Distinct tribological endotypes of pathological human synovial fluid reveal characteristic biomarkers and variation in efficacy of viscosupplementation at reducing local strains in articular cartilage.

OBJECTIVE: Viscosupplementation has been used for decades to treat mild to moderate osteoarthritis, yet it is unknown if the lubricating function of different pathological synovial fluids (SF) vary, or if they respond differentially to viscosupplementation. The objectives of this study were to (i) evaluate the friction coefficients and induced shear strains in articular cartilage when lubricated with pathological SF, (ii) identify the effect of hyaluronic acid (HA) supplementation on friction coefficients and shear strains, and (iii) identify SF biomarkers that correlate with lubricating function. METHOD: Human pathological SF was grouped by white blood cell count (inflammatory: >2000 cells/mm3, n = 6; non-inflammatory: <2000 cells/mm3, n = 6). Compositional analyses for lubricin and cytokines were performed. Friction coefficients and local tissue shear strain measurements were coupled using new, microscale rheological analyses by lubricating neonatal bovine cartilage explants with SF alone and in a 1:1 ratio with HA (Hymovis®). RESULTS: Friction coefficients were not significantly different between the inflammatory and non-inflammatory pathologies (p = 0.09), and were poorly correlated with peak tissue strains at the cartilage articular surface (R2 = 0.34). A subset of inflammatory SF samples induced higher tissue strains, and HA supplementation was most effective at lowering friction and tissue strains in this inflammatory subset. Across all pathologies there were clear relationships between polymorphonuclear neutrophil (PMN), IL-8, and lubricin concentrations with cartilage tissue strains. CONCLUSION: These results suggest that pathological SF is characterized by distinct tribological endotypes where SF lubricating behaviors are differentially modified by viscosupplementation and are identifiable by biomarkers.

How can 50 years of solute transport data in articular cartilage inform the design of arthritis therapeutics?

OBJECTIVE: For the last half century, transport of nutrients and therapeutics in articular cartilage has been studied with various in vitro systems that attempt to model in vivo conditions. However, experimental technique, tissue species, and tissue storage condition (fresh/frozen) vary widely and there is debate on the most appropriate model system. Additionally, there is still no clear overarching framework with which to predict solute transport properties based on molecular characteristics. This review aims to develop such a framework, and to assess whether experimental procedure affects trends in transport data. METHODS: Solute data from 31 published papers that investigated transport in healthy articular cartilage were obtained and analyzed for trends. RESULTS: Here, we show that diffusivity of spherical and globular solutes in cartilage can be predicted by molecular weight (MW) and hydrodynamic radius via a power-law relationship. This relationship is robust for many solutes, spanning 5 orders of magnitude in MW and was not affected by variations in cartilage species, age, condition (fresh/frozen), and experimental technique. Traditional models of transport in porous media exhibited mixed effectiveness at predicting diffusivity in cartilage, but were good in predicting solute partition coefficient. CONCLUSION: Ultimately, these robust relationships can be used to accurately predict and improve transport of solutes in adult human cartilage and enable the development of better optimized arthritis therapeutics.

Joint-dependent response to impact and implications for post-traumatic osteoarthritis.

OBJECTIVE: The prevalence of osteoarthritis (OA) varies between joints. Cartilage in eight different joints was evaluated to elucidate the disparate susceptibilities between joints to post-traumatic OA (PTOA) and provide evidence for joint-specific clinical treatments. The hypothesis was that cartilage in different joints would have varying cell death and anabolic gene expression profiles after injury. METHODS: Adult equine cartilage explants were harvested from shoulder (SH), elbow (EL), carpal (CA), metacarpophalangeal (MC), patellofemoral (FP), tarsal (TA), metatarsophalangeal (MT), and proximal interphalangeal (PP) joints, and injured by loading with 30 MPa within 1 s. Fractional dissipated energy, cell density, cell death, and gene expression were quantified. RESULTS: PP had the highest fractional dissipated energy (94%, 95% confidence interval [CI] 88 to 101%). Cell density was highest in the superficial zone in all samples, with MC and MT having the highest peak density. Injured samples had significantly increased cell death (13.5%, 95% CI 9.1 to 17.9%) than non-injured samples (6.8%, 95% CI 2.5 to 11.1%, P = 0.016); however, cell death after injury was not significantly different between joints. Gene expression was significantly different between joints. CD-RAP expression in normal cartilage was lowest in FP (Cp = 21, 95% CI -80 to 122). After injury, the change in CD-RAP expression increased and was highest in FP (147% relative increase after injury, 95% CI 64 to 213). CONCLUSION: Different joints have different baseline characteristics, including cell density and gene expression, and responses to injury, including energy dissipation and gene expression. These unique characteristics may explain differences in OA prevalence and suggest differences in susceptibility to PTOA. CLINICAL RELEVANCE: Understanding differences in the response to injury and potential susceptibility to OA can lead to the development of preventative or treatment strategies. KEY TERMS: Gene expression, cartilage injury, chondrocyte, multiphoton microscopy, cartilage biomechanical properties, PTOA. WHAT IS KNOWN ABOUT THE SUBJECT: The prevalence of OA is variable among joints; however, most laboratory studies are performed on a single joint - most commonly the knee, and extrapolated to other joints such as the ankle or shoulder. A small number of studies have compared knee and ankle cartilage and reported differences in mechanical properties and gene expression. WHAT THIS STUDY ADDS TO EXISTING KNOWLEDGE: There are differences in baseline cell density and gene expression, and differences in response to injury, including gene expression and cell death. This suggests that there are inherent differences leading to varying susceptibilities in OA prevalence among joints. Joint-specific treatments may improve OA therapies.

Identification of cartilage injury using quantitative multiphoton microscopy.

OBJECTIVE: Cartilage injury can lead to post-traumatic osteoarthritis (PTOA). Immediate post-trauma cellular and structural changes are not widely understood. Furthermore, current cellular-resolution cartilage imaging techniques require sectioning of cartilage and/or use of dyes not suitable for patient imaging. In this study, we used multiphoton microscopy (MPM) data with FDA-approved sodium fluorescein to identify and evaluate the pattern of chondrocyte death after traumatic injury. METHOD: Mature equine distal metacarpal or metatarsal osteochondral blocks (OCBs) were injured by 30 MPa compressive loading delivered over 1 s. Injured and control sites were imaged unfixed and in situ 1 h post-injury with sodium fluorescein using rasterized z-scanning. MPM data was quantified in MATLAB, reconstructed in 3-D, and projected in 2-D to determine the damage pattern. RESULTS: MPM images (600 per sample) were reconstructed and analyzed for cell death. The overall distribution of cell death appeared to cluster into circular (n = 7) or elliptical (n = 4) patterns (p = 0.006). Dead cells were prevalent near cracks in the matrix, with only 26.3% (SE = 5.0%, p < 0.0001) of chondrocytes near cracks being viable. CONCLUSION: This study demonstrates the first application of MPM for evaluating cellular-scale cartilage injury in situ in live tissue, with clinical potential for detecting early cartilage damage. With this technique, we were able to uniquely observe two death patterns resulting from the same compressive loading, which may be related to local variability in matrix structure. These results also demonstrate proof-of-concept MPM diagnostic use in detecting subtle and early cartilage damage not detectable in any other way.

Boundary mode frictional properties of engineered cartilaginous tissues.

Despite the fact that lubrication is a primary function of articular cartilage, there is little information on the frictional properties of cartilaginous engineered tissues. A biochemical mediator of cartilage frictional properties in boundary lubrication, lubricin, has been shown to be secreted from chondrocyte-hydrogel constructs. In the current studies we utilized articular chondrocytes (CON), meniscal fibrochondrocytes (MEN), and mesenchymal stem cells (MSC) in alginate cultures to determine lubricin localization and the inherent boundary lubrication friction coefficient. Additionally, we investigated the ability of these tissues to be lubricated by synovial fluid and the reversibility of this lubrication. Cell-alginate constructs were cultured over six weeks, culture medium assayed for lubricin release by ELISA and constructs analyzed with immunohistochemical (IHC) methods to investigate the localization of lubricin. Engineered tissues were tested in a custom friction instrument to determine the equilibrium friction coefficient (microeq) in boundary lubrication mode, following incubation with equine synovial fluid (SF), and subsequent extraction in l.5M NaCl. MSCs released 10 fold more lubricin than CON or MEN cultures. IHC analysis showed no localization of lubricin to alginate, minimal focal staining of engineered constructs at six weeks in culture, and the ability of all engineered tissues to localize lubricin when exogenously treated with SF. Frictional characterization showed no difference in microeq over culture for all engineered tissues, while incubation in SF decreased microeq for all tissues over culture duration, and extraction of lubricin resulted in a loss of lubrication of all engineered tissues.

Cell and protein compatible 3D bioprinting of mechanically strong constructs for bone repair.

Rapid prototyping of bone tissue engineering constructs often utilizes elevated temperatures, organic solvents and/or UV light for materials processing. These harsh conditions may prevent the incorporation of cells and therapeutic proteins in the fabrication processes. Here we developed a method for using bioprinting to produce constructs from a thermoresponsive microparticulate material based on poly(lactic-co-glycolic acid) at ambient conditions. These constructs could be engineered with yield stresses of up to 1.22 MPa and Young's moduli of up to 57.3 MPa which are within the range of properties of human cancellous bone. Further study showed that protein-releasing microspheres could be incorporated into the bioprinted constructs. The release of the model protein lysozyme from bioprinted constructs was sustainted for a period of 15 days and a high degree of protein activity could be measured up to day 9. This work suggests that bioprinting is a viable route to the production of mechanically strong constructs for bone repair under mild conditions which allow the inclusion of viable cells and active proteins.

Minimally invasive technique of auricular cartilage harvest for tissue engineering.

Tissue engineered human cartilage is presently being utilized in clinical research programs in a variety of medical disciplines including otolaryngology, urology, and orthopedics. In this study, we present a new methodology for auricular cartilage harvest that can be applied to tissue engineering. Eight 16-week-old pigs were subjected to a traditional open cartilage harvest technique involving suture closure, while the other ear was subjected to the closed stitchless cartilage harvest, using a 12-gauge core biopsy needle. Surgical time was significantly (p < 0.0001) shorter (3.5 +/- 2.8 min for closed vs. 14.4 +/- 5 min for open), and no sutures where utilized in the closed technique. Sample weights were significantly (p < 0.00001) greater (0.115 +/- 0.028 g vs. 0.045 +/- 0.005 g) for the closed techniques. However, the minimally invasive closed technique had fewer incidents of bruising, hematoma, long-term stitch abscess, and scarring. Cell culture data shows no disadvantage to either technique with regards to cell growth characteristics. Final histological data from donor ears indicates favorable results with the minimally invasive technique. This technique preserves cell viability and isolation efficiency while decreasing surgical time and lessening postoperative complications.

Biomechanical analysis of a chondrocyte-based repair model of articular cartilage.

The objective of this study was to evaluate the biomechanical properties of newly formed cartilaginous tissue synthesized from isolated chondrocytes. Cartilage from articular joints of lambs was either digested in collagenase to isolated chondrocytes or cut into discs that were devitalized by multiple freeze-thaw cycles. Isolated cells were incubated in suspension culture in the presence of devitalized cartilage matrix for 3 weeks. Multiple chondrocyte/matrix constructs were assembled with fibrin glue and implanted subcutaneously in nude mice for up to 6 weeks. Testing methods were devised to quantify integration of cartilage pieces and mechanical properties of constructs. These studies showed monotonic increase with time in tensile strength, fracture strain, fracture energy, and tensile modulus to values 5-10% of normal articular cartilage by 6 weeks in vivo. Histological analysis indicated that chondrocytes grown on dead cartilage matrix produced new matrix that integrated individual cartilage pieces with mechanically functional tissue.

Tissue-engineered human auricular cartilage demonstrates euploidy by flow cytometry.

Transforming growth factor-beta (TGF-beta) and basic fibroblast growth factor (bFGF) are known to stimulate the rate of chondrocyte proliferation. The theoretical risk of malignant transformation associated with growth factor stimulation of chondrocytes should be addressed; aneuploidy has been found to occur in human cartilaginous tumors. In this study, chondrocytes were obtained from six human auricles and cultured in vitro for 6 weeks in the presence or absence of TGF-beta and bFGF. Cells were analyzed for DNA at 3-, 4-, 5-, and 6-week intervals by flow cytometry (FACScan), which demonstrated no evidence of aneuploidy. A persistent increase in S-phase was noted in cells cultured only with TGF-beta. Cells were implanted in athymic mice, and after 8 weeks of implantation, the cartilage constructs formed were examined histologically. The tissue-engineered cartilage cultured originally in bFGF most resembled normal, native cartilage. Specimens cultured in TGF-beta produced suboptimal cartilage morphology. Flow cytometry shows no evidence of aneuploidy, with chondrocytes maintaining their normal diploid state. Further studies incorporating additional methods of analysis need to be done.

The effect of dynamic compression on the response of articular cartilage to insulin-like growth factor-I.

Articular cartilage is routinely subjected to mechanical forces and to cell-regulatory molecules. Previous studies have shown that mechanical stimuli can influence articular chondrocyte metabolic activity, and biochemical studies have shown that growth factors and cytokines control many of the same cell functions. Little is known, however, of the relationships or interplay, if any, between these two key components of the articular environment. This study investigated the comparative and interactive effects of low amplitude, sinusoidal, dynamic compression and insulin-like growth factor-I (IGF-I), a polypeptide in synovial fluid that is anabolic for cartilage. In bovine patellofemoral cartilage explants, IGF-I increased protein and proteoglycan synthesis 90% and 120%, respectively while dynamic compression increased protein and proteoglycan synthesis 40% and 90%, respectively. Stimulation by IGF-I was significantly greater than by dynamic compression for both protein and proteoglycan synthesis. When applied together, the two stimuli enhanced protein and proteoglycan synthesis by 180% and 290%, respectively, a degree greater than that achieved by either stimulus alone. IGF-I augmented protein synthesis with a time constant of 12.2 h. Dynamic compression increased protein synthesis with a time constant of 2.9 h, a rate significantly faster than that of IGF-I, suggesting that these signals act via distinct cell activation pathways. When used together, dynamic compression and IGF-I acted with a time constant of 5.6 h. Thus, dynamic compression accelerated the biosynthetic response to IGF-I and increased transport of IGF-I into the articular cartilage matrix, suggesting that, in addition to independently stimulating articular chondrocytes, cyclic compression may improve the access of soluble growth factors to these relatively isolated cells.

Comparison of chondrogensis in static and perfused bioreactor culture.

As a result of the low yield of cartilage from primary patient harvests and a high demand for autologous cartilage for reconstructive surgery and structural repair, primary explant cartilage must be augmented by tissue engineering techniques. In this study, chondrocytes seeded on PLLA/PGA scaffolds in static culture and a direct perfusion bioreactor were biochemically and histologically analyzed to determine the effects of fluid flow and media pH on matrix assembly. A gradual media pH change was maintained in the bioreactor within 7.4-6.96 over 2 weeks compared to a more rapid decrease from 7.4 to 6.58 in static culture over 3 days. Seeded scaffolds subjected to 1 microm/s flow demonstrated a 118% increase (p < 0.05) in DNA content, a 184% increase (p < 0.05) in GAG content, and a 155% (p < 0.05) increase in hydroxyproline content compared to static culture. Distinct differences were noted in tissue morphology, including more intense staining for proteoglycans by safranin-O and alignment of cells in the direction of media flow. Culture of chondrocyte seeded matrices thus offers the possibility of rapid in vitro expansion of donor cartilage for the repair of structural defects, tracheal injury, and vascularized tissue damage.

The role of cartilage streaming potential, fluid flow and pressure in the stimulation of chondrocyte biosynthesis during dynamic compression.

The effects of streaming potential, fluid flow and hydrostatic pressure on chondrocyte biosynthesis were studied by comparing the spatial profiles of these physical stimuli to the profiles of biosynthesis within cartilage disks subjected to dynamic unconfined compression. The radial streaming potential was measured using compression frequencies and disk sizes relevant to studies of physical modulation of cartilage metabolism; a general analytical solution to the unconfined compression of a poroelastic cylinder with impermeable, rigid, adhesive platens was derived using potential theory. The solution with adhesive platen boundary conditions, using measured values of cartilage material properties, predicted streaming potentials that were much closer to experimental results between 0.001 and 1 Hz than a solution using frictionless platen boundary conditions. The predicted radial profiles of streaming potential gradient and fluid velocity (but not hydrostatic pressure) were similar to the previously reported radial dependence of proteoglycan synthesis induced by dynamic unconfined compression. Changes in stiffness associated with reduction of disk diameter suggested that the relative contributions of collagen and proteoglycans to cartilage mechanical properties may be a function of loading frequency in unconfined compression; such anisotropies may explain the remaining discrepencies between measured stiffness and stiffness predicted by the present model.

Direct perfusion measurements of cancellous bone anisotropic permeability.

More extensive characterization of trabecular connectivity and intertrabecular space will be instrumental in understanding disease states and designing engineered bone. This project presents an experimental protocol to define the directional dependence of transport properties as measured from healthy cancellous bone when considered as a biologic, porous medium. In the initial design phases, mature bovine bone was harvested from the femoral neck (n=6 cylinders) and distal condyle (n=4 cubes) regions and used for "proof of concept" experimentation. A power study on those results led to the presented work on 20 cubic samples (mean volume=4.09cm(3)) harvested from a single bovine distal femur. Anisotropic intrinsic permeabilities (k(i)) were quantified along the orthogonal anatomic axes (i=medial-lateral, anterior-posterior, and superior-inferior) from each individual cubic bone sample. Using direct perfusion measurements, permeability was calculated based upon Darcy's Law describing flow through porous media. The maximum mean value was associated with the superior-inferior orientation (4.65x10(-10)m(2)) in comparison with the mean anterior-posterior (4.52x10(-10)m(2)) and medial-lateral (2.33x10(-10)m(2)) direction values. The results demonstrate the anisotropic (p=0.0143) and heterogeneous (p=0.0002) nature of the tissue and encourage the ongoing quantification of parameters within the established poroelastic models.

Age dependence of cellular properties of human septal cartilage: implications for tissue engineering.

BACKGROUND: The persistent need for cartilage replacement material in head and neck surgery has led to novel cell culture methods developed to engineer cartilage. Currently, there is no consensus on an optimal source of cells for these endeavors. OBJECTIVES: To evaluate human nasal cartilage as a potential source of chondrocytes and to determine the effect of donor age on cellular and proliferation characteristics. SUBJECTS: Nasal cartilage specimens were obtained after reconstructive surgery from 46 patients ranging in age from 15 to 60 years. METHODS: Specimens were weighed and chondrocytes were isolated by digestion in 0.2% collagenase type II for 16 hours. Cells were maintained in primary cultures until confluency, then seeded onto polylactic acid-polyglycolic acid scaffolds. Seeding efficiency was determined by quantification of DNA content of seeded constructs by means of Hoechst dye 33258. Specimen weights, cell yields, cell content, and doubling time were also measured and correlated to donor age. RESULTS: Mean (+/-SD) cartilage mass obtained (648 +/- 229 mg) is higher than from typical biopsy specimens of auricular cartilage, and the cellular characteristics show a higher proliferation rate than auricular chondrocytes. Cell yield increased with age, while doubling time decreased with age in samples from patients ranging from 15 to 60 years old. CONCLUSIONS: The use of nasal septal cartilage as a source of cells for tissue engineering may be valid over a wide range of patient ages. The large tissue yield and consequent cell yield make this tissue a potential starting source of chondrocytes for large-volume tissue-engineered implants.

Rapid 3D printing of anatomically accurate and mechanically heterogeneous aortic valve hydrogel scaffolds.

The aortic valve exhibits complex three-dimensional (3D) anatomy and heterogeneity essential for the long-term efficient biomechanical function. These are, however, challenging to mimic in de novo engineered living tissue valve strategies. We present a novel simultaneous 3D printing/photocrosslinking technique for rapidly engineering complex, heterogeneous aortic valve scaffolds. Native anatomic and axisymmetric aortic valve geometries (root wall and tri-leaflets) with 12-22 mm inner diameters (ID) were 3D printed with poly-ethylene glycol-diacrylate (PEG-DA) hydrogels (700 or 8000 MW) supplemented with alginate. 3D printing geometric accuracy was quantified and compared using Micro-CT. Porcine aortic valve interstitial cells (PAVIC) seeded scaffolds were cultured for up to 21 days. Results showed that blended PEG-DA scaffolds could achieve over tenfold range in elastic modulus (5.3±0.9 to 74.6±1.5 kPa). 3D printing times for valve conduits with mechanically contrasting hydrogels were optimized to 14 to 45 min, increasing linearly with conduit diameter. Larger printed valves had greater shape fidelity (93.3±2.6, 85.1±2.0 and 73.3±5.2% for 22, 17 and 12 mm ID porcine valves; 89.1±4.0, 84.1±5.6 and 66.6±5.2% for simplified valves). PAVIC seeded scaffolds maintained near 100% viability over 21 days. These results demonstrate that 3D hydrogel printing with controlled photocrosslinking can rapidly fabricate anatomical heterogeneous valve conduits that support cell engraftment.

Biomechanical characterisation of equine laryngeal cartilage.

REASONS FOR PERFORMING THE STUDY: Upper airway obstruction is a common problem in the performance horse as the soft tissues of the larynx collapse into the airway, yet there is a paucity of information on biomechanical properties for the structural cartilage components. OBJECTIVE: To measure the geometry and compressive mechanical properties of the hyaline cartilage to improve understanding of laryngeal function and morphology. METHODS: A total of 11 larynges were harvested from Thoroughbred and Standardbred racehorses. During gross dissection, linear dimensions of the cricoid were obtained. From both the cricoid and arytenoid, specimens were cored to obtain 6 mm disc samples from 3 sites within the dorsal cricoid (caudal, middle and rostral) and 2 central sites in the arytenoids (inner, outer). The specimens were mechanically tested using radial confined compression to calculate the aggregate modulus and permeability of the tissue. The biomechanical data were analysed using a nested mixed effects model. RESULTS: Geometrically, the cricoid has relatively straight walls compared to the morphology of human, ovine and canine larynges. There were significant observations of higher modulus with increasing age (0.13 MPa per year; P = 0.007) and stiffer cricoid cartilage (2.29 MPa) than the arytenoid cartilage (0.42 MPa; P<0.001), but no difference was observed between the left and right sides. Linear contrasts showed that the rostral aspect (2.51 MPa) of the cricoid was 20% stiffer than the caudal aspect (2.09 MPa; P = 0.025), with no difference between the arytenoid sites. CONCLUSIONS: The equine larynx is a well supported structure due to both the geometry and material properties of the cricoid cartilage. The hyaline structure is an order of magnitude higher in compressive modulus compared to the arytenoids and other hyaline-composed tissues. POTENTIAL RELEVANCE: These characterisations are important to understand the biomechanics of laryngeal function and the mechanisms involved with surgical interventions.

Interaction of epidermal growth factor and insulin-like growth factor-I in the regulation of growth plate chondrocytes.

The action of growth factors on the cells of the epiphyseal growth plate is an important mechanism in the regulation of skeletal growth. Insulin-like growth factor-I (IGF-I) is known to play a central role in the regulation of bone growth. In contrast, the role, if any, of epidermal growth factor (EGF) is not yet clear. In these studies, we tested the hypothesis that EGF interacts with IGF-I in the regulation of growth plate chondrocyte mitotic and metabolic activities. Chondrocytes isolated from bovine radioulnar growth plates and incubated in suspension culture were analyzed for their responsiveness to EGF with respect to synthesis of DNA, proteins, and proteoglycans, responsiveness to IGF-I, and ability to specifically bind [125I]IGF-I. Treatment of growth plate chondrocytes with maximally effective concentrations (10-100 ng/ml) of EGF produced a 16-27% increase in specific binding of [125I]IGF-I. Scatchard analysis indicated that this increase in specific binding was due to an increase in the number of receptors/cell with no change in receptor affinity. EGF stimulated protein synthesis by 30-35%. Pretreatment with EGF increased the responsiveness of chondrocytes to IGF-I, resulting in 90 and 60% augmentation of IGF-I-stimulated mitotic activity and proteoglycan synthesis, respectively. Given the prominent role of IGF-I in skeletal development and the presence of EGF in the growth plate, this study suggests an important role for interactions between these growth factors in the regulation of skeletal growth.