Erythrocyte concentration had a significant effect on ACL fibroblasts within a 3D scaffold. The samples with an erythrocyte concentration at or above physiologic concentration showed a suppression of cell proliferation over time when compared to those with lower erythrocyte concentrations. Interestingly, even with lower cell proliferation, the samples with supraphysiologic erythrocyte concentration showed a significantly higher rate of procollagen/DNA production over 21 days than the samples with physiologic or lower concentration of erythrocytes. In addition, erythrocyte concentration at or above physiologic levels caused a slower rate of scaffold contraction in the collagen gels.
The results in the study support the findings previously reported by Fredriksson et al.2
and Jacobson et al.3
regarding erythrocyte suppression of fibroblast proliferation. Specifically, Fredriksson et al. determined that cell proliferation of human lung fibroblasts was significantly inhibited when cultured with erythrocytes above concentrations 5 × 105
and 5 × 108
erythrocyte/ml DMEM (2). Similarly, Jacobson et al.3
determined that cell proliferation was significantly less in a 3D collagen–platelet hydrogel with 1.5 × 109
erythrocyte/ml than the control collagen–platelet hydrogel with no erythrocytes. Here, in a 3D collagen gel containing PPP and varying concentrations of erythrocytes, cellular proliferation was significantly suppressed in the two groups at or above 1 × 109
erythrocyte/ml. The gels used in the Jacobson et al. study match our physiologic gels both in erythrocyte concentration and fibroblast seeding density, which serves to support the validity of our findings for that group. Our model expands on the Jacobson et al. study to show the proliferation response of the same gels with higher and lower erythrocyte concentrations as well, which suggests the concentration of erythrocytes in whole blood may be a threshold value below which cell proliferation is not suppressed in a 3D collagen gel. We see that in both a 2D and 3D model in the presence and absence of other blood components, erythrocytes at or above physiologic concentrations suppress fibroblast proliferation.
The procollagen/DNA production over time by the fibroblasts was highest in the supraphysiologic samples. Linear regression analysis revealed that only 6% of the change in procollagen over time could be predicted by DNA levels. Since fibroblast concentration was consistent between groups at the time of gel seeding and no significant levels of other blood components were present in the gels, it is likely that the high erythrocyte concentration was a major factor in causing this effect. Accumulation of procollagen is often attributed to ascorbic acid deficiency,11
but in this study media containing 1% ascorbic acid was replenished for all samples every 3–4 days. Nonetheless, our study shows that although cell proliferation was suppressed in the supraphysiologic gels, the remaining fibroblasts in those samples produced the highest levels of procollagen.
From our DNA measurements, it appears that fibroblast proliferation in the PPP and SUB groups began between days 1 and 14 and was accelerated between days 14 and 21. In contrast we observed a maintenance and decline in fibroblast count in the PHYS and SUPRA gels, respectively, during those time frames. We hypothesize that the high levels of hemoglobin released between days 4 and 11 in the PHYS and SUPRA gels result in damage or death of the fibroblasts resulting in suppression of further proliferation. One of the main products of hemoglobin degradation is bilirubin. Previous studies by Chuniaud et al.12
have shown that fibroblasts are subject to mitochondrial activity suppression and cell lysis in the presence of increasing concentrations of bilirubin. Such adverse effects begin within 24 h of exposure and result in near complete elimination of activity and viability within 48 h. The higher concentrations of erythrocytes in the SUPRA and PHYS groups compared to SUB and PPP groups may have resulted in higher concentrations of hemoglobin release after lysis, which then may have resulted in high concentrations of biproducts (bilirubin plus perhaps others) toxic to fibroblasts.
Interestingly, a suppression of procollagen release from fibroblasts in the SUPRA gels was not observed even as fibroblast proliferation was inhibited. We hypothesize that this result could also be related to the large amounts of hemoglobin released from lysed erythrocytes in the SUPRA gels. In a series of studies, Stamler et al. began to elucidate the mechanism by which hemoglobin, in addition to binding oxygen, also binds and releases nitric oxide (NO) in order to regulate vasodilation in vivo.13,14
Through an oxygen-dependant allosteric transition, the position and reactivity of the heme and cysteine residues within the hemoglobin molecule are changed in order to allow different binding conformations of NO. In the presence of oxygen, NO is bound to the cysteine residue and remains active, whereas in a deoxygenated state hemoglobin scavenges free NO through binding to the heme residues, inactivating NO.13
Furthermore, several studies have shown that increased levels of NO stimulate collagen synthesis by fibroblasts in vitro.15,16
Therefore, we postulate that the increase in procollagen levels in the SUPRA group after day 14 may be related to the high levels of hemoglobin release from lysed erythrocytes through day 11. NO specifically bound to the cysteine residue of released hemoglobin molecules could stimulate procollagen synthesis from remaining viable fibroblasts within the gel. Further studies measuring NO within the gels and correlation with collagen production are needed to validate this hypothesis.
In the current study, contraction over time was suppressed in samples with physiologic and higher erythrocyte concentrations over 21 days. This is in contrast to another study by Fredriksson et al.17
which showed an increase in contraction in fibroblast-containing collagen gels with addition of erythrocytes in a time and concentration-dependent manner. Significant increase was shown compared to gels without erythrocytes on all days between 1 and 5, and the augmentation was dependent on the number of erythrocytes added. In the Fredriksson study, the gels were free-floating at the time of measurement, while the gels in the current study were anchored. In addition, we did not view any gel contraction until the day 14 time point, while they observed contraction after 24 h.
Gel contraction has previously been found to be a function of whether the gels are attached, or anchored, to stable points. One study supportive of the differences in time of contraction observed between the current study and the Fredriksson study was done by Galois et al.18
They found that free-floating collagen gels seeded with chondrocytes contracted more readily than anchored gels over 12 days. Furthermore, in an interesting series of studies, Grinnell et al. determined that fibroblasts in anchored collagen gels develop isometric tension after several hours of culture, whereas fibroblasts in floating gels remain mechanically unloaded. That work also suggested that gels in both models will eventually contract the same amount, but the contraction occurs via different mechanisms and at different times. The floating gels contract immediately while the anchored gels contract upon release from the anchoring surface as cell extensions collapse.19
In our experiment, the collagen gels were firmly anchored with mesh on two ends, but likely also experienced temporary tension from the silicon tubing along the other two long edges. As a result, the measured contraction was delayed until the gels were released from the construct along the long edges. It is possible that further contraction would have continued if the gels were released from the mesh anchors.
One limitation to this study is the use of animal models. As it is very difficult to obtain ACL cells from a patient without an ACL injury or osteoarthritis, we worked to mitigate this limitation by using an animal model that is thought to most closely approach the human condition from a biomechanical and wound healing standpoint. The same animal model (porcine) was used to obtain both the fibroblasts and blood components used in the study to minimize any cross-species complications. The porcine model is commonly used for wound healing studies20
and also has similar anatomy and biomechanics to the human knee.21
The fibroblasts used in this study were passaged twice prior to the experiment. We chose a low passage because it has been shown that late passaged fibroblasts display an increased nuclear size, which correlates with slow and nondividing cells and thus, higher passage cells may be a less accurate representation of the in vivo condition.22,23
The chief limitation of this study is its in vitro nature. The environment of the culture dish is very different from that of the in vivo environment, and although the in vitro model is useful to begin to understand intrinsic ACL cell migration behaviors, there are likely many other cell types involved in governing the wound response, including inflammatory cells found in the blood. These cells are excluded from this in vitro assay, and therefore future studies should include in vivo studies where the effects of the intrinsic ACL cells are more difficult to isolate and ascertain, but the cumulative invasion characteristics of all the cells in the in vivo wound environment can be determined as a function of erythrocyte concentration.
In summary, the inclusion of physiologic or supraphysiologic concentrations of erythrocytes in a simulated 3D provisional scaffold resulted in protection of the scaffold during the first few weeks of remodeling. Procollagen expression was increased and the scaffold had significantly less contraction. These features suggest the presence of erythrocytes in a tissue-engineered construct may be beneficial in maintaining a scaffold in a wound site. Additional in vivo studies are warranted to study this hypothesis.