Patient-specific 3D models aid planning for triplane proximal femoral osteotomy in slipped capital femoral epiphysis.

PURPOSE: Slipped capital femoral epiphysis (SCFE) can result in a complex three-dimensional (3D) deformity of the proximal femur. A three-plane proximal femoral osteotomy (TPFO) has been described to improve hip mechanics. The purpose of this study was to evaluate the benefits of using 3D print technology to aid in surgical planning. PATIENTS AND METHODS: Fifteen children treated with TPFO for symptomatic proximal femoral deformity due to SCFE were included in this study. Ten patients were treated by a single surgeon with (model group, n = 5) or without (no-model group, n = 5) a 3D model for pre-operative planning, and compared with patients treated by two senior partners without the use of a model (senior group, n = 5) to evaluate for a learning curve. Peri-operative data including patient body mass index (BMI), surgical time and fluoroscopy time were recorded. RESULTS: Children in all three groups had similar BMIs at the time of the TPFO. Post-operative radiographic parameters were equally improved in all three groups. On average, surgical time decreased by 45 minutes and 38 minutes, and fluoroscopy time decreased by 50% and 25%, in the model group compared with the no-model and senior groups, respectively. CONCLUSIONS: Patient-specific 3D models aid in surgical planning for complex 3D orthopaedic deformities by enabling practice of osteotomies. Results suggest that 3D models may decrease surgical time and fluoroscopy time while allowing for similar deformity correction. These models may be especially useful to overcome steep learning curves for complex procedures or in trainee education through mock surgical procedures.

Conditional Alpl Ablation Phenocopies Dental Defects of Hypophosphatasia.

Loss-of-function mutations in ALPL result in hypophosphatasia (HPP), an inborn error of metabolism that causes defective skeletal and dental mineralization. ALPL encodes tissue-nonspecific alkaline phosphatase, an enzyme expressed in bone, teeth, liver, and kidney that hydrolyzes the mineralization inhibitor inorganic pyrophosphate. As Alpl-null mice die before weaning, we aimed to generate mouse models of late-onset HPP with extended life spans by engineering a floxed Alpl allele, allowing for conditional gene ablation (conditional knockout [cKO]) when crossed with Cre recombinase transgenic mice. The authors hypothesized that targeted deletion of Alpl in osteoblasts and selected dental cells ( Col1a1-cKO) or deletion in chondrocytes, osteoblasts, and craniofacial mesenchyme ( Prx1-cKO) would phenocopy skeletal and dental manifestations of late-onset HPP. Col1a1-cKO and Prx1-cKO mice were viable and fertile, and they did not manifest the epileptic seizures characteristic of the Alpl-/- model of severe infantile HPP. Both cKO models featured normal postnatal body weight but significant reduction as compared with wild type mice by 8 to 12 wk. Plasma alkaline phosphatase for both cKO models at 24 wk was reduced by approximately 75% as compared with controls. Radiography revealed profound skeletal defects in cKO mice, including rachitic changes, hypomineralized long bones, deformations, and signs of fractures. Microcomputed tomography confirmed quantitative differences in cortical and trabecular bone, including decreased cortical thickness and mineral density. Col1a1-cKO mice exhibited classic signs of HPP dentoalveolar disease, including short molar roots with thin dentin, lack of acellular cementum, and osteoid accumulation in alveolar bone. Prx1-cKO mice exhibited the same array of periodontal defects but featured less affected molar dentin. Both cKO models exhibited reduced alveolar bone height and 4-fold increased numbers of osteoclast-like cells versus wild type at 24 wk, consistent with HPP-associated periodontal disease. These novel models of late-onset HPP can inform on long-term skeletal and dental manifestations and will provide essential tools to further studies of etiopathologies and therapeutic interventions.

Periodontal Defects in the A116T Knock-in Murine Model of Odontohypophosphatasia.

Mutations in ALPL result in hypophosphatasia (HPP), a disease causing defective skeletal mineralization. ALPL encodes tissue nonspecific alkaline phosphatase (ALP), an enzyme that promotes mineralization by reducing inorganic pyrophosphate, a mineralization inhibitor. In addition to skeletal defects, HPP causes dental defects, and a mild clinical form of HPP, odontohypophosphatasia, features only a dental phenotype. The Alpl knockout (Alpl (-/-)) mouse phenocopies severe infantile HPP, including profound skeletal and dental defects. However, the severity of disease in Alpl (-/-) mice prevents analysis at advanced ages, including studies to target rescue of dental tissues. We aimed to generate a knock-in mouse model of odontohypophosphatasia with a primarily dental phenotype, based on a mutation (c.346G>A) identified in a human kindred with autosomal dominant odontohypophosphatasia. Biochemical, skeletal, and dental analyses were performed on the resulting Alpl(+/A116T) mice to validate this model. Alpl(+/A116T) mice featured 50% reduction in plasma ALP activity compared with wild-type controls. No differences in litter size, survival, or body weight were observed in Alpl(+/A116T) versus wild-type mice. The postcranial skeleton of Alpl(+/A116T) mice was normal by radiography, with no differences in femur length, cortical/trabecular structure or mineral density, or mechanical properties. Parietal bone trabecular compartment was mildly altered. Alpl(+/A116T) mice featured alterations in the alveolar bone, including radiolucencies and resorptive lesions, osteoid accumulation on the alveolar bone crest, and significant differences in several bone properties measured by micro-computed tomography. Nonsignificant changes in acellular cementum did not appear to affect periodontal attachment or function, although circulating ALP activity was correlated significantly with incisor cementum thickness. The Alpl(+/A116T) mouse is the first model of odontohypophosphatasia, providing insights on dentoalveolar development and function under reduced ALP, bringing attention to direct effects of HPP on alveolar bone, and offering a new model for testing potential dental-targeted therapies in future studies.

Microstructural remodeling of articular cartilage following defect repair by osteochondral autograft transfer.

OBJECTIVE: To assess collagen network alterations occurring with flow and other abnormalities of articular cartilage at medial femoral condyle (MFC) sites repaired with osteochondral autograft (OATS) after 6 and 12 months, using quantitative polarized light microscopy (qPLM) and other histopathological methods. DESIGN: The collagen network structure of articular cartilage of OATS-repaired defects and non-operated contralateral control sites were compared by qPLM analysis of parallelism index (PI), orientation angle (α) relative to the local tissue axes, and retardance (Γ) as a function of depth. qPLM parameter maps were also compared to ICRS and Modified O'Driscoll grades, and cell and matrix sub-scores, for sections stained with H&E and Safranin-O, and for Collagen-I and II. RESULTS: Relative to non-operated normal cartilage, OATS-repaired regions exhibited structural deterioration, with low PI and more horizontal α, and unique structural alteration in adjacent host cartilage: more aligned superficial zone, and reoriented deep zone lateral to the graft, and matrix disorganization in cartilage overhanging the graft. Shifts in α and PI from normal site-specific values were correlated with histochemical abnormalities and co-localized with changes in cell organization/orientation, cloning, or loss, indicative of cartilage flow, remodeling, and deterioration, respectively. CONCLUSIONS: qPLM reveals a number of unique localized alterations of the collagen network in both adjacent host and implanted cartilage in OATS-repaired defects, associated with abnormal chondrocyte organization. These alterations are consistent with mechanobiological processes and the direction and magnitude of cartilage strain.

Statistical shape modeling of proximal femoral shape deformities in Legg-Calvé-Perthes disease and slipped capital femoral epiphysis.

INTRODUCTION: The current understanding of morphological deformities of the hip such as femoroacetabular impingement (FAI), Legg-Calvé-Perthes disease (LCPD), and slipped capital femoral epiphysis (SCFE) is based on two-dimensional metrics, primarily involving the femoral head, that only partially describe the complex skeletal morphology. OBJECTIVE: This study aimed to improve the three-dimensional (3-D) understanding of shape variations during normal growth, and in LCPD and SCFE, through statistical shape modeling. DESIGN: Thirty-two patients with asymptomatic, LCPD, and SCFE hips, determined from physical and radiographic examinations, were scanned using 3-D computed tomography (CT) at a voxel size of (0.5-0.9 mm)(2) in-plane and 0.63 mm slice thickness. Statistical shape modeling was performed on segmented proximal femoral surfaces to determine modes of variation and shape variables quantifying 3-D shape. In addition, conventional variables were determined for all femora. RESULTS: Proximal femur shape was described by eight modes of variation and corresponding shape variables. Statistical shape variables were distinct with age and revealed coordinated, growth-associated differences in neck length-to-width ratio, femoral head medialization, and trochanter protrusion. After size and age-based shape adjustment, diseased proximal femora were characterized by shape variables distinct from those of asymptomatic hips. The shape variables defined morphology in health and disease, and were correlated with certain conventional variables of shape, including neck-shaft angle, head diameter, and neck diameter. CONCLUSION: 3-D quantitative analyses of proximal femoral bone shape during growth and in disease are useful for furthering the understanding of normal and abnormal shape deviations which affect cartilage biomechanics and risk of developing osteoarthritis.

Cartilage shear dynamics during tibio-femoral articulation: effect of acute joint injury and tribosupplementation on synovial fluid lubrication.

OBJECTIVE: To determine the effects of acute injury and tribosupplementation by hyaluronan (HA) on synovial fluid (SF) modulation of cartilage shear during tibio-femoral articulation. METHODS: Human osteochondral blocks from the lateral femoral condyle (LFC) and tibial plateau (LTP) were apposed, compressed 13%, and subjected to sliding under video microscopy. Tests were conducted with equine SF from normal joints (NL-SF), SF from acutely injured joints (AI-SF), and AI-SF to which HA was added (AI-SF+HA). Local and overall shear strain (E(xz)) and the lateral displacement (Deltax) at which E(xz) reached 50% of peak values (Deltax(1/2)) were determined. RESULTS: During articulation, LFC and LTP cartilage E(xz) increased with Deltax and peaked when surfaces slid, with peak E(xz) being maintained during sliding. With AI-SF as lubricant, surface and overall Deltax(1/2) were approximately 40% and approximately 20% higher, respectively, than values with NL-SF and AI-SF+HA as lubricant. Also, peak E(xz) was markedly higher with AI-SF as lubricant than with NL-SF as lubricant, both near the surface (approximately 80%) and overall (50-200%). Following HA supplementation to AI-SF, E(xz) was reduced from values with AI-SF alone by 30-50% near the surface and 20-30% overall. Magnitudes of surface and overall E(xz) were markedly (approximately 50 to 80%) higher in LTP cartilage than LFC cartilage for all lubricants. CONCLUSION: Acute injury impairs SF function, elevating cartilage E(xz) markedly during tibio-femoral articulation; such elevated E(xz) may contribute to post-injury associated cartilage degeneration. Since HA partially restores the function of AI-SF, as indicated by E(xz), tribosupplements may be beneficial in modulating normal cartilage homeostasis.

Biomechanical, structural, and biochemical indices of degenerative and osteoarthritic deterioration of adult human articular cartilage of the femoral condyle.

OBJECTIVE: To compare the tensile biomechanical properties of age-matched adult human knee articular cartilage exhibiting distinct stages of degenerative or osteoarthritic deterioration and to determine the relationships between tensile properties and biochemical and structural properties hypothesized to underlie functional biomechanical deterioration. METHODS: Age-matched articular cartilage samples, obtained from the lateral and medial femoral condyles (LFC and MFC), exhibited (1) minimal fibrillation, characteristic of normal aging (NLA), (2) overt fibrillation associated with degeneration (DGN), or (3) overt fibrillation associated with osteoarthritis (OA). DGN samples were from knees that exhibited degeneration but not osteophytes while OA samples were from fragments removed during total knee arthroplasty. Cartilage samples were analyzed for tensile properties, cell and matrix composition, and histopathological structure. RESULTS: Differences in tensile, compositional and surface structural properties were indicative of distinct stages of cartilage degeneration, early (OA) advanced (DGN) and late (OA) with early degenerative changes in NLA samples being more advanced in the MFC than the LFC, including higher surface fibrillation, lower intrinsic fluorescence, and lower mechanical integrity. The transition from early to advanced degeneration involved a diminution in mechanical function, surface integrity, and intrinsic fluorescence. The transition from advanced to late degeneration involved an increase in cartilage water content, an increase in degraded collagen, and loss of collagen. CONCLUSIONS: These results provide evidence of coordinated mechanical dysfunction, collagen network remodeling, and surface fibrillation. Even in the cartilage of knees exhibiting overt fibrillation but not extensive erosions characteristic of clinical osteoarthritis, most features of advanced cartilage degeneration were present.

Wear-lines and split-lines of human patellar cartilage: relation to tensile biomechanical properties.

BACKGROUND: Articular cartilage undergoes age-associated degeneration, resulting in both structural and functional biomechanical changes. At early stages of degeneration, wear-lines develop in the general direction of joint movement. With aging, cartilage exhibits a decrease in tensile modulus. The tensile modulus of cartilage has also been related to the orientation of the collagen network, as revealed by split-lines. OBJECTIVE: To determine the relative contribution of wear-line and split-line orientation on the tensile biomechanical properties of human patellar cartilage from different depths. METHODS: In human patellar cartilage, wear- and split-lines are aligned parallel to each other at the proximal facet, and perpendicular to each other at the medial facet. Using superficial, middle, and deep cartilage sections from these two sites, tensile samples were prepared in two orthogonal orientations. Thus, for each depth, there were four groups of samples, with their long axes were aligned either parallel or perpendicular to wear-line direction and also aligned parallel or perpendicular to split-line direction. Uniaxial tensile tests were performed to assess equilibrium and ramp moduli. RESULTS: Tensile equilibrium moduli varied with wear-line orientation (P<0.05) and depth (P<0.001), in an interactive manner (P<0.05), and tended to vary with split-line orientation (P=0.16). In the superficial layer, equilibrium and ramp modulus were higher when the samples were loaded parallel to wear-lines (P<0.05). CONCLUSION: These results indicate that mild wear (i.e., wear-line formation) at the articular surface has deleterious functional effects on articular cartilage and represent an early aging-associated degenerative change. The identification and recognition of functional biomechanical consequences of wear-lines are useful for planning and interpreting tensile biomechanical tests in human articular cartilage.

Cartilage growth and remodeling: modulation of balance between proteoglycan and collagen network in vitro with beta-aminopropionitrile.

OBJECTIVE: To examine the effect of beta-aminopropionitrile (BAPN), an inhibitor of lysyl oxidase, on growth and remodeling of immature articular cartilage in vitro. DESIGN: Immature bovine articular cartilage explants from the superficial and middle layers were cultured for 13 days in serum-containing medium with or without BAPN. Variations in tissue size, accumulation of proteoglycan and collagen (COL), and tensile mechanical properties were assessed. RESULTS: The inclusion of serum resulted in expansive tissue growth, stimulation of proteoglycan and COL deposition, and a diminution of tensile integrity. Supplementation of medium with BAPN accentuated this phenotype in terms of a further increase in tissue size in explants from the superficial layer and further diminution of tensile integrity, without affecting the contents of proteoglycan and COL in explants from both the superficial and middle layers. CONCLUSION: COL crosslinking is a major factor in modulating the phenotype of cartilage growth and the associated balance between proteoglycan content and integrity of the COL network.

Differential regulation of proteoglycan 4 metabolism in cartilage by IL-1alpha, IGF-I, and TGF-beta1.

OBJECTIVES: To determine (1) if interleukin-1 alpha (IL-1alpha), insulin like growth factor I (IGF-I), and transforming growth factor-beta 1 (TGF-beta1) regulate proteoglycan 4 (PRG4) metabolism in articular cartilage, in terms of chondrocytes expressing PRG4 and PRG4 bound at the articular surface, and (2) if these features of cartilage PRG4 metabolism correlate with its secretion. METHODS: Articular cartilage explants were harvested and cultured for 6 days with or without 10% fetal bovine serum (FBS), alone, or with the addition of 10ng/ml IL-1alpha, 300ng/ml IGF-I, or 10ng/ml TGF-beta1. PRG4 expression by chondrocytes in the cartilage disks was assessed by immunohistochemistry (IHC). PRG4 bound to the articular surface of disks was quantified by extraction and enzyme-linked immunosorbent assay (ELISA). PRG4 secreted into culture medium was quantified by ELISA and characterized by Western Blot. RESULTS: PRG4 expression by chondrocytes near the articular surface was markedly decreased by IL-1alpha, stimulated by TGF-beta1, and not affected by IGF-I. The level of PRG4 accumulation in the culture medium was correlated with the number of chondrocytes expressing PRG4. The amount of PRG4 bound at the articular surface was modulated by incubation in medium including FBS, but did not correlate with levels of PRG4 secretion. CONCLUSIONS: Cartilage secretion of PRG4 is highly regulated by certain cytokines and growth factors, in part through alteration of the number of PRG4-secreting chondrocytes near the articular surface. The biochemical milieu may regulate the PRG4 content of synovial fluid during cartilage injury or repair.

PRG4 exchange between the articular cartilage surface and synovial fluid.

The boundary lubrication function of articular cartilage is mediated in part by proteoglycan 4 (PRG4) molecules, found both in synovial fluid (SF) and bound to the articular cartilage surface. Currently the mechanism by which PRG4 binds to the articular surface is not well understood. The objectives of this study were to determine (1) the effect of bathing fluid contents on PRG4 concentration at the articular surface ([PRG4](cart)), and (2) whether native PRG4 can be removed from the surface and subsequently repleted with PRG4 from synovial fluid. In one experiment, cylindrical cartilage disks were stored in solutions of various PRG4 concentrations, either in phosphate-buffered saline (PBS) or SF as the carrier fluid. In a separate experiment, cartilage disks were stored in solutions expected to remove native PRG4 from the articular surface and allow subsequent repletion with PRG4 from SF. [PRG4](cart) was independent of PRG4 concentration of the bathing fluid, and was similar for both carrier fluids. PRG4 was removed from cartilage by treatment with hyaluronidase, reduction/alkylation, and sodium dodecyl sulphate, and was repleted fully by subsequent bathing in SF. These results suggest that the articular surface is normally saturated with tightly bound PRG4, but this PRG4 can exchange with the PRG4 in SF under certain conditions. This finding suggests that all tissues surrounding the joint cavity that secrete PRG4 into the SF may help to maintain lubrication function at the articular surface.

Age- and site-associated biomechanical weakening of human articular cartilage of the femoral condyle.

OBJECTIVE: To determine the time sequence of biochemical and structural events associated with, and hypothesized to underlie, age-associated tensile weakening of macroscopically normal adult human articular cartilage of the knee. METHODS: Macroscopically normal human articular cartilage of the lateral and medial femoral condyles (LFC and MFC) from Young (21-39 yrs), Middle (40-59 yrs), and Old (>/=60 yrs) age donors were analyzed for tensile properties, surface wear, and cell and matrix composition. RESULTS: Variations in tensile, compositional, and surface structural properties were indicative of early, intermediate, and late stages of age-associated cartilage deterioration, occurring at an earlier age in the MFC than the LFC. Differences between Young and Middle age groups (indicative of early-to-intermediate stage changes) included decreased mechanical function in the superficial zone, with a loss of (or low) tensile integrity, and surface wear, with faint striations and mild staining on the articular surface after application of India ink. Differences between Middle and Old age groups (indicative of intermediate-to-late stage changes) included maintenance of moderate level biomechanical function, a decrease in cellularity, and a decrease in matrix glycosaminoglycan content. Tissue fluorescence increased steadily with age. CONCLUSIONS: Many of these age-associated differences are identical to those regarded as pathological features of cartilage degeneration in early osteoarthritis. These findings provide evidence for the roles of mechanical wear, cell death, and enzymatic degradation in mediating the progression through successive and distinguishable stages of early cartilage deterioration.

A model of synovial fluid lubricant composition in normal and injured joints.

The synovial fluid (SF) of joints normally functions as a biological lubricant, providing low-friction and low-wear properties to articulating cartilage surfaces through the putative contributions of proteoglycan 4 (PRG4), hyaluronic acid (HA), and surface active phospholipids (SAPL). These lubricants are secreted by chondrocytes in articular cartilage and synoviocytes in synovium, and concentrated in the synovial space by the semi-permeable synovial lining. A deficiency in this lubricating system may contribute to the erosion of articulating cartilage surfaces in conditions of arthritis. A quantitative intercompartmental model was developed to predict in vivo SF lubricant concentration in the human knee joint. The model consists of a SF compartment that (a) is lined by cells of appropriate types, (b) is bound by a semi-permeable membrane, and (c) contains factors that regulate lubricant secretion. Lubricant concentration was predicted with different chemical regulators of chondrocyte and synoviocyte secretion, and also with therapeutic interventions of joint lavage and HA injection. The model predicted steady-state lubricant concentrations that were within physiologically observed ranges, and which were markedly altered with chemical regulation. The model also predicted that when starting from a zero lubricant concentration after joint lavage, PRG4 reaches steady-state concentration approximately 10-40 times faster than HA. Additionally, analysis of the clearance rate of HA after therapeutic injection into SF predicted that the majority of HA leaves the joint after approximately 1-2 days. This quantitative intercompartmental model allows integration of biophysical processes to identify both environmental factors and clinical therapies that affect SF lubricant composition in whole joints.

Effect of synovial fluid on boundary lubrication of articular cartilage.

OBJECTIVES: The lubrication of articulating cartilage surfaces in joints occurs through several distinct modes. In the boundary mode of lubrication, load is supported by surface-to-surface contact, a feature that makes this mode particularly important for maintenance of the normally pristine articular surface. A boundary mode of lubrication is indicated by a kinetic friction coefficient being invariant with factors that influence formation of a fluid film, including sliding velocity and axial load. The objectives of this study were to (1) implement and extend an in vitro articular cartilage-on-cartilage lubrication test to elucidate the dependence of the friction properties on sliding velocity, axial load, and time, and establish conditions where a boundary mode of lubrication is dominant, and (2) determine the effects of synovial fluid (SF) on boundary lubrication using this test. METHODS: Fresh bovine osteochondral samples were analyzed in an annulus-on-disk rotational configuration, maintaining apposed articular surfaces in contact, to determine static (mu(static) and mu(static),(N(eq)) and kinetic ([mu(kinetic)] and [mu(kinetic),(N(eq))]) friction coefficients, each normalized to the instantaneous and equilibrium (N(eq)) normal loads, respectively. RESULTS: With increasing pre-sliding durations, mu(static) and mu(static),(N(eq)) were similar, and increased up to 0.43 +/- 0.03 in phosphate buffered saline (PBS) and 0.19 +/- 0.01 in SF, whereas [mu(kinetic)] and [mu(kinetic),(N(eq))] were steady. Over a range of sliding velocities of 0.1-1 mm/s and compression levels of 18% and 24%, [mu(kinetic)] was 0.072 +/- 0.010 in PBS and 0.014 +/- 0.003 in SF, and [mu(kinetic),(N(eq))] was 0.093 +/- 0.005 in PBS and 0.018 +/- 0.002 in SF. CONCLUSIONS: A boundary mode of lubrication was achieved in a cartilage-on-cartilage test configuration. SF functioned as an effective friction-lowering boundary lubricant for native articular cartilage surfaces.

Indentation probing of human articular cartilage: Effect on chondrocyte viability.

BACKGROUND: Clinical arthroscopic probes based on indentation testing are being developed. However, the biological effects of certain design parameters (i.e., tip geometry and size) and loading protocols (i.e., indentation depth, rate, and repetition) on human articular cartilage are unclear. OBJECTIVE: Determine if indenter design and indentation protocol modulate mechanical injury of probed cartilage samples. METHODS: The objectives of this study were to determine the effects of indentation testing using clinically applicable tips (0.4mm radius, plane- or sphere-ended) and protocols (indentation depths of 100, 200, or 300 microm, applied at a rate of 50 or 500 microm/s) on the extent and the pattern of chondrocyte death, should it occur. Grossly normal osteochondral blocks were harvested from human talar dome, indented, stained with live/dead dyes, and imaged en face on a fluorescence microscope. RESULTS: The occurrence and the extent of cell death generally increased with indentation depth, being undetected at an indentation depth of 100 microm but marked at 300 microm. In addition, tip geometry affected the pattern of cell death: ring- and solid circle-shaped areas of cell deaths were apparent when compressed to 300 microm using plane- and sphere-ended indenters. CONCLUSION: Indenter design and indentation protocol modulated the extent and the pattern of chondrocyte death. These results have implications for designing indentation probes and protocols, as well as clinicians performing arthroscopic probing.

Modulation of depth-dependent properties in tissue-engineered cartilage with a semi-permeable membrane and perfusion: a continuum model of matrix metabolism and transport.

The functional properties of cartilaginous tissues are determined predominantly by the content, distribution, and organization of proteoglycan and collagen in the extracellular matrix. Extracellular matrix accumulates in tissue-engineered cartilage constructs by metabolism and transport of matrix molecules, processes that are modulated by physical and chemical factors. Constructs incubated under free-swelling conditions with freely permeable or highly permeable membranes exhibit symmetric surface regions of soft tissue. The variation in tissue properties with depth from the surfaces suggests the hypothesis that the transport processes mediated by the boundary conditions govern the distribution of proteoglycan in such constructs. A continuum model (DiMicco and Sah in Transport Porus Med 50:57-73, 2003) was extended to test the effects of membrane permeability and perfusion on proteoglycan accumulation in tissue- engineered cartilage. The concentrations of soluble, bound, and degraded proteoglycan were analyzed as functions of time, space, and non-dimensional parameters for several experimental configurations. The results of the model suggest that the boundary condition at the membrane surface and the rate of perfusion, described by non-dimensional parameters, are important determinants of the pattern of proteoglycan accumulation. With perfusion, the proteoglycan profile is skewed, and decreases or increases in magnitude depending on the level of flow-based stimulation. Utilization of a semi-permeable membrane with or without unidirectional flow may lead to tissues with depth-increasing proteoglycan content, resembling native articular cartilage.

Continuous passive motion applied to whole joints stimulates chondrocyte biosynthesis of PRG4.

UNLABELLED: Continuous passive motion (CPM) is currently a part of patient rehabilitation regimens after a variety of orthopedic surgical procedures. While CPM can enhance the joint healing process, the direct effects of CPM on cartilage metabolism remain unknown. Recent in vivo and in vitro observations suggest that mechanical stimuli can regulate articular cartilage metabolism of proteoglycan 4 (PRG4), a putative lubricating and chondroprotective molecule found in synovial fluid and at the articular cartilage surface. OBJECTIVES: (1) Determine the topographical variation in intrinsic cartilage PRG4 secretion. (2) Apply a CPM device to whole joints in bioreactors and assess effects of CPM on PRG4 biosynthesis. METHODS: A bioreactor was developed to apply CPM to bovine stifle joints in vitro. Effects of 24h of CPM on PRG4 biosynthesis were determined. RESULTS: PRG4 secretion rate varied markedly over the joint surface. Rehabilitative joint motion applied in the form of CPM regulated PRG4 biosynthesis, in a manner dependent on the duty cycle of cartilage sliding against opposing tissues. Specifically, in certain regions of the femoral condyle that were continuously or intermittently sliding against meniscus and tibial cartilage during CPM, chondrocyte PRG4 synthesis was higher with CPM than without. CONCLUSIONS: Rehabilitative joint motion, applied in the form of CPM, stimulates chondrocyte PRG4 metabolism. The stimulation of PRG4 synthesis is one mechanism by which CPM may benefit cartilage and joint health in post-operative rehabilitation.

Static and dynamic compression regulate cartilage metabolism of PRoteoGlycan 4 (PRG4).

The boundary lubrication function of articular cartilage is mediated in part by molecules at the articular surface and in synovial fluid, encoded by Prg4. The objective of this study was to determine whether static and dynamic compression regulate PRG4 biosynthesis by cartilage explants. Articular cartilage disks were harvested to include the articular surface from immature bovines. Some disks were subjected to 24 h (day 1) of loading, followed by 72 h (days 2-4) of free-swelling culture to assess chondrocyte responses following unloading. Loading consisted of 6 or 100 kPa of static compression, with or without superimposed dynamic compression (10 or 300 kPa peak amplitude, 0.01 Hz). Other disks were cultured free-swelling as controls. PRG4 secretion into culture medium was inhibited by all compression protocols during day 1. Following unloading, cartilage previously subjected to dynamic compression to 300 kPa exhibited a rebound effect, secreting more PRG4 than did controls, while cartilage previously subjected to 100 kPa static loading secreted less PRG4. Immunohistochemistry revealed that all compression protocols also affected the number of cells expressing PRG4. The paradigm that mechanical stimuli regulate biosynthesis in cartilage appears operative not only for load bearing matrix constituents, but also for PRG4 molecules mediating lubrication.

Platelet-rich plasma stimulates porcine articular chondrocyte proliferation and matrix biosynthesis.

OBJECTIVE: Platelet-rich plasma (PRP) is a fraction of plasma that contains high levels of multiple growth factors. The purpose of this study was to examine the effects of PRP on cell proliferation and matrix synthesis by porcine chondrocytes cultured in alginate beads, conditions that promote the retention of the chondrocytic phenotype, in order to determine the plausibility of using this plasma-derived material for engineering cartilage. DESIGN: PRP and platelet-poor plasma (PPP) were prepared from adult porcine blood. Adult porcine chondrocytes were cultured in the presence of 10% PRP, 10% PPP or 10% fetal bovine serum (FBS) for 3 days. Cell proliferation, proteoglycan (PG) and collagen synthesis were quantified, and the structure of newly synthesized PG and collagen was characterized. RESULTS: Treatment with 10% PRP resulted in a small but significant increase in DNA content (+11%, vs FBS; P<0.01; vs PPP; P<0.001). PG and collagen syntheses by the PRP-treated chondrocytes were markedly higher than those by chondrocytes treated by FBS or PPP (PG; PRP: +115% vs FBS; +151% vs PPP, both P<0.0001, collagen; PRP: +163% vs FBS; +163% vs PPP, both P<0.0001). Biochemical analyses revealed that treatment with PRP growth factors did not markedly affect the types of PGs and collagens produced by porcine chondrocytes, suggesting that the cells remained phenotypically stable in the presence of PRP. CONCLUSION: PRP isolated from autologous blood may be useful as a source of anabolic growth factors for stimulating chondrocytes to engineer cartilage tissue.

Osteogenic protein-1 promotes the formation of tissue-engineered cartilage using the alginate-recovered-chondrocyte method.

OBJECTIVE: This study examined the effects of a growth factor, recombinant human osteogenic protein-1 (rhOP-1), on the formation of tissue-engineered cartilaginous tissue by adult bovine articular chondrocytes using the alginate-recovered-chondrocyte (ARC) method. DESIGN: To ascertain if rhOP-1 enhances the formation of the cell-associated matrix (CM) and the characteristics of CM formation, bovine articular chondrocytes were first cultured for up to 14 days in alginate beads in medium supplemented with serum, with or without rhOP-1. Then, the recovered chondrocytes and their associated CM were resuspended in medium, with or without OP-1, seeded onto culture inserts, and incubated for an additional 14 days. The fabricated ARC tissues were subjected to biochemical and histological analyses. RESULTS: The addition of rhOP-1 to the medium in the alginate bead culture step resulted in an increased accumulation of both proteoglycan (PG) and collagen, with a ratio of PG to collagen that was higher than that found in native adult cartilage. The addition of rhOP-1 in the second step had a similar stimulatory effect during 14 days of culture. Histological examination of the tissue formed under all conditions revealed a cartilage-like matrix, stained strongly by toluidine blue. The thickness of the tissues obtained from culture conditions that included the addition of rhOP-1 was four times greater than that of the tissues cultured without rhOP-1. CONCLUSIONS: Using the ARC method, rhOP-1 enhanced the formation of matrix and generated a voluminous tissue-engineered cartilaginous construct. These characteristics may be beneficial in generating constructs that can cover large defects.