Population doublings and percentage of S100-positive cells as predictors of in vitro chondrogenicity of expanded human articular chondrocytes

The aim of this study was to investigate the interconnection between the processes of proliferation, dedifferentiation, and intrinsic redifferentiation (chondrogenic) capacities of human articular chondrocyte (HAC), and to identify markers linking HAC dedifferentiation status with their chondrogenic potential. Cumulative population doublings (PD) of HAC expanded in monolayer culture were determined, and a threshold range of 3.57-4.19 PD was identified as indicative of HAC loss of intrinsic chondrogenic capacity in pellets incubated without added chondrogenic factors. While several specific gene and surface markers defined early HAC dedifferentiation process, no clear correlation with the loss of intrinsic chondrogenic potential could be established. CD90 expression during HAC monolayer culture revealed two subpopulations, with sorted CD90-negative cells showing lower proliferative capacity and higher chondrogenic potential compared to CD90-positive cells. Although these data further validated PD as critical for in vitro chondrogenesis, due to the early shift in expression, CD90 could not be considered for predicting chondrogenic potential of HAC expanded for several weeks. In contrast, an excellent mathematically modeled correlation was established between PD and the decline of HAC expressing the intracellular marker S100, providing a direct link between the number of cell divisions and dedifferentiation/loss of intrinsic chondrogenic capacity. Based on the dynamics of S100-positive HAC during expansion, we propose asymmetric cell division as a potential mechanism of HAC dedifferentiation, and S100 as a marker to assess chondrogenicity of HAC during expansion, of potential value for cell-based cartilage repair treatments.

T1 assessment of hip joint cartilage following intra-articular gadolinium injection: a pilot study

This pilot study defines the feasibility of cartilage assessment in symptomatic femoroacetabular impingement patients using intra-articular delayed gadolinium-enhanced MRI of cartilage (ia-dGEMRIC). Nine patients were scanned preliminary to study the contrast infiltration process into hip joint cartilage. Twenty-seven patients with symptomatic femoroacetabular impingement were subsequently scanned with intra-articular delayed gadolinium-enhanced MRI of cartilage. These T(1) findings were correlated to morphological findings. Zonal variations were studied. This pilot study demonstrates a significant difference between the pre- and postcontrast T(1) values (P < 0.001) remaining constant for 45 min. We noted higher mean T(1) values in morphologically normal-appearing cartilage than in damaged cartilage, which was statistically significant for all zones except the anterior-superior zone. Intraobserver (0.972) and interobserver correlation coefficients (0.933) were statistically significant. This study outlines the feasibility of intra-articular delayed gadolinium-enhanced MRI of cartilage for assessment of cartilage changes in patients with femoroacetabular impingement. It can also define the topographic extent and differing severities of cartilage damage.

Intravenous versus intra-articular delayed gadolinium-enhanced magnetic resonance imaging in the hip joint: a comparative analysis

The purpose of this study was to investigate whether T1-mapping of hip joint with intra-articular delayed gadolinium-enhanced magnetic resonance imaging (MRI) of cartilage (ia-dGEMRIC) is comparable to the already established intravenous (iv)-technique for assessing different grades of cartilage degeneration.

The in vivo effects of unloading and compression on T1-Gd (dGEMRIC) relaxation times in healthy articular knee cartilage at 3.0 Tesla

The purpose was to investigate the in vivo effects of unloading and compression on T1-Gd relaxation times in healthy articular knee cartilage.

Micromass co-culture of human articular chondrocytes and human bone marrow mesenchymal stem cells to investigate stable neocartilage tissue formation in vitro

Cell therapies for articular cartilage defects rely on expanded chondrocytes. Mesenchymal stem cells (MSC) represent an alternative cell source should their hypertrophic differentiation pathway be prevented. Possible cellular instruction between human articular chondrocytes (HAC) and human bone marrow MSC was investigated in micromass pellets. HAC and MSC were mixed in different percentages or incubated individually in pellets for 3 or 6 weeks with and without TGF-beta1 and dexamethasone (±T±D) as chondrogenic factors. Collagen II, collagen X and S100 protein expression were assessed using immunohistochemistry. Proteoglycan synthesis was evaluated applying the Bern score and quantified using dimethylmethylene blue dye binding assay. Alkaline phosphatase activity (ALP) was detected on cryosections and soluble ALP measured in pellet supernatants. HAC alone generated hyaline-like discs, while MSC formed spheroid pellets in ±T±D. Co-cultured pellets changed from disc to spheroid shape with decreasing number of HAC, and displayed random cell distribution. In -T-D, HAC expressed S100, produced GAG and collagen II, and formed lacunae, while MSC did not produce any cartilage-specific proteins. Based on GAG, collagen type II and S100 expression chondrogenic differentiation occurred in -T-D MSC co-cultures. However, quantitative experimental GAG and DNA values did not differ from predicted values, suggesting only HAC contribution to GAG production. MSC produced cartilage-specific matrix only in +T+D but underwent hypertrophy in all pellet cultures. In summary, influence of HAC on MSC was restricted to early signs of neochondrogenesis. However, MSC did not contribute to the proteoglycan deposition, and HAC could not prevent hypertrophy of MSC induced by chondrogenic stimuli.

T2 star relaxation times for assessment of articular cartilage at 3 T: a feasibility study

T2 mapping techniques use the relaxation constant as an indirect marker of cartilage structure, and the relaxation constant has also been shown to be a sensitive parameter for cartilage evaluation. As a possible additional robust biomarker, T2* relaxation time is a potential, clinically feasible parameter for the biochemical evaluation of articular cartilage.

The structure of articular cartilage

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Evaluation of articular cartilage in patients with femoroacetabular impingement (FAI) using T2* mapping at different time points at 3.0 Tesla MRI: a feasibility study

To define the feasibility of utilizing T2* mapping for assessment of early cartilage degeneration prior to surgery in patients with symptomatic femoroacetabular impingement (FAI), we compared cartilage of the hip joint in patients with FAI and healthy volunteers using T2* mapping at 3.0 Tesla over time.

Extra-articular fractures of the proximal phalanges of the fingers: a comparison of 2 methods of functional, conservative treatment

For nonsurgical treatment of fractures of the proximal phalanges of the triphalangeal fingers, different dynamic casts have been described. The main principle behind these casts is advancement and tightening of the extensor hood, caused by a combination of blocking the metacarpophalangeal joints in flexion and actively flexing the proximal interphalangeal joints. In contrast to established treatment protocols using functional forearm casts, the Lucerne cast allows for free mobilization of the wrist joint. The purpose of the current multicenter study was to compare the results of conservative, functional treatment using 2 different methods, either a forearm cast or a Lucerne cast.

Antinociceptive activity of pre- versus post-operative intra-articular bupivacaine in goats undergoing stifle arthrotomy

OBJECTIVE: To evaluate the peri-operative analgesic efficacy of intra-articular bupivacaine administered before or after stifle arthrotomy. STUDY DESIGN: Prospective, randomized, blind, placebo-controlled experimental trial. ANIMALS: Thirty-nine healthy goats. METHODS: The goats were allocated randomly to one of three intra-articular treatment groups: group PRE (bupivacaine before and saline after surgery), group POST (saline before and bupivacaine after surgery) and group CON (saline before and after surgery). Anaesthesia was maintained with a constant end-tidal sevoflurane of 2.5%. Intra-operatively heart rate (HR), respiratory rate and mean arterial blood pressure (MAP) after critical surgical events (CSE) were recorded and compared with pre-incision values. Propofol requirements to maintain surgical anaesthesia were recorded. Flunixin was administered for 5 days. Post-operative pain assessment at 20 minutes, 2 hours, 4 hours after recovery and on day 2 and 3 included a multidimensional pain score (MPS), a lameness score and mechanical nociceptive threshold (MNT) testing. Rescue analgesia consisted of systemic opioids. Data were analysed using Kruskal-Wallis, Mann-Whitney, Friedman or chi-square tests as appropriate. RESULTS: Intra-operatively, group PRE had lower HR and MAP at several CSEs than groups POST/CON and required less propofol [0 mg kg(-1) (0-0 mg kg(-1))] than group POST/CON [0.3 mg kg(-1) (0-0.6 mg kg(-1))]. Post-operatively, group POST had significantly higher peri-articular MNTs than groups PRE and CON up to 4 hours after recovery. No treatment effect was detected for MPS, lameness scores and rescue analgesic consumption at any time point. CONCLUSIONS AND CLINICAL RELEVANCE: Pre-operative intra-articular bupivacaine provided notable intra-operative analgesia in goats undergoing stifle arthrotomy but did not reduce post-operative pain. Post-operative intra-articular bupivacaine provided a short lasting reduction of peri-articular hyperalgesia without affecting the requirements for systemic analgesia. Multimodal perioperative pain therapy is recommended to provide adequate analgesia for stifle arthrotomy in goats.

Does bone wax induce a chronic inflammatory articular reaction?

Bone wax is used to control femoral neck bleeding during open femoroacetabular impingement (FAI) surgery. Despite its widespread use, only a few case reports and small case series describe side effects after extraarticular use. It is unclear whether intraarticular use of bone wax leads to such complications. However, during revision FAI surgery, we have observed various degrees of articular inflammatory reactions.

The structural architecture of adult mammalian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal development

OBJECTIVE: During postnatal development, mammalian articular cartilage acts as a surface growth plate for the underlying epiphyseal bone. Concomitantly, it undergoes a fundamental process of structural reorganization from an immature isotropic to a mature (adult) anisotropic architecture. However, the mechanism underlying this structural transformation is unknown. It could involve either an internal remodelling process, or complete resorption followed by tissue neoformation. The aim of this study was to establish which of these two alternative tissue reorganization mechanisms is physiologically operative. We also wished to pinpoint the articular cartilage source of the stem cells for clonal expansion and the zonal location of the chondrocyte pool with high proliferative activity. METHODS: The New Zealand white rabbit served as our animal model. The analysis was confined to the high-weight-bearing (central) areas of the medial and lateral femoral condyles. After birth, the articular cartilage layer was evaluated morphologically at monthly intervals from the first to the eighth postnatal month, when this species attains skeletal maturity. The overall height of the articular cartilage layer at each juncture was measured. The growth performance of the articular cartilage layer was assessed by calcein labelling, which permitted an estimation of the daily growth rate of the epiphyseal bone and its monthly length-gain. The slowly proliferating stem-cell pool was identified immunohistochemically (after labelling with bromodeoxyuridine), and the rapidly proliferating chondrocyte population by autoradiography (after labelling with (3)H-thymidine). RESULTS: The growth activity of the articular cartilage layer was highest 1 month after birth. It declined precipitously between the first and third months, and ceased between the third and fourth months, when the animal enters puberty. The structural maturation of the articular cartilage layer followed a corresponding temporal trend. During the first 3 months, when the articular cartilage layer is undergoing structural reorganization, the net length-gain in the epiphyseal bone exceeded the height of the articular cartilage layer. This finding indicates that the postnatal reorganization of articular cartilage from an immature isotropic to a mature anisotropic structure is not achieved by a process of internal remodelling, but by the resorption and neoformation of all zones except the most superficial (stem-cell) one. The superficial zone was found to consist of slowly dividing stem cells with bidirectional mitotic activity. In the horizontal direction, this zone furnishes new stem cells that replenish the pool and effect a lateral expansion of the articular cartilage layer. In the vertical direction, the superficial zone supplies the rapidly dividing, transit-amplifying daughter-cell pool that feeds the transitional and upper radial zones during the postnatal growth phase of the articular cartilage layer. CONCLUSIONS: During postnatal development, mammalian articular cartilage fulfils a dual function, viz., it acts not only as an articulating layer but also as a surface growth plate. In the lapine model, this growth activity ceases at puberty (3-4 months of age), whereas that of the true (metaphyseal) growth plate continues until the time of skeletal maturity (8 months). Hence, the two structures are regulated independently. The structural maturation of the articular cartilage layer coincides temporally with the cessation of its growth activity - for the radial expansion and remodelling of the epiphyseal bone - and with sexual maturation. That articular cartilage is physiologically reorganized by a process of tissue resorption and neoformation, rather than by one of internal remodelling, has important implications for the functional engineering and repair of articular cartilage tissue.

Intra-articular calcium phosphate cement: Its fate and impact on joint tissues in a rabbit model

Clinical application of injectable ceramic cement in comminuted fractures revealed penetration of the viscous paste into the joint space. Not much is known on the fate of this cement and its influence on articular tissues. The purpose of this experimental study was to assess these unknown alterations of joint tissues after intra-articular injection of cement in a rabbit knee. Observation periods were from 1 week up to 24 months, with three rabbits per group. Norian SRS cement was injected into one knee joint, the contralateral side receiving the same volume of Ringers' solution. Light microscopic evaluation of histologic sections was performed, investigating the appearance of the cement, inflammatory reactions, and degenerative changes of the articular surface. No signs of pronounced acute or chronic inflammation were visible. The injected cement was mainly found as a single particle, anterior to the cruciate ligaments. It became surrounded by synovial tissues within 4 weeks and showed signs of superficial resorption. In some specimens, bone formation was seen around the cement. Degeneration of the articular surface showed no differences between experimental and control side, and no changes over time became apparent. No major degenerative changes were induced by the injected cement. The prolonged presence of cement still seems to make it advisable to remove radiologically visible amounts from the joint space.

Nitric oxide and metalloproteinases in canine articular ligaments: a comparison between the cranial cruciate, the medial genual collateral and the femoral head ligament

Osteoarthritis due to cranial cruciate ligament (CCL) rupture or hip dysplasia is one of the most important causes of chronic lameness in dogs. This study aimed at comparing nitric oxide (NO) production by the CCL with that of the femoral head ligament (FHL) and the medial collateral ligament (MCL), and investigating the pathway of NO production and the concomitant metalloproteinase (MMP) activity in the presence or absence of an inflammatory stimulus. Ligaments of normal dogs were subjected to different stimuli, and NO and MMP activity from explant culture supernatants were compared. The results showed that in explant cultures of the canine CCL more NO was produced than in those of the other two ligaments. A higher level of NO was produced when CCLs were exposed to the inducible nitric oxide synthase (iNOS)-inducing cocktail TNF/IL-1/LPS, and NO synthesis could be inhibited by both l-NMMA, a general nitric oxide synthase (NOS) inhibitor and l-NIL, a specific iNOS inhibitor. However, a correlation between NO synthesis and iNOS expression levels as determined by immunohistochemistry was not observed. In contrast to CCL, no evidence for iNOS-dependent NO synthesis was observed for MCL and FHL. The CCL produced less MMP than MCL and FHL, and no correlation between MMP and NO could be demonstrated. MMP activity in the CCL increased significantly after 48 h of incubation with the inflammatory stimulus. The results suggest that in canine osteoarthritis NO synthesized by canine CCL plays a more important role in the pathogenesis of osteoarthritis of the stifle than that synthesized by FHL and MCL.

Cartilage tissue engineering by expanded goat articular chondrocytes

In this study we investigated whether expanded goat chondrocytes have the capacity to generate cartilaginous tissues with biochemical and biomechanical properties improving with time in culture. Goat chondrocytes were expanded in monolayer with or without combinations of FGF-2, TGF-beta1, and PDGFbb, and the postexpansion chondrogenic capacity assessed in pellet cultures. Expanded chondrocytes were also cultured for up to 6 weeks in HYAFF-M nonwoven meshes or Polyactive foams, and the resulting cartilaginous tissues were assessed histologically, biochemically, and biomechanically. Supplementation of the expansion medium with FGF-2 increased the proliferation rate of goat chondrocytes and enhanced their postexpansion chondrogenic capacity. FGF-2-expanded chondrocytes seeded in HYAFF-M or Polyactive scaffolds formed cartilaginous tissues with wet weight, glycosaminoglycan, and collagen content, increasing from 2 days to 6 weeks culture (up to respectively 2-, 8-, and 41-fold). Equilibrium and dynamic stiffness measured in HYAFF M-based constructs also increased with time, up to, respectively, 1.3- and 16-fold. This study demonstrates the feasibility to engineer goat cartilaginous tissues at different stages of development by varying culture time, and thus opens the possibility to test the effect of maturation stage of engineered cartilage on the outcome of cartilage repair in orthotopic goat models.