The development of comparative animal models to study the biomechanical and biological factors involved in flexor tendon adhesions is important for advancing our understanding of this debilitating problem and for designing therapeutic and rehabilitation treatment programs. A number of elegant studies in multiple human and animal models have identified passive controlled gliding motion as the most important factor in reducing the risk of adhesion formation.3, 15–18
Other studies have focused on molecular treatment of the flexor tendon injury to provide adhesion-free healing via the delivery of anti-scarring adjuvants that inhibit the effects of TGF-β and bFGF among other factors.19–23
Despite their promise, these approaches remain experimental and have yet to yield a clinical application,3
largely because our understanding of the molecular mechanisms involved in the formation of adhesions after flexor tendon injury and grafting remains incomplete.
The novel mouse model of FDL tendon grafts offers a quantitative tool to not only examine the biomechanical aspects of flexor tendon grafts, but also to potentially elucidate the molecular events involved in repair and subsequent adhesion formation via the use of transgenic mouse models of gain and loss of function. However, this model has a number of inherent limitations. The mouse model is admittedly challenging due to the small size of the FDL tendon, which requires microsurgical reconstruction under magnifying lens, however the reproducibility of the data in our study strongly supports the feasibility of this model. In addition, larger animal models (e.g.
canine) that more closely resemble the size and anatomy of human flexor tendons allow for testing the effects of passive motion/loading protocols in reducing adhesions,6, 24, 26
which we were unable to reproduce in this small animal model for obvious technical reasons. Furthermore, while the mouse FDL tendon graft model does not represent a true
zone II reconstruction model, we deliberately immobilized the flexor mechanism by severing the proximal FDL tendon insertion in the flexor muscle to abolish early tendon gliding. This resulted in impairment of MTP joint flexion via mechanisms that have the hallmarks of adhesions including histological evidence of fibrotic scar tissue especially around the live autografts similar to those observed in larger animals such as dogs which have been used for years in flexor tendon repair research.
To quantify the effects of adhesions on the biomechanics of the flexor mechanism in our murine FDL tendon model, we developed an innovative Gliding Coefficient as a measure of the resistance to tendon gliding and MTP joint flexion. Since we did not measure the MTP joint flexion angle by another method that would constitute a golden standard against which to assess the accuracy of the measurements, we computed the intra- and inter-observer reproducibility of the joint angle measurements and determined that the average intra- and inter-observer errors were < 1% which provides confidence about the reproducibility of the MTP joint flexion angle measurements. Furthermore, the MTP joint flexion test is non-destructive and allows for subsequent biomechanical testing of the grafts since the maximum applied excursion load of 19 grams was about 10% of the failure force of the 14 day grafts which was the earliest healing time point we tested.
As a measure of adhesions, previous studies have reported the digital range of motion upon the application of a single defined load to cause tendon excursion.21, 24
By contrast, the Gliding Coefficient is based on joint flexion data over a range of applied loads that would cause a maximum 75° flexion in a normal unoperated MTP joint. The test offers information about the joint ROM (the plateau) and the resistance to flexion with increased loading (the gliding coefficient). The Gliding Coefficient is similar to the Work of Flexion which measures the resistance to flexion over a range of applied excursions. The work of flexion test is feasible in larger animal models that allow flexion testing under displacement control without the risk of damaging the graft. However, due to the small size of the tendon and low levels of force required to effect flexion, our test was conducted under load control to ensure that we do not induce loading that would be damaging to the tendons. There are other advantages to using the Gliding Coefficient rather than reporting a single joint flexion angle. First, if the “single” flexion angle or ROM is incorrectly reported due to measurement error or due to an error in the determination of the neutral position, it would be difficult to observe this error as an outlier. Instead, by recording and plotting the flexion angle over a range of applied loads, and computing the Gliding Coefficient based on the mathematical model as a “rate” constant for joint flexion under controlled loading, we can easily identify those measurements that deviate from the model and provide erroneous estimation of the joint function. Since this is a novel measure to assess the resistance to joint flexion under load control, we examined the correlation between the GC and the maximum MTP joint flexion angle range of motion (ROM) and reported a strong negative correlation (r=−0.97) which corroborates the GC as a measure of the resistance to joint flexion sensitive to the effects of adhesions and less prone to the effects of errors inherent in measuring a single angle as the ROM.
As hypothesized, at 14 and 28 days post-grafting both live autografts and reconstituted freeze-dried allografts had significantly greater Gliding Coefficients and hence more adhesions than normal unoperated tendons or time zero repairs. Interestingly, by 42 days post-operatively and thereafter the Gliding Coefficient was not different than time zero repairs for both autografts and allografts. Histologically, the amount of fibrotic tissue surrounding the 28-day autografts and allografts is markedly reduced by 42 days. There are two possible explanations for these improvements. First, the noted improvement in joint flexion may be a result of the resumption of tendon excursion after the proximal tendon-muscle insertion had been allowed sufficient time to heal and restore the flexor mechanism. This theory is based on the anecdotal observation that the mice more actively used their operated limbs by 28 days. Previous studies have suggested that small flexor tendon excursions following injury may be sufficient for full restoration of the flexion range of motion.24
Regardless, this feature of our model is different than the clinical experience which suggests that the onset of fibrotic adhesions does not resolve spontaneously and might require meticulous tenolysis surgery.3
Second, we hypothesized that the marked increase in the expression of Gdf5
mRNA might be involved in the improvements in joint flexion after 28 days. Whether this increased mRNA expression translates into increased GDF5 and VEGF protein synthesis at the repair site after 28 days remains to be verified in future experiments using immunohistochemistry.
In agreement with the limited data in the literature, we found that reconstituted freeze-dried allografts did not cause increased adhesions compared to live autografts. To the contrary, 28-day live autografts experienced a significant fivefold increase in their Gliding Coefficients compared to the processed allografts. Previous studies compared the healing of flexor tendon autografts and freeze-dried allografts implanted in the paws of dogs and reported that: 1) the implanted allografts were tolerated well by the host; and 2) the implanted allografts allowed flexor tendon function similar to that allowed by autografts.10
Others reported similar observation in bovine flexor tendons suggesting that acellular allografts induced minimal adhesion formation.11, 12
It has been recognized for quite some time that even minor manipulations of a live tendon graft such as the passing of a suture through the tendon induces an “intrinsic” inflammatory stimulation of the resident cells. Since freeze-dried allografts are acellular the intrinsic inflammatory response is not expected. It is conceivable therefore that autograft transplantation may exacerbate the adhesion tissues, presumably resulting from the surgical manipulation of a live graft that might lead to inflammatory stimulation of tenocyte proliferation and migration from the live graft in addition to other intrinsic and extrinsic factors.5
A number of factors may have been responsible for the observation that murine FDL tendon allografts and autografts were similar in terms of their failure tensile properties, but remained significantly weaker than normal unoperated tendons despite modest increases over time. While both grafts initially provided a scaffold to bridge the experimental defect, the two grafts supposedly heal with different mechanisms. Live autografts likely heal via intrinsic and extrinsic mechanisms that involve the graft tenocytes as well as the influx of synovial fibroblasts, precursor cells, and inflammatory cells, respectively.3, 25
As a result, autografts underwent extensive remodeling that negatively affected the rate of accrual of biomechanical strength over time as has been reported for flexor tendon gap defects.26
By contrast, the acellular allografts can only heal by extrinsic mechanisms. Potenza et al demonstrated that extrinsic cells from the synovial capsule of the joint populated and contributed to the healing of lacerations within freeze-dried allografts implanted in canine and rabbit knee joints.27, 28
In our model, we observed modest scarring around the mostly acellular middle segment of the allograft at 14 and 28 days that remained isolated resembling a foreign body response. However at the interface with the host tendon stubs, hypercellular scarring was exuberant in bridging and remodeling the allograft-host juncture, resulting in cellular infiltration into the graft. While the allografts appeared to undergo little remodeling compared to live autografts, the accrual of biomechanical strength was still as slow as live autografts possibly due to the localization of the repair response to the graft ends. In both groups, however, the abolishment of tendon gliding and loading due to the deliberate severing of the proximal tendon-muscle insertion is likely the factor that slows return toward normal biomechanical properties. Therefore, clinical interpretations about the biomechanical equivalency of live autograft and freeze-dried allografts from this model should only be made with the limitations of the model in mind.
Admittedly, the clinical utility of freeze-dried (lyophilized) tendon allografts is debatable. There are clinical reports that suggest that freeze-dried allografts are of no significant value in the surgical management of certain indications such as chronic massive rotator cuff tears,29
and may induce intraarticular reaction when used in ACL reconstruction,30, 31
for example. However, other clinical reports indicate that freeze-dried allografts used for ligament and tendon repairs and arthroscopic reconstruction of ACL deficient knees provide satisfactory clinical results.32, 33
Animal studies including our own data indicate that implanted freeze-dried tendon/ligament allografts are similar in (biomechanical) strength to live tendon/ligament autografts.10
Furthermore, a freeze-dried animal or human tendon rehydrates easily before surgical implantation without adverse effects on their biomechanical properties as we () and others have reported.34
The patterns of growth factor gene expression have been previously described in flexor tendon healing,35–37
but not for autograft and allograft flexor tendon reconstruction models. In our study we evaluated the expression of Tgfb1
, and Vegfa
transcripts on days 14 and 28 post-surgery, which corresponded to the earliest time point where adhesions were observed. We found a twofold increase in these transcript levels (compared to normal tendon) in 28-day allografts. By contrast, the level of Tgfb1
mRNA expression was not upregulated in the live autografts at either 14 or 28 days. It is quite possible that the upregulation in Tgfb1
expression might have been an earlier event in the repair response of autografts and allografts since previous studies suggested that Tgfb1
mRNA levels are nearly 3.5-fold increased in a rabbit flexor tendon healing model as early as 3 days and remain upregulated through 12 days of healing before returning to normal levels at 24 days.35
Interestingly, we observed that the level of expression of Vegfa
was increased by 20-fold in the autografts at 28 days. These observations are somewhat similar to reports that demonstrated that Vegfa
mRNA levels more than double at 7 and 10 days of healing following canine flexor tendon injury.36, 37
The differences in the levels and temporal kinetics of Vegfa
upregulation maybe related to the relative size of the graft compared to primary healing. While local and direct GDF-5 protein delivery on collagen sponge implants has been shown to increase the tensile strength of rat Achilles tendon repair tissues,38
to the best of our knowledge the effects of this growth factor on flexor tendon adhesion formation are unknown. In our model, we observed that Gdf5
mRNA levels were sevenfold increased in 28-day autografts, which experienced the highest levels of adhesions. This increase in Gdf5
transcription was concomitant with the observed increases in VEGF expression. This observation is consistent with recent reports that suggest that GDF-5 promotes angiogenic activity of stromal cells by increasing VEGF gene expression in vitro.39
How GDF-5 and VEGF might be implicated in the observed adhesions in our murine model of flexor tendon grafts remains to be carefully evaluated.
In conclusion, we developed the first murine model of flexor tendon grafts along with an innovative outcome measure for the quantitative assessment of joint flexion function. Despite its limitations, our model has the potential to enable systematic testing of the cellular and molecular events involved in repair and adhesion formation through the utilization of transgenic mouse models in future studies. Furthermore, the model can potentially aid in rapid and inexpensive screening of novel molecular treatments of flexor tendon adhesions.