Blood and cartilage: friend or foe?

Introduction: Blood has been considered an armful factor for the articular cartilage. Previous studies have demonstrated that recurrent intra-articular bleedings represent a negative factor for articular cartilage, inducing a deterioration of the tissue. This could ultimately lead to degenerative osteoarthritis, even though the mechanism is not yet entirely understood (1-3). Other authors have investigated the effect of peripheral blood on articular cartilage both in vitro and in vivo: Roosendal and Hooiveld have shown that a shortterm exposure of human articular cartilage to whole blood in vitro induced an irreversible dose-dependent inhibition of proteoglycan synthesis and it was accompanied by cell apoptosis (4,5). However, when a short-term exposure was performed in vivo after injection of autologous blood into the canine knee, the initially adverse changes in cartilage proteoglycan synthesis turned into normalization after 10 weeks (6). Recently, the same group has tested the threshold of blood exposure time and concentration that lead to irreversible joint damage (7). However, many current surgical procedures for articular cartilage repair, like subchondral bone drilling (8), abrasion artrhoplasty (9) and microfracture (10), are based on the capacity of bone marrow cells to produce a fibrocartilaginous tissue when migrated in a joint environment (11). In contrast, in the performance of the techniques based on the transplantation of autologous chondrocytes, the presence of blood has been considered a disturbing factor for the development of the new cartilage tissue (12). The more recent techniques for cartilage repair and reconstruction utilize autologous chondrocytes seeded onto a biocompatible scaffold, in which they can duplicate, mature and produce new cartilage matrix in vitro and in vivo after surgical implantation. Also in this approach, it is recommended care in protecting the reparative cells from the contact with blood, which could derive from the subchondral bone or any other part of the joint injured during the surgical implantation (13), both in open and arthroscopic approach. However, the nature of engineered cartilage differs from that of cartilage explants examined in the studies mentioned above. Therefore, we believe that the influence of the contact of peripheral blood to the engineered cartilage represents an important but still unclear issue that needs to be investigated. The engineered cartilage, however, is structurally and biologically different from native articular cartilage, as it is supposed to complete the maturation in vivo. Therefore, it is probably more susceptible to the adverse effects of an articular bleeding, as indicated by studies of other authors, who investigated the effect of blood on immature joint (14). The effect of blood on engineered cartilage was only recently investigated (15). We have developed an in vitro model to investigate the effect of blood contact on the tissue-engineered implant and demonstrated that a 3-day exposure of cartilage to 50% (volume/ volume) blood results in a temporary and reversible effect on engineered cartilage tissue obtained from chondrocytes seeded onto collagen scaffold. However, some important issues remain to be clarified: could the different blood concentration negatively affect the chondrocytes’ vitality and synthetic properties? Is the negative effect of blood on the engineered cartilage due to the toxic effect of the peripheral blood or to the lack of nutrients occurring during the exposure to blood? The aim of this study was to investigate the effect of different concentrations of blood and of the lack of nutrients on the morphological, biochemical and biomechanical properties of engineered cartilage, synthesized by articular chondrocytes seeded onto a biological scaffold. Additionally, we have analyzed the effect of the main pro-inflammatory chemokine IL-1β on engineered cartilage based on human articular chondrocytes cultured for two different culture times. Experimental data Tissue engineered cartilage was developed combining expanded chondrocytes with a collagen membrane scaffold. Two sets of experiments were performed: one testing swine chondrocytes and allogeneic blood as inflammatory factor (“Blood study”); the other testing human adult chondrocytes and IL-1β (0.05 ng/ml) (“IL-1 study”). In the “Blood study”, articular chondrocytes were isolated from swine joints, expanded in monolayer culture, seeded onto collagen membranes and cultured for 2 weeks. During this period, an immature extracellular matrix, produced by cells, stabilized the chondrocytes to the biological membrane. This method generally duplicates the protocol for membrane seeding used in current clinical practice. After 2 weeks (t0), some samples were retrieved for analysis; others were exposed for 3 days to the contact with swine peripheral blood diluted with culture medium at 2 different concentrations (B50% group = 50% blood / 50% medium; B80% group = 80% blood / 20% medium); others were exposed for 3 days to the contact with a PBS solution. Following these 3 days (t3), some samples were retrieved for analysis, others were returned to standard culture conditions for 21 additional days (t3+21), in order to investigate the “long term effect” of the blood contact. For all the listed experimental times, some samples belonging to the control group were left in standard culture conditions without having any contact with blood or PBS. All groups of seeded membranes were analyzed grossly, by optic microscopy (OM), biochemically, histologically, and by biomechanical test. In particular, for morphological analysis, samples were analyzed macroscopically and by OM at all experimental times. Samples were weighed and sized with a calliper (products of axis) upon retrieve from culture. The edge of membranes was evaluated by OM analysis. For biochemical analysis, rate of cellular proliferation was evaluated by mitochondrial redox reaction to the tetrazolium salt (MTT) at all experimental times. For histological analysis, samples were processed and stained with safranin-o. Few sections (only at the time t3+21) were processed for immunohistochemical analysis and stained for type II collagen. For biomechanical analysis, samples were tested under unconfined geometry for compression using an electromagnetic testing machine. In the IL-1 study, human articular chondrocytes were harvested post-morted, expanded in monolayer, seeded onto collagen I/III scaffolds and cultured for 2 and 4 weeks. IL-1 was added during the last 3 days of culture. Samples were analyzed with histology, immunohistochemistry for collagen II, and biochemistry. In the “Blood study”, all seeded samples showed an increase in the weight and an evident cartilage-like matrix production. The evaluation of the samples with an optic microscope showed similar results for all study groups. It demonstrated the presence of spherically-shaped cells homogeneously distributed around the edge of the samples and firmly attached to the membranes. A specific concentration-dependent reduction of the mitochondrial activity due to blood contact was evident at the earlier culture time, followed by a partial recover at the longer culture time. An initial reduction of the biomechanical properties of the membranes, followed by a late stabilization, was recorded, regardless the presence of blood. In the “Il-1 study”, all samples exposed to IL-1 demonstrated a reduction in the intensity of Safranin-o and collagen II staining and in the quality of the biochemical composition. This reduction was more marked for the samples cultured for 2 weeks only, while samples cultured for 4 weeks had a better response to the pro-inflammatory stimulus. Conclusion The results from this study demonstrated that isolated chondrocytes could be seeded onto a biological scaffold, producing cartilagelike matrix with tissue specific morphology, composition, and biomechanical integrity. The blood contact seemed to produce a delay in the weight increase of the samples with respect to the control group. The analysis of the mitochondrial activity seemed to indicate a negative effect of the blood contact on the seeded membranes. In fact, samples exposed to a medium diluted with 80% and 50% blood recorded a depression of the MTT values with respect to group C. The same negative effect was recorded for PBS group where the lack of medium nutrients was probably the cause of the reduction of the chondrocytes’ vitality. However, the negative effect of the blood contact was specific, because the samples exposed to a medium diluted with different quantity of blood showed different depression of the MTT values. At the longest time period (t3+21), the cellular activity of the blood groups increased almost reaching the values achieved before the blood contact. This indicates that the toxic effect on the chondrocytes was temporary and reversible. The analysis of the biomechanical data did not support these evidences, because the biomechanical results were not affected by blood contact. It could be hypothized that exposure time was not long enough to produce differences in the biomechanical properties of the constructs. We can conclude that the negative effect of the blood on the engineered cartilage is evident at the cellular level. However, it does not seem to be perceptible at the (engineered) tissue level, with the model utilized here. Three days of exposure did not entirely devitalize the tissue cells and did not seem to influence the synthetic properties of the chondrocytes and the biomechanical integrity of the immature cartilage at the longest time point. In the “IL-1 study” the negative effect of IL-1 seemed to be more marked than that of blood in the “Blood study”, although a direct comparison of the 2 model results very speculative. Moreover, this negative effect was clearly dependent on the level of maturation of the construct. A further in vivo study is probably needed to investigate the potential facilitation of the biological environment in reversing the negative effects of bleeding on the cells of the engineered cartilage as shown by other authors for native articular cartilage (14). Future studies are also desirable to test the effect of the different level of maturation of the engineered cartilage on its capacity of surviving and integrating in a joint environment exposed to bleeding.