Synovial Fluid Lubricant Properties are Transiently Deficient after Arthroscopic Articular Cartilage Defect Repair with Platelet-Enriched Fibrin Alone and with Mesenchymal Stem Cells.

BACKGROUND: Following various types of naturally-occurring traumatic injury to an articular joint, the lubricating ability of synovial fluid is impaired, with a correlated alteration in the concentration and/or structure of lubricant molecules, hyaluronan and proteoglycan-4. However, the effect of arthroscopic cartilage repair surgery on synovial fluid lubricant function and composition is unknown. HYPOTHESIS: Arthroscopic treatment of full-thickness chondral defects in horses with (1) platelet-enriched fibrin or (2) platelet-enriched fibrin+mesenchymal stem cells leads to equine synovial fluid with impaired lubricant function and hyaluronan and proteoglycan-4 composition. STUDY DESIGN: Controlled Laboratory Study. METHODS: Equine synovial fluid was aspirated from normal joints at a pre-injury state (0 days) and at 10 days and 3 months following fibrin or fibrin+mesenchymal stem cell repair of full thickness chondral defects. Equine synovial fluid samples were analyzed for friction-lowering boundary lubrication of normal articular cartilage (static and kinetic friction coefficients) and concentrations of hyaluronan and proteoglycan-4, as well as molecular weight distribution of hyaluronan. Experimental groups deficient in lubrication function were also tested for the ability of exogenous high-molecular weight hyaluronan to restore lubrication function. RESULTS: Lubrication and biochemical data varied with time after surgery but generally not between repair groups. Relative to pre-injury, kinetic friction was higher (+94%) at 10 days but returned to baseline levels at 3 months while static friction was not altered. Correspondingly, hyaluronan concentration was transiently lower (-64%) and shifted towards lower molecular weight forms, while proteoglycan-4 concentration was increased (+210%) in 10-day samples relative to pre-injury levels. Regression analysis revealed that kinetic friction decreased with increasing total and high molecular weight hyaluronan. Addition of high molecular weight hyaluronan to bring 10-day hyaluronan levels to 2.0mg/ml restored kinetic friction to pre-injury levels. CONCLUSION: Following arthroscopic surgery for cartilage defect repair, synovial fluid lubrication function is transiently impaired, in association with decreased hyaluronan concentration. This functional deficiency in synovial fluid lubrication can be counteracted in vitro by addition of high molecular weight hyaluronan. CLINICAL RELEVANCE: Synovial fluid lubrication is deficient shortly following arthroscopic cartilage repair surgery, and supplementation with high molecular weight hyaluronan may be beneficial.

In vivo oxygen tension in human septal cartilage increases with age.

OBJECTIVES/HYPOTHESIS: Tissue-engineered septal cartilage may provide a source of autologous cartilage for repair of nasal defects. Production of clinically useful neocartilage involves multiple steps that include manipulating the culture environment. Partial pressure of oxygen (ppO(2) ) is a property that has been shown to influence cartilage development. Specifically, studies suggest low ppO(2) augments in vitro growth of articular cartilage. Although in vivo measurements of articular cartilage ppO(2) have demonstrated hypoxic conditions, measurements have not been performed in septal cartilage. The objective of this study was to determine the ppO(2) of septal cartilage in vivo. STUDY DESIGN: Prospective, basic science. METHODS: The ppO(2) was measured in 14 patients (mean ± standard deviation age, 35.9 ± 14.5 years; range, 18-63 years) during routine septoplasty or septorhinoplasty using the OxyLab pO(2) monitor (Oxford Optronix Ltd., Oxford, UK). Measurements were taken from the septum and inferior turbinate. Each patient's age and sex were recorded. RESULTS: The average ppO(2) measured at the septum and inferior turbinate was 10.5 ± 10.1 mm Hg (1.4 ± 1.3%) and 27.6 ± 12.4 mm Hg (3.6 ± 1.6%), respectively. The ppO(2) of these locations was significantly different (P < .005). Advancing age was positively correlated with septal ppO(2) (R(2) = 0.42; P < .05). Septal ppO(2) showed no significant sex variation. CONCLUSIONS: This is the first report of in vivo measurement of ppO(2) in septal cartilage. The data demonstrate reduced oxygenation of septal cartilage relative to the inferior turbinate. This elucidates an important characteristic of the in vivo milieu that can be applied to septal cartilage tissue engineering.

Culture of human septal chondrocytes in a rotary bioreactor.

OBJECTIVES: (1) To show that extracellular matrix deposition in 3-dimensional culture of human septal chondrocytes cultured in a rotary bioreactor is comparable to the deposition achieved under static culture conditions. (2) To demonstrate that the biomechanical properties of human septal chondrocytes cultured in a bioreactor are enhanced with time and are analogous to beads cultured under static culture. STUDY DESIGN: Prospective, basic science. SETTING: Research laboratory. METHODS: Human septal chondrocytes from 9 donors were expanded in monolayer and seeded in alginate beads. The beads were cultured in a rotary bioreactor for 21 days in media supplemented with growth factors and human serum, using static culture as the control. Biochemical and biomechanical properties of the beads were measured. RESULTS: Glycosaminoglycan (GAG) accumulation significantly increased during 2 measured time intervals, 0 to 21 days and 10 to 21 days (P < .01). No significant difference was seen between the static and bioreactor conditions. Substantial type II collagen production was demonstrated in the beads terminated at day 21 of culture in both conditions. In addition, the biomechanical properties of the beads were significantly improved at 21 days in comparison to beads from day 0. CONCLUSION: Human septal chondrocytes cultured in alginate beads exhibit significant matrix deposition and improved biomechanical properties after 21 days. Alginate bead diameter and stiffness positively correlated with GAG and type II collagen accretion. Matrix production in beads is supported by the use of a rotary bioreactor.

Effect of tibial plateau fracture on lubrication function and composition of synovial fluid.

BACKGROUND: Intra-articular fractures may hasten posttraumatic arthritis in patients who are typically too active and too young for joint replacement. Current orthopaedic treatment principles, including recreating anatomic alignment and establishing articular congruity, have not eliminated posttraumatic arthritis. Additional biomechanical and biological factors may contribute to the development of arthritis. The objective of the present study was to evaluate human synovial fluid for friction-lowering function and the concentrations of putative lubricant molecules following tibial plateau fractures. METHODS: Synovial fluid specimens were obtained from the knees of eight patients (twenty-five to fifty-seven years old) with a tibial plateau fracture, with five specimens from the injured knee as plateau fracture synovial fluid and six specimens from the contralateral knee as control synovial fluid. Each specimen was centrifuged to obtain a fluid sample, separated from a cell pellet, for further analysis. For each fluid sample, the start-up (static) and steady-state (kinetic) friction coefficients in the boundary mode of lubrication were determined from a cartilage-on-cartilage biomechanical test of friction. Also, concentrations of the putative lubricants, hyaluronan and proteoglycan-4, as well as total protein, were determined for fluid samples. RESULTS: The group of experimental samples were obtained at a mean (and standard deviation) of 11 ± 9 days after injury from patients with a mean age of 45 ± 13 years. Start-up and kinetic friction coefficients demonstrated similar trends and dependencies. The kinetic friction coefficients for human plateau fracture synovial fluid were approximately 100% higher than those for control human synovial fluid. Hyaluronan concentrations were ninefold lower for plateau fracture synovial fluid compared with the control synovial fluid, whereas proteoglycan-4 concentrations were more than twofold higher in plateau fracture synovial fluid compared with the control synovial fluid. Univariate and multivariate regression analysis indicated that kinetic friction coefficient increased as hyaluronan concentration decreased. CONCLUSIONS: Knees afflicted with a tibial plateau fracture have synovial fluid with decreased lubrication properties in association with a decreased concentration of hyaluronan.

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.

Compaction enhances extracellular matrix content and mechanical properties of tissue-engineered cartilaginous constructs.

Many cell-based tissue-engineered cartilaginous constructs are mechanically softer than native tissue and have low content and abnormal proportions of extracellular matrix (ECM) constituents. We hypothesized that the load-bearing mechanical properties of cartilaginous constructs improve with the inclusion of collagen (COL) and proteoglycan (PG) during assembly. The objectives of this work were to determine (1) the effect of addition of PG, COL, or COL+PG on compressive properties of 2% agarose constructs and (2) the ability of mechanical compaction to concentrate matrix content and improve the compressive properties of such constructs. The inclusion of COL+PG improved the compressive properties of hydrogel constructs compared with PG or COL alone. Mechanical compaction increased the PG and COL concentrations in and compressive stiffness of the constructs. Chondrocytes included in the constructs maintained high viability after compaction. These results support the concepts that the assembly of cartilaginous constructs with COL+PG and application of mechanical compaction enhance the ECM content and compressive properties of engineered cartilaginous constructs.

In vivo implantation of tissue-engineered human nasal septal neocartilage constructs: a pilot study.

OBJECTIVE: To determine the in vivo biocompatibility of septal neocartilage constructs developed in vitro by an alginate intermediate step. STUDY DESIGN: Prospective, animal model. SETTING: Research laboratory. SUBJECTS AND METHODS: A murine model was used to examine the maturation of neocartilage constructs in vivo. Chondrocytes collected from patients undergoing septoplasty were expanded in monolayer and suspended in alginate beads for 3-dimensional culture in media containing human serum and growth factors. After in vitro incubation for 5 weeks, the constructs were implanted in the dorsum of athymic mice for 30 and 60 days (n = 9). After the mice were sacrificed, the constructs were recovered for assessment of their morphological, histochemical, biochemical, and biomechanical properties. RESULTS: The mice survived and tolerated the implants well. Infection and extrusion were not observed. Neocartilage constructs maintained their general shape and size and demonstrated cell viability after implantation. The implanted constructs were firm and opaque, sharing closer semblance to native septal tissue relative to the gelatinous, translucent preimplant constructs. Histochemical staining with hematoxylin and eosin (H&E) revealed that the constructs exhibited distinct morphologies characteristic of native tissue, which were not observed in preimplant constructs. DNA and type II collagen increased with duration of implantation, whereas type I collagen and glycoaminoglycans (GAG) decreased. Mechanical testing of a 60-day implanted construct demonstrated characteristics similar to native human septal cartilage. CONCLUSIONS: Neocartilage constructs are viable in an in vivo murine model. The histologic, biochemical, and biomechanical features of implanted constructs closely resemble native septal tissue when compared with preimplant constructs.

Tissue engineering by molecular disassembly and reassembly: biomimetic retention of mechanically functional aggrecan in hydrogel.

In vitro assembly of key functional extracellular matrix constituents for tissue-engineered constructs may provide a tool to modulate the retention of proteoglycan (PG) aggregates, which are crucial to compressive biomechanical properties of connective tissues. This study tested the hypotheses that (1) biomimetic molecular reassembly of PG aggregates (native aggrecan [AGC] with hyaluronan [HA] ± link protein [LP]) affects AGC retention kinetics in hydrogel constructs, (2) the compressive properties of such hydrogel constructs are related to the content of retained AGC, and (3) the reassembly method is compatible with chondrocytes. Addition of HA to AGC in hydrogel constructs increased AGC retention in a dose-dependent manner, and the addition of LP to AGC + HA further enhanced AGC retention. The level of AGC retention, in turn, was associated with increased equilibrium compressive stress of the constructs. Chondrocytes could be included in the process, and maintained expression of the chondrogenic phenotype, secreting type II collagen but little type I collagen. Thus, by altering the assembly of PG aggregates with HA ± LP, which affects AGC retention, it may be possible to achieve the targeted levels of PG components to modulate the mechanical properties of the engineered construct for cartilage as well as other tissues containing PG and PG aggregates.

Insulin-like growth factor-I and growth differentiation factor-5 promote the formation of tissue-engineered human nasal septal cartilage.

INTRODUCTION: Tissue engineering of human nasal septal chondrocytes offers the potential to create large quantities of autologous material for use in reconstructive surgery of the head and neck. Culture with recombinant human growth factors may improve the biochemical and biomechanical properties of engineered tissue. The objectives of this study were to (1) perform a high-throughput screen to assess multiple combinations of growth factors and (2) perform more detailed testing of candidates identified in part I. METHODS: In part I, human nasal septal chondrocytes from three donors were expanded in monolayer with pooled human serum (HS). Cells were then embedded in alginate beads for 2 weeks of culture in medium supplemented with 2% or 10% HS and 1 of 90 different growth factor combinations. Combinations of insulin-like growth factor-I (IGF-1), bone morphogenetic protein (BMP)-2, BMP-7, BMP-13, growth differentiation factor-5 (GDF-5), transforming growth factor ß (TGFß)-2, insulin, and dexamethasone were evaluated. Glycosaminoglycan (GAG) accumulation was measured. A combination of IGF-1 and GDF-5 was selected for further testing based on the results of part I. Chondrocytes from four donors underwent expansion followed by three-dimensional alginate culture for 2 weeks in medium supplemented with 2% or 10% HS with or without IGF-1 and GDF-5. Chondrocytes and their associated matrix were then recovered and cultured for 4 weeks in 12 mm transwells in medium supplemented with 2% or 10% HS with or without IGF-1 and GDF-5 (the same medium used for alginate culture). Biochemical and biomechanical properties of the neocartilage were measured. RESULTS: In part I, GAG accumulation was highest for growth factor combinations including both IGF-1 and GDF-5. In part II, the addition of IGF-1 and GDF-5 to 2% HS resulted in a 12-fold increase in construct thickness compared with 2% HS alone (p < 0.0001). GAG and type II collagen accumulation was significantly higher with IGF-1 and GDF-5. Confined compression modulus was greatest with 2% HS, IGF-1, and GDF-5. CONCLUSION: Supplementation of medium with IGF-1 and GDF-5 during creation of neocartilage constructs results in increased accumulation of GAG and type II collagen and improved biomechanical properties compared with constructs created without the growth factors.

Growth of human septal chondrocytes in fibrin scaffolds.

BACKGROUND: Tissue engineering of nasal septal cartilage has been the focus of research owing to its superior structural rigidity and ease of harvest. In vitro constructs formed from septal chondrocytes using fibrin glue within a polyglycolic acid (PGA) scaffold have been shown to be viable, but their cellular growth and expression of differentiated features still have not been quantified. In this study, we evaluated cellular proliferation and production of cartilaginous extracellular matrix (ECM) components in fibrin glue preparations within a PGA scaffold. METHODS: Human chondrocytes were expanded for one passage in monolayer in culture medium. The cells were then grown in (1) fibrinogen, (1/2)x-thrombin, (1/2)x (F/2:T/2); (2) fibrinogen, 1/10x-thrombin, 1/10x (F/10:T/10); (3) fibrinogen, 1x-thrombin, 1/100x (F/1:T/100). RESULTS: Cellular proliferation and glycosaminoglycan (GAG) production per cell were highest in the F/2:T/2 preparations. Greater proliferation was seen in chondrocyte-fibrin composites seeded onto the PGA scaffold when compared with chondrocytes seeded onto the PGA scaffold alone. No significant difference in GAG production was seen. CONCLUSION: The addition of fibrin glue to chondrocytes seeded onto a PGA scaffold results in increased cellular proliferation while maintaining production of ECM components. Long-term stable fibrin gels in combination with PGA scaffolds may facilitate generation of cartilaginous tissue for use in reconstructive surgery.

Interactive cytokine regulation of synoviocyte lubricant secretion.

Cytokine regulation of synovial fluid (SF) lubricants, hyaluronan (HA), and proteoglycan 4 (PRG4) is important in health, injury, and disease of synovial joints, and may also provide powerful regulation of lubricant secretion in bioreactors for articulating tissues. This study assessed lubricant secretion rates by human synoviocytes and the molecular weight (MW) of secreted lubricants in response to interleukin (IL)-1beta, IL-17, IL-32, transforming growth factor-beta 1 (TGF-beta1), and tumor necrosis factor-alpha (TNF-alpha), applied individually and in all combinations. Lubricant secretion rates were assessed using ELISA and binding assays, and lubricant MW was assessed using gel electrophoresis and Western blotting. HA secretion rates were increased approximately 40-fold by IL-1beta, and increased synergistically to approximately 80-fold by the combination of IL-1beta + TGF-beta1 or TNF-alpha + IL-17. PRG4 secretion rates were increased approximately 80-fold by TGF-beta1, and this effect was counterbalanced by IL-1beta and TNF-alpha. HA MW was predominantly <1 MDa for controls and individual cytokine stimulation, but was concentrated at >3 MDa after stimulation by IL-1beta + TGF-beta1 + TNF-alpha to resemble the distribution in human SF. PRG4 MW was unaffected by cytokines and similar to that in human SF. These results contribute to an understanding of the relationship between SF cytokine and lubricant content in health, injury, and disease, and provide approaches for using cytokines to modulate lubricant secretion rates and MW to help achieve desired lubricant composition of fluid in bioreactors.

Cartilage outgrowth in fibrin scaffolds.

BACKGROUND: Fibrin glue has been a favorable hydrogel in cartilage tissue engineering, but implantation of chondrocyte-fibrin suspensions have resulted in volume loss. In this study, human septal cartilage chips were seeded onto a fibrin scaffold, and cellular proliferation and production of cartilaginous extracellular matrix (ECM) were evaluated. METHODS: Human septal cartilage was diced into cartilage chips and encased with and without fibrin glue. Four conditions were initially tested for DNA content and glycosaminoglycan (GAG) production: (1) control medium in tissue culture, (2) control medium with fibrin glue, (3) collagenase-supplemented medium in tissue culture, and (4) collagenase-supplemented medium seeded in fibrin glue. Cartilage chips cultured in collagenase-treated medium were then seeded onto either cell culture plates, suspended in alginate, or mixed with fibrin. Cellular proliferation, GAG production, and histochemistry were evaluated. RESULTS: Fibrin preparations increased cellular proliferation and DNA content. GAG levels were highest in collagenase-treated samples encased in fibrin. Cartilage chips treated with collagenase showed increased cellular proliferation in the fibrin preparations compared with preparations without fibrin. GAG increased with the addition of fibrin when compared with explant. Histochemistry revealed increased GAG accumulation in the regions between the cartilage chips with the addition of fibrin. CONCLUSION: Adding fibrin glue to collagenase-treated cartilage chips results in increased proliferation and maintains ECM production and, therefore, may facilitate generation of cartilaginous tissue for use in reconstructive surgery.

Expansion and redifferentiation of chondrocytes from osteoarthritic cartilage: cells for human cartilage tissue engineering.

OBJECTIVE: To determine if selected culture conditions enhance the expansion and redifferentiation of chondrocytes isolated from human osteoarthritic cartilage with yields appropriate for creation of constructs for treatment of joint-scale cartilage defects, damage, or osteoarthritis. METHODS: Chondrocytes isolated from osteoarthritic cartilage were analyzed to determine the effects of medium supplement on cell expansion in monolayer and then cell redifferentiation in alginate beads. Expansion was assessed as cell number estimated from DNA, growth rate, and day of maximal growth. Redifferentiation was evaluated quantitatively from proteoglycan and collagen type II content, and qualitatively by histology and immunohistochemistry. RESULTS: Using either serum or a growth factor cocktail (TFP: transforming growth factor beta1, fibroblast growth factor 2, and platelet-derived growth factor type bb), cell growth rate in monolayer was increased to 5.5x that of corresponding conditions without TFP, and cell number increased 100-fold within 17 days. In subsequent alginate bead culture with human serum or transforming growth factor beta1 and insulin-transferrin-selenium-linoleic acid-bovine serum albumin, redifferentiation was enhanced with increased proteoglycan and collagen type II production. Effects of human serum were dose dependent, and 5% or higher induced formation of chondron-like structures with abundant proteoglycan-rich matrix. CONCLUSION: Chondrocytes from osteoarthritic cartilage can be stimulated to undergo 100-fold expansion and then redifferentiation, suggesting that they may be useful as a cell source for joint-scale cartilage tissue engineering.

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.

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.

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.

Boundary lubrication of articular cartilage: role of synovial fluid constituents.

OBJECTIVE: To determine whether the synovial fluid (SF) constituents hyaluronan (HA), proteoglycan 4 (PRG4), and surface-active phospholipids (SAPL) contribute to boundary lubrication, either independently or additively, at an articular cartilage-cartilage interface. METHODS: Cartilage boundary lubrication tests were performed with fresh bovine osteochondral samples. Tests were performed using graded concentrations of SF, HA, and PRG4 alone, a physiologic concentration of SAPL, and various combinations of HA, PRG4, and SAPL at physiologic concentrations. Static (mu(static, Neq)) and kinetic () friction coefficients were calculated. RESULTS: Normal SF functioned as an effective boundary lubricant both at a concentration of 100% ( = 0.025) and at a 3-fold dilution ( = 0.029). Both HA and PRG4 contributed independently to a low mu in a dose-dependent manner. Values of decreased from approximately 0.24 in phosphate buffered saline to 0.12 in 3,300 mug/ml HA and 0.11 in 450 mug/ml PRG4. HA and PRG4 in combination lowered mu further at the high concentrations, attaining a value of 0.066. SAPL at 200 mug/ml did not significantly lower mu, either independently or in combination with HA and PRG4. CONCLUSION: The results described here indicate that SF constituents contribute, individually and in combination, both at physiologic and pathophysiologic concentrations, to the boundary lubrication of apposing articular cartilage surfaces. These results provide insight into the nature of the boundary lubrication of articular cartilage by SF and its constituents. They therefore provide insight regarding both the homeostatic maintenance of healthy joints and pathogenic processes in arthritic disease.

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.

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.

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.