity and histology. RESULTS: Matrix content was highest with concentrations of BMP-2 and -7 of 100 ng/ml and 20 ng/ml, respectively, with serum. Adding both BMP-2 and -7, with serum, increased matrix content by factors of 5.1 versus serum-only cultures, 2.7 versus only BMP-2 with serum, and 2.4 versus only BMP-7 with serum. All comparisons were statistically significant. CONCLUSION: BMP-2 and -7 significantly increase production of extracellular matrix by septal chondrocytes suspended in alginate. The presence of serum improves matrix production. SIGNIFICANCE: BMP-2 and -7 have great potential for use in cartilage tissue engineering.

Tailoring secretion of proteoglycan 4 (PRG4) in tissue-engineered cartilage.

Articular cartilage provides a low-friction surface for joint articulation, with boundary lubrication facilitated by proteoglycan 4 (PRG4), which is secreted by chondrocytes of the superficial zone. Chondrocytes from different zones are phenotypically distinct, and their phenotypes in vitro are influenced by the system in which they are cultured. We hypothesized that culturing cells from the superficial (S) zone in two-dimensional monolayer or three-dimensional alginate would affect their synthesis of PRG4, and that subsequently seeding them atop alginate-recovered cells from the middle/ deep (M) zone in various proportions would result in tissue-engineered constructs with varying levels of PRG4 secretion and matrix accumulation. During monolayer culture, S cells retained their PRG4-secreting phenotype, whereas in alginate culture the percentage of cells secreting PRG4 decreased with time. Constructs formed with increasing percentages of S cells decreased in thickness and matrix accumulation, depending on both the culture conditions before construct formation and the S-cell density. PRG4-secreting cells were localized to the S-cell seeded construct surface, with secretion rates of 0.1-4 pg/cell/day or 0.1-1 pg/cell/day for constructs formed with monolayer-recovered or alginate-recovered S cells, respectively. Tailoring secretion of PRG4 in cartilage constructs may be useful for enhancing low-friction properties at the articular surface, while maintaining other surfaces free of PRG4 for enhancing integration with surrounding tissues.

Tracking chondrocytes and assessing their proliferation with PKH26: effects on secretion of proteoglycan 4 (PRG4).

Distinguishing between implanted and host-derived cells, as well as between distinct cell phenotypes, would be useful in assessing the mechanisms of cell-based repair of cartilage. The fluorescent tracker dye, PKH26, was previously applied to several cell types to assess proliferation in vitro and to track cells in vivo. The objectives of this study were to assess the utility of PKH26 for tracking chondrocytes from superficial and middle zones and their proliferation, and determine the effects of PKH26 on chondrocyte functions, in particular, proliferation and secretion of Proteoglycan 4 (PRG4). PKH26-labeled and unlabeled superficial and middle zone chondrocytes were plated in either low- or high-density monolayer culture and analyzed for retention of PKH26 by flow cytometry and fluorescence microscopy at days 0 and 7. Cell suspensions and conditioned media were analyzed for DNA and secretion of PRG4, respectively. Flow cytometric histograms were deconvolved so that the number of cells in each doubling generation contributing to the final cell population could be estimated. Chondrocytes were consistently and intensely labeled with PKH26 through 7 cycles of division. At day 7 of culture, >97% of superficial zone cells seeded at low or high density could be distinguished as fluorescent, as could middle zone cells seeded at high density. Retention of cell fluorescence after PKH26 labeling and lack of adverse effects on cell proliferation and synthesis of PRG4 suggest that PKH26 can be useful in determining the fate and function of implanted chondrocytes in vivo, as well as monitoring proliferation in vitro.

Stored human septal chondrocyte viability analyzed by confocal microscopy.

OBJECTIVES: To analyze the effects of prolonged storage time, at warm and cold temperatures, on the viability of human nasal septal chondrocytes and to understand the implications for tissue engineering of septal cartilage. DESIGN: Basic science. SUBJECTS: Septal cartilage was obtained from 10 patients and placed in bacteriostatic isotonic sodium chloride solution. Four specimens were kept at 23 degrees C, and 4 were kept at 4 degrees C. The viability of the chondrocytes within the cartilage was assessed using confocal laser scanning microscopy every 5 days. The 2 other specimens were assessed for viability on the day of harvest. RESULTS: Viability on the day of harvest was 96%, implying minimal cell death from surgical trauma. After 1 week, cell survival in all specimens was essentially unchanged from the day of harvest. At 23 degrees C, the majority (54%) of cells were alive after 20 days. At 4 degrees C, 70% of cells survived 1 month and 38% were alive at 2 months. Qualitatively, chondrocytes died in a topographically uniform distribution in warm specimens, whereas cold specimens displayed a more irregular pattern of cell death. CONCLUSION: Septal chondrocytes remain viable for prolonged periods when stored in simple bacteriostatic isotonic sodium chloride solution, and such survival is enhanced by cold storage.

Human serum for tissue engineering of human nasal septal cartilage.

OBJECTIVE: To compare the chondrogenic and proliferative effects of pooled human serum (HS) and fetal bovine serum (FBS) on tissue-engineered human nasal septal chondrocytes. STUDY DESIGN AND SETTING: Human chondrocytes were expanded for one passage in monolayer in medium supplemented with 10% FBS, 2% HS, 10% HS, or 20% HS. Cells were then suspended in alginate beads for 3D culture for 2 weeks with 10% FBS, 2% HS, 10% HS, or 20% HS. RESULTS: Monolayer cell yields were greater with HS than FBS. In alginate, cellular proliferation, glycosaminoglycan production per cell, and type II collagen were significantly higher with 10% HS compared to 10% FBS controls. CONCLUSION: HS results in increased proliferation and production of cartilaginous extracellular matrix by tissue-engineered human nasal septal chondrocytes, compared to FBS controls. SIGNIFICANCE: Culture with human serum may facilitate creation of neocartilage constructs that more closely resemble native tissue.

Depth-varying density and organization of chondrocytes in immature and mature bovine articular cartilage assessed by 3d imaging and analysis.

Articular cartilage is a heterogeneous tissue, with cell density and organization varying with depth from the surface. The objectives of the present study were to establish a method for localizing individual cells in three-dimensional (3D) images of cartilage and quantifying depth-associated variation in cellularity and cell organization at different stages of growth. Accuracy of nucleus localization was high, with 99% sensitivity relative to manual localization. Cellularity (million cells per cm3) decreased from 290, 310, and 150 near the articular surface in fetal, calf, and adult samples, respectively, to 120, 110, and 50 at a depth of 1.0 mm. The distance/angle to the nearest neighboring cell was 7.9 microm/31 degrees , 7.1 microm/31 degrees , and 9.1 microm/31 degrees for cells at the articular surface of fetal, calf, and adult samples, respectively, and increased/decreased to 11.6 microm/31 degrees , 12.0 microm/30 degrees , and 19.2 microm/25 degrees at a depth of 0.7 mm. The methodologies described here may be useful for analyzing the 3D cellular organization of cartilage during growth, maturation, aging, degeneration, and regeneration.

Characterization of human nasal septal chondrocytes cultured in alginate.

BACKGROUND: After serial passages in monolayer, chondrocytes dedifferentiate into a fibroblast-like phenotype. Our objective was to determine if culture in alginate affects the phenotype of dedifferentiated human nasal septal chondrocytes. STUDY DESIGN: Human nasal septal chondrocytes were seeded at low density and passaged in monolayer culture. At passages (P) 1, 2, and 3 a portion of cells were cultured in alginate. Collagen, glycosaminoglycan (GAG), and DNA production were assessed. RESULTS: Chondrocytes in alginate proliferated less yet produced higher levels of GAG and collagen than those in monolayer culture. Alginate encapsulated P1 chondrocytes stained strongly for GAG and collagen type II, and minimally for collagen type I. Monolayer cells at P0 and P1 stained positively for collagen type II. All monolayer passages stained positive for collagen type I with minimal GAG staining. CONCLUSIONS: Compared with monolayer culture, alginate stimulates deposition of GAG and collagen type II, and supports the chondrocyte phenotype through P1, but does not promote redifferentiation.

Proteoglycan 4 (PRG4) synthesis and immunolocalization in bovine meniscus.

Proteoglycan 4 (PRG4) is synthesized and secreted into the synovial fluid by articular chondrocytes and synovial cells, lining the cavity of joints. A thin layer of PRG4 is also present at the articular surface, where it appears to be involved in boundary lubrication. This study investigated if PRG4 is also synthesized and secreted by the cells within meniscus, and if PRG4 is also present in, and at the surface of, meniscus. PRG4 was visualized in sections of bovine calf menisci by immunohistochemistry. PRG4 was detected in two regions: (1) at the femoral and tibial surfaces of the meniscus, and within cells below these surfaces; and (2) within and near cells along the radial tie fibers and circumferential fibers. From meniscus tissue harvested from these surfaces, PRG4 was extracted with 4M GuHCl and quantified by ELISA. There was 0.20 +/- 0.01 and 0.25 +/- 0.04 microg PRG4/cm(2) area of lateral and medial meniscus surface, respectively. ELISA analysis of spent medium from other samples of meniscus surface tissue incubated in medium supplemented with serum and ascorbate showed that 8.1 +/- 1.1 microg PRG4/cm(2) area of meniscus surface was secreted over six days. These results demonstrate that PRG4 is synthesized and secreted by certain cell populations in the meniscus, and that PRG4 is present in the meniscus at surfaces and also internal fibers where it may contribute to boundary lubrication.

Effect of growth factors on cell proliferation, matrix deposition, and morphology of human nasal septal chondrocytes cultured in monolayer.

OBJECTIVES: Tissue engineering of septal cartilage provides ex vivo growth of cartilage from a patient's own septal chondrocytes for use in craniofacial reconstruction. To become clinically applicable, it is necessary to rapidly expand a limited population of donor chondrocytes and then stimulate the production of extracellular matrix on a biocompatible scaffold. The objective of this study was to determine favorable serum-free culture conditions for proliferation of human septal chondrocytes using various concentrations and combinations of four growth factors. STUDY DESIGN: Prospective, randomized, controlled study. METHODS: Nasal septal chondrocytes from six patient donors were isolated by enzymatic digestion and expanded in monolayer culture in both serum-free media (SFM) and 2% fetal bovine serum (FBS). Both of these groups were exposed to varying concentrations and combinations of transforming growth factor (TGF)-beta1, basic fibroblast growth factor (FGF)-2 both at 1, 5, and 25 ng/mL, and bone morphogenetic protein (BMP)-2 and insulin-like growth factor (IGF)-1, both at 5, 25, and 125 ng/mL in the medium during the expansion phase. Cell morphology was assessed throughout the culture duration. After 7 days of monolayer growth, cultures were assessed for cellularity and glycosaminoglycan (GAG) content. RESULTS: The addition of low-dose FBS in culture media consistently led to significantly greater cell proliferation and matrix deposition than the SFM cell cultures. FGF-2 and TGF-beta1 both alone and in combination led to the greatest proliferative effect compared with the other growth factors. In contrast, BMP-2 and IGF-1 led to the least cell proliferation although was most effective in retaining chondrocyte cell morphology. CONCLUSIONS: With the addition of TGF-beta1 and FGF-2 to culture media, the concentration of serum can be greatly decreased and possibly eliminated altogether without jeopardizing cell proliferation.

Growth and phenotype of low-density nasal septal chondrocyte monolayers.

OBJECTIVE: To analyze the growth patterns and differentiation of human septal chondrocyte monolayers of different seeding densities. STUDY DESIGN: Chondrocytes from 8 donors were plated at densities ranging from 20,000 cells/cm(2) (high density) to 300 cells/cm(2) (very low density). Confluency, cellularity, and glycosaminoglycan content were determined from days 1 to 15. RESULTS: Confluency was attained at 5.8, 8.3, 11.0, and 14.8 days for high-, intermediate-, low-, and very low-density monolayers, respectively (P < 0.001). Regression growth curves showed typical lag, logarithmic, and stationary phases. Confluent monolayers attained similar cellularity (power = 0.94) and differentiation (power = 0.88), regardless of initial density. CONCLUSIONS: Human septal chondrocyte monolayers reach confluency from very low initial densities. Growth patterns, cellularity, and differentiation are similar to other starting densities. SIGNIFICANCE: Very low-density monolayers expanded cell number 838-fold in 1 passage and therefore are sufficient for tissue-engineering purposes. This is important because of the requirement of maintaining differentiation and the limitation of small tissue harvest specimens.

Human knee and ankle cartilage explants: catabolic differences.

The prevalence of osteoarthritis (OA) is lower in some joints, i.e., the ankle, than in the knee. We have compared the cartilages from these two joints of the same limb in adult donors (matched pairs). Our data to date suggest that there are metabolic, biochemical and biomechanical differences between the cartilages of the two joints. The current study has focused on extending the metabolic studies comparing the response of chondrocytes to Interleukin-1beta (IL-1beta) and osteogenic protein 1 (OP-1) by analyzing changes in sulfate incorporation into glycosaminoglycans (GAGs) as a measure of proteoglycan (PG) synthesis. Human adult chondrocytes from normal knees (tibiofemoral) and ankles (talocrural) joints cultured as explants both responded to IL-1beta after 72 h by decreasing PG synthesis; however, the IC50 for the knee chondrocytes was 6.2 pg/ml, while that for the ankle was 35 pg/ml. When the explants were incubated for 72 h with IL-1beta and allowed to rebound without IL-1beta, synthesis of PG was significantly elevated by ankle chondrocytes within five days; knee chondrocytes were unable to significantly increase synthesis even after eight days. However, in both knee and ankle, application of OP-I enhanced PG synthesis in the rebound phase. In response to IL-1, an upregulation of proteinase activity was detectable by an increase in the neoepitopes proteolytically-generated by both aggrecanase and matrix metalloproteinases (MMPs), in the deep zone of the knee cartilage. Stromelysin and collagenase were upregulated as well. The data emerging from these studies confirm that the ankle is less responsive to catabolic stimulation and more responsive to anabolic stimulation following IL-1 removal. These differences in metabolic activity between the cartilages of the two joints could in part help to explain their differences in susceptibility to OA.

Tissue-engineered human nasal septal cartilage using the alginate-recovered-chondrocyte method.

OBJECTIVES: Tissue engineering of nasal septal cartilage has numerous potential applications in craniofacial reconstruction. Chondrocytes suspended in alginate gel have been shown to produce a substantial cell-associated matrix. The objective of this study was to determine whether cartilage tissue could be generated using the alginate-recovered-chondrocyte (ARC) method, in which chondrocytes are cultured in alginate as an intermediate step in tissue fabrication. METHODS: Nasal septal chondrocytes from five patient donors were isolated by enzymatic digestion, then expanded in monolayer culture. At confluency, a portion of those cells were seeded at high density onto a semipermeable membrane and cultured for 14, 21, or 28 days (monolayer group). The remaining cells were suspended in alginate and cultured until a cell-associated matrix was observed (10-17 days). Cells and their associated matrix were released from alginate (ARC group), seeded onto a semipermeable membrane, and cultured as already described. DNA (Hoechst 33258 Assay), glycosaminoglycan (GAG; dimethylmethylene blue assay), and collagen (hydroxyproline assay) were analyzed biochemically. Immunohistochemistry was performed to assess expression of collagens type I and type II. Histochemistry was performed to localize cells accumulating sulfated GAG (Alcian blue stain). RESULTS: The ARC constructs, in contrast to the monolayer constructs, had substantial structural stability and the histologic and gross appearance of cartilaginous tissue. ARC constructs demonstrated significantly greater GAG and collagen accumulation than monolayer constructs (P <.05). Histologic analysis revealed substantial GAG and collagen type II production and only moderate collagen type I production. The composition of the matrix was thus similar to that of native human septal cartilage. CONCLUSIONS: Tissue-engineered human nasal septal cartilage using the ARC method has the histologic and gross appearance of native cartilage and has biochemical composition more like that of native cartilage than monolayer constructs. This is the first report of human nasal septal neocartilage formation without the use of biodegradable scaffolds.

Human septal chondrocyte redifferentiation in alginate, polyglycolic acid scaffold, and monolayer culture.

OBJECTIVES/HYPOTHESIS: Tissue engineering laboratories are attempting to create neocartilage that could serve as an implant material for structural support during reconstructive surgery. One approach to forming such tissue is to proliferate chondrocytes in monolayer culture and then seed the expanded cell population onto biodegradable scaffolds. However, chondrocytes are known to dedifferentiate after this type of monolayer growth and, as a result, decrease their production of cartilaginous extracellular matrix components such as sulfated glycosaminoglycans. The resultant tissue lacks the biomechanical properties characteristic of cartilage. The objective of the study was to determine whether different culture systems could induce monolayer-expanded human septal chondrocytes to redifferentiate and form extracellular matrix. STUDY DESIGN: Laboratory research. METHODS: Chondrocytes were isolated from human nasal septal cartilage of five donor patients (age, 35.8 +/- 9.3 y). Cell populations were seeded at low density (30,000 cells/cm2) into monolayer culture and expanded for 4 to 6 days. Following trypsin release, chondrocytes were placed into three different systems for neocartilage formation: alginate beads, polyglycolic acid scaffolds, and monolayer. After 7 and 14 days of growth, neocartilage was analyzed using histological and quantitative biochemical assessment of cellularity (Hoechst 33258 assay) and sulfated glycosaminoglycan content (dimethyl methylene blue assay). RESULTS: Histologically, alginate beads contained spherical chondrocytes surrounded by dense extracellular matrix, an appearance similar to that of native cartilage. In contrast, polyglycolic acid scaffolds and monolayer cultures contained elongated cells with scant staining for matrix sulfated glycosaminoglycans, which are features that are characteristic of dedifferentiated chondrocytes. Biochemical analysis demonstrated a lower level of cell proliferation (P <.001) in scaffolds (+52% over baseline) and alginate (+96% over baseline) than in monolayer (+366% over baseline), as well as a higher content of sulfated glycosaminoglycans per cell (P <.001), after 14 days of growth in alginate culture than in either polyglycolic acid scaffolds (19-fold difference) or monolayer (98-fold difference). CONCLUSIONS: Of the systems compared, monolayer-expanded human septal chondrocytes demonstrated the greatest accumulation of sulfated glycosaminoglycans per cell when grown in alginate beads. Future research on cartilage tissue engineering may use alginate culture for reverting dedifferentiated cells back to the chondrocytic phenotype.

Mechanical characterization of native and tissue-engineered cartilage.

Cartilage functions as a low-friction, wear-resistant, load-bearing tissue. During a normal gait cycle, one cartilage surface rolls and slides against another, all the while being loaded and unloaded. The durability of the tissue also makes for an impressive material to study. However, when cartilage is damaged or diseased, the tissue has little capacity to repair itself. The goal of cell-based repair strategies to replace damaged or diseased tissue requires that the functional biomechanical properties of normal (developing or mature), diseased, and repair cartilage be restored. This chapter addresses some of the major methods used to assess the biomechanical properties of native and tissue-engineered cartilage. First, the traditional methods of testing by compression, tension, shear, and indentation are reviewed. Next, additional methods to evaluate interfacial mechanics and lubrication are described. Thus, a variety of mechanical tests may be used to assess functional properties for normal, diseased, and tissue-engineered cartilage.