The use of collagen, mostly of bovine and porcine origin, as a biomaterial for intra-articular tissue engineering applications in orthopedic surgery has shown much promise in vitro
and in vivo
]. However, there still are concerns being uttered regarding the safety of such applications, and, to date, there is a lack of evidence to counter such claims. Collagen is highly similar in the amino acid sequence and epitope structure across species and generally considered safe [15
]. The incidence of immunologic reactions to xenogenic collagenous implants ranges between 2-4% in clinical observation of extra-articular application of bovine and porcine collagen and these reactions mostly are trivial and inconsequential [16
]. It is to be expected that these observations overstate the incidence of intra-articular reactions, which should be lower due to the immuno-privileged nature of the intra-articular environment. It was the objective of this study to test for clinically relevant, adverse reactions to the intra-articular use of a collagenous biomaterial, more specifically atelocollagen, in an in vivo
model. Additionally, we included a platelet concentrate as a cofactor into our model, since most intra-articular current tissue engineering applications strive to harness the regenerative powers of such agents.
Atelocollagen is considered an even safer alternative to generic collagen, because the telopeptides of the collagen molecule, which are thought to be the major agent of immunogenicity, are removed [6
]. However, it has been shown that, in addition to the telopeptides, central and helical domains of collagen play an important role in the immunologic interaction between bovine and porcine collagen and human host tissue as well [6
]. It should be considered that any such interaction might be due to either antigenicity, which is the interaction between collagen and pre-existing antibodies, or immunogenicity, which involves the de-novo formation of antibodies against specific epitopes. The latter might also result in an auto-immune response if similar epitopes as presented by the implant are also presented in host tissues too. This process is the bedrock of collagen-induced arthritis (CIA), an animal model of rheumatoid arthritis [20
]. In CIA, animals are injected with collagen emulsified in an immunological adjuvant – a reagent that boosts immune responses [22
]. Fears of initiating CIA by using collagen implants in patients, however, are ungrounded because CIA requires at the very least an adjuvant, and – despite investigators’ best efforts - has thus far only been accomplished for types II and XI collagen in a limited number of species [6
]. There remains, however, the risk of an immune reaction of unspecified severity towards a collagenous implant itself. Thus it was the objective of this study to explicitly assess such potential reactions in a relevant animal model. Any such reaction might be facilitated or exacerbated by cytokines secreted after the addition of PRP, and this factor was included as covariate in our study. Finally, we chose a time point at 15 weeks to make sure that none of any observed effects are caused by perioperative events.
Our study has some potential limitations. First of all, our definition of an adverse immunological reaction is rather non-specific, but since type and extent of a possible host reaction to a collagen implant cannot be reliably predicted we deliberately chose these more general endpoints. Of note, the objective of this study was to search for adverse effects that might affect clinical outcome due to rejection, inflammation, adhesions, et cetera, allowing for more in depth research into any found reaction later on. By no means did we intend to give a complete, comprehensive, and chronological description of all immunological processes involved in the intra-articular use of collagen. External validity is a final concern. Although the pig is a validated model of human immunity [23
], there might be some relevant differences in the interaction of a bovine collagen with a porcine versus a human host [6
]. Additional studies in humans will be necessary to definitively confirm our conclusions.
The introduction of atelocollagen into the knee joint as either a sponge or platelet gel to stimulate healing of the ACL did not appear cause a significant joint effusion when compared to intact knees. Interestingly, there were some significant differences between those knees treated with sutures alone and intact knees, on outcomes scales, MRI, and range of motion. However, the rather small absolute values of these differences minimize their clinical importance. There was a significant difference in the effusion length of the suture group and intact group, with the suture group having a smaller effusion length than the intact group (Fig. ). One possible explanation for this could be the efflux of synovial fluid through the femoral or tibial tunnels into the extraarticular space, therefore decreasing the effusion length of the suture group. The femoral tunnel of those in suture group is perhaps less obstructed than those of other groups (intact animals are without a femoral tunnel; collagen may block the femoral tunnel of other groups) which may allow for increased fluid passage in this particular group. It is also possible that the failure of healing of the ACL in the suture group led to increased stress on the secondary capsular structures, resulting in hypertrophy and contracture of the capsule and elimination of some of the normal joint space. Future studies on the mechanism behind this observed effect are planned.
Synovial reaction and inflammatory changes in the synovium may be more sensitive markers of the immunogenic process than joint effusion. We found no evidence that the addition of a collagen-platelet composite affected the thickness of the synovium or was associated with the development of synovial villi. Nor did we observe increased vascularity or lymphocytic infiltrates when comparing ACL treated with a collagen-platelet composite with those treated with suture repair alone. Other materials that caused immunologic rejection when previously used for ACL replacement, such as Carbon Fiber Ligament [25
], Goretex [28
], or Kennedy LAD [31
] showed considerable to massive monocytic infiltrates.
No differences were seen in synovial fluid between knees or in systemic leukocyte counts over time to suggest significant humoral effects occurring as a result of the implantation of atelocollagen. Contrary to effusion and synovial thickening, leukocyte counts are able to reflect even minor immunological reactions. Although such low-grade reactions might not cause further immune responses, they might very well affect the outcome of the procedure and thus might be a more important endpoint than joint effusion since they are independent from biomechanics. Quite the contrary, any synovial leukocytosis is a highly specific and sensitive sign for intra-articular immunological processes.
We wanted to address the question of whether addition of a platelet concentrate would affect potential immune responses. Platelets, oftentimes in the form of platelet rich plasma, have shown much promise as stimulators of regenerative processes. However, the amply released cytokines that cause the beneficial effects of platelet concentrates might also provoke or amplify immune responses. Despite previous findings of an association between increased platelet concentration and cytokine release [32
], we did not observe an association between any of the endpoints and the presence or absence of PRP. TNF-α levels were similar in both the PRP-exposed and non-exposed groups. However, TNF-α has been reported to be released from platelet-rich fibrin matrices [33
]. Therefore, in the knees where PRP was added, an intrinsic decrease in TNF-α expression by the synoviocytes may have been masked by the TNF-α release from the PRP. Further experiments to determine the geography of TNF-α release from an injured joint are planned.
While we did not observe systemic differences in TNF-α between PRP and non-PRP groups, animals treated with PRP were found to have significantly lower levels of IL-1β than those not treated with PRP. These findings are consistent with prior reports that have shown that PRP does not have a significant effect on TNF-a release [34
], but that PRP does play a role in the suppression of IL-1β release from chondrocytes [35
]. In addition, IL-1β production is stimulated by TNF-α, it is possible that a later increase in IL-1β may follow an earlier increase in TNF-α at time points not studied here. Additional time points and further research are planned to investigate this hypothesis.
In addition, a limitation of this study was that we did not look at the cytokine levels of the synovial fluid inside the joint. Future studies are planned to evaluate this in more depth given the interesting findings here, including the lower synovial fluid WBC in the CPC group. While it can not be stated for certain why the WBC count in the joint was lower for the CPC group than the INTACT group, prior reports state that PRP suppresses the inflammatory cascade [36
] and therefore might have reduced WBC extravasation, a process we are interested in exploring further.
In conclusion, the use of either a collagen sponge or gel made little difference in the resulting changes induced in the joint or surrounding tissues. The reduction of IL1-β with the use of PRP suggests the possibility of a decrease in the occurrence of inflammation with enhanced repair, a preliminary finding that deserves additional study to determine whether suture repair enhanced with a collagen-platelet gel may lead to decreased inflammation in the knee when compared with suture repair alone. These findings suggest that the use of Type I soluble collagen is relatively safe, although additional safety studies in humans will be required if these techniques prove efficacious in animal models.