The objectives of this study were to find the most effective and least destructive procedure among three ACL decellularization protocols and to assess the capacity of ACL tissue treated with this protocol to serve as a biomaterial for human ACL tissue engineering. The results demonstrated that treatment with TRITON-X combined high effectiveness in decellularization with minimal changes in tissue biochemistry. Also, we could observe that ACL tissue decellularized with TRITON-X could be successfully reseeded with ACL fibroblasts showing persistent DNA content and increasing procollagen production.
It has been shown that implanted grafts undergo an initial phase of cellular necrosis, followed by an inflammatory reaction, and finally tissue remodeling. The course of these events can result in both remodeling as well as tunnel enlargement or frank graft rejection.34,35
It has been hypothesized that decellularized grafts could optimize ACL tissue engineering, and even ACL replacement, since they avoid the abovementioned problems by removing the intrinsic cells and thus the stimulus for inflammation. In 2004, Cartmell and colleagues published a study on decellularization of patellar tendon grafts with Tri(n-butyl)phosphate (TBP) or SDS and found 70%–90% reduction of intrinsic cells and similar biomechanics despite morphological changes in the tissue.23
Woods et al. studied the effectiveness of TRITON-X combined with either SDS or TBP and found the former to be most effective in decellularization, but also most destructive in terms of glycosaminoglycan and collagen depletion of samples and increase in tensile stiffness.24
Unfortunately, this study used TRITON-X as a baseline treatment in all groups, thus rendering a direct comparison of SDS, TRITON-X, and TBP impossible. Also, this study showed that SDS-treated samples were only poorly repopulated by fibroblasts, and Gratzer et al., in 2006, demonstrated this was due to such matrix alterations as mentioned above and not remnants of SDS.36
What stands out from these studies is the need for a systematic, direct comparison of those decellularization methods that have been developed in the literature. The effectiveness of such a procedure needs to be measured by the three parameters: completeness of cell removal, preservation of the structural and compositional nature of the tissue, and the capacity for repopulation by fibroblasts. Of note, since the biomaterial is meant to primarily act as a scaffold for cells, its biomechanical properties are not as important as they would be in a tissue-engineered whole ACL graft.24
Our findings showed that all treatments are highly effective in decellularization, and given the absolute values and fairly narrow confidence intervals of remaining DNA, there is no reason to suspect a different result in a larger study. We used DNA measurement to determine decellularization effectiveness, since this is a very sensitive method to test for the presence of cells. We did not assess decellularization histologically, since such assessment might yield a false negative result due to oversight of remaining cells in the physiologically already rather hypocellular ACL. Furthermore, findings concerning structural alterations due to decellularization showed no significant changes in the contents of collagen or total protein, but did show a reduction in glycosaminoglycan content, with the potential of a complete depletion of GAG by SDS or trypsin, which is in accordance with findings from previous studies.15,36
For GAG content, however, the relatively wide confidence intervals would allow for considerably different results in future studies. Of the protocols tested in this study, TRITON-X had the least detrimental effect on GAG content. In summary, we interpreted these findings as a recommendation for the use of TRITON-X to decellularize ACL tissue effectively and with the least adverse effects.
Reseeding of the decellularized ACL with human fibroblasts was successful, and our measurements showed persistent DNA contents and increasing procollagen measures over time, depicting the typical behavior of highly differentiated cells with high biosynthetic activity but low mitotic rates. Lower seeding density could lead to higher rates of mitosis, which might be beneficial in a defect healing environment. Yet it is important to consider that orchestrated biosynthesis is more important than rapid cell growth, since the latter would lead to a functionally and mechanically inferior scar tissue.37-39
Our histological specimens showed that the cells formed clusters on the surface of the biomaterial, as is often seen in static seeding.40,41
Deeper infiltration into the tissue might be seen at later follow-ups, but since our study was designed to test the feasibility of reseeding in general and not the behavior of the reseeded cells, data on time points later than 2 weeks are not available. Another reason for poor infiltration is the rather high density of the material. In fact, a previous study showed that ultrasonic modification of decellularized tendon increases recellularization.42
Finally, we interpret the decreasing content of free soluble collagen in the culture medium despite increasing procollagen production as an indirect proof of ongoing incorporation of newly synthesized collagen into the extracellular matrix. It should be remembered that procollagen contents should only be seen as a surrogate of collagen, but, as pointed out earlier, we decided to use procollagen rather than collagen measurement because we wanted to study cellular behavior independent from extracellular processes.
Our study has some shortcomings. Firstly, the question emerges whether it is necessary to decellularize tissue for ACL tissue engineering at all. Disease transmission with ACL allografts seems almost negligible,43
and studies have suggested that enzymatic removal of cell surface epitopes reduces immunogenicity of xenografts.44
However, as mentioned above, it has been shown that the presence of intrinsic cells, and their necrosis after graft harvest and implantation, can delay host cell infiltration and affect ligamentization.34,35,45
Although the specifics of donor–host cell interaction, both beneficial and detrimental, remain widely unknown, it is rather unlikely that the implantation of allogenic or even xenogenic cells will have any benefit to the host. Future studies comparing these methodologically different, yet technically equivalent, methods such as decellularization or epitope removal will be needed to definitively answer this question. Secondly, there is currently no reason to choose xenogenic over allogenic tissue for ACL tissue engineering other than availability. However, a xenogenic material is more easily and abundantly available, which would prove most valuable in a clinical application. Also, it is likely that the findings obtained from porcine tissue are also valid for human ACL. Lastly, there might be additional confounding factors we did not include in our analysis, yet the fairly narrow confidence intervals and findings consistent with the literature support the precision and validity of our findings, while the size of our sample discourages further sub-grouping.46
In summary, given the lack of prior comparative studies, the character of our study is more hypothesis-building in nature than hypothesis-testing.
In conclusion, our findings commend decellularization with TRITON-X over the use of SDS or trypsin, measured by effectiveness of cell removal and minimization of biochemical alterations. Also, porcine ACL tissue decellularized with TRITON -X can be successfully reseeded with human fibroblasts, which produce procollagen in increasing quantities. Future studies addressing the in vivo efficacy of decellularized ACL and other available scaffolds would be of great interest.