Tissue engineering holds much promise for regenerative approaches to ACL treatment[2
]. Fibrin is a logical candidate as a biomaterial for such ACL tissue engineering. The objective of this study was to assess the effects of fibrin concentration on fibroblast proliferation and biosynthetic activity, effects that will support or hinder the outcome of any potential fibrin-based procedure. We chose to study an adolescent model to account for the age-dependence of ACL fibroblast behavior[23
], and the fact that adolescent individuals, a group characterized by a sharply increasing incidence of ACL injuries and a lack of treatment options, might profit most from a tissue-engineered approach to ACL treatment[25
]. Our findings show that while all groups showed increasing DNA and collagen content over time, a lower than physiological concentration of fibrin showed the highest DNA and collagen production
Our study has some potential weaknesses. One is that our gels have a physiological concentration of fibrin, but that does not necessarily mean they have a physiological structure. As a matter of fact, previous in-vitro research in fibrin clot structure has shown structurally homogenous, porous clots at lower concentrations and rapidly polymerizing, non-homogenously dense clots with very small pores at higher concentrations[19
]. Thus our findings are perfectly valid for fibrin-based biomaterials, but not necessarily for biologic wound healing. Finally, we chose a rather narrow range of fibrin, and a wider range would have shown differences in effects more clearly. However, we wanted to build our model on a biological important parameters, thus we chose the physiological concentration of 3 mg/mL[21
] and a biologically plausible range around this value[27
]. Finally, we chose to use a porcine model, rather than a human model. Beyond the obvious problem of availability of human tissue, most of the basic science in ACL tissue engineering is based on porcine models[2
], thus results that pertain to such models and allow for interpretation in their context seem more important immediately, despite the fact that eventually a human application is aimed at. From what is known, extraction of the results of procine models and application in human disease is possible and valid given the considerable commonalities in cell behavior, tissue composition, and anatomy[2
Our results demonstrated steady increases in DNA and collagen over time in all of the gels. The increase rate in DNA (25% per day) matches commonly accepted doubling times of approximately 3 days for mesenchymal cells. We furthermore interpreted the fact that collagen content in the culture medium decreased after peaking at 6 days while collagen content in the cell-fibrin constructs rose unabatedly as an indicator of incorporation of collagen into newly formed extra-cellular matrix during tissue remodeling. The assumption of high tissue remodeling is supported by the considerably fast rate of degradation seen in most fibrin gels.
In summary, our data suggest that all groups showed ongoing cellular growth and proliferation as well as tissue remodeling, suggesting the fibroblasts in the gels were viable and active. In this model we observed attenuated DNA synthesis and collagen production with increasing fibrin concentration. A very likely reason for such an effect is an increase in biomaterial density beyond the limits of permeability and nutrient transfer. This explanation, rather than a direct suppressive effect of fibrin on cells, is supported by the non-linear nature of this effect as was shown by the larger differences between 1 mg and 3 mg compared to 3 mg and 6 mg and earlier published evidence for increased porosity in gels from diluted fibrin[18
]. Our own microscopic analysis, in accordance with data put forward by Cox et al. and Ho et al., showed fairly similar structural characteristics for 1 and 3 mg/mL and a much denser, less porous appearance at 6 mg/ml.
Our interpretation of the effects of fibrin concentration is supported by both statistically significant results and substantive absolute differences. Finally, it is very interesting that there are no significant differences in type I and type III collagen mRNA contents, suggesting that the cellular genetic programming to produce collagen is unaffected, but that biosynthesis may be inhibited on the post-translational level. This finding, too, supports our interpretation of the effects of tissue density, which might inhibit anything form nutrient transfer to extracellular protein assembly, rather than direct cellular effects. From our regression model, we could show that fibrin concentration is not a significant predictor of collagen mRNA production, thus our results may be valid even beyond our chosen range of concentrations. Finally, we interpret the non-significant reduction in mRNA levels over time as a negative feed-back mechanism, which is visible due to the short duration of our observation, and, again, may suggest accumulation of collagen precursor product intracellularly because of inhibit post-translational processing.
Findings from earlier studies corroborate our results. Ho et al studied the combined effects of fibrin concentration and thrombin concentration on fibroblast behavior, yet at much higher than physiological concentrations of fibrin[32
]. This group reported decreased cell proliferation with increases in fibrin concentration as well. Additionally, it could be demonstrated that this effect could be countered by increases in thrombin concentration, which might result in more organized clot formation. Cox and colleagues presented similar findings in 2006 for mesenchymal stem cells, but could also show that thrombin effects are most meaningful at low fibrin concentrations. Murray et al., finally, demonstrated that high concentrations of thrombin inhibit cellular migration and reduce biomaterial strength. These findings were included in our study design to avoid interaction with the effects of fibrin concentration.