The objective of this study was to investigate changes in spatial Col1 and Col2 gene expression, tissue morphology and natural healing biomechanics over time following creation of a full-length, central PT injury. The natural healing tissue did not generate normal cellular or matrix organization by 5 weeks post-surgery or normal tissue biomechanics by 8 weeks post-surgery. A typical wound healing response was seen with hemostasis, inflammation, proliferation and remodeling stages.15
We found an influx of inflammatory cells at 1 week consistent with previous work in a lamb central-third PT defect model.16
Also consistent with our study, Sanchis-Alfonso et. al. found a disorganized matrix and hypercellular tissue at 2 weeks post-surgery with increased collagen matrix and reduced cellularity by 3 weeks. Although both studies showed further matrix remodeling and collagen fiber alignment along the primary axis of the tendon between 3 and 5 weeks, our results also revealed inferior repair biomechanics.
Most studies, including the current results, report linear stiffness and ultimate load to failure rather than repair properties in a lower-force, more functional region of loading. We have recorded peak in vivo
forces of 21% and 40% of normal PT failure forces in the rabbit11
models, respectively, for activities of daily living (ADLs) to create benchmarks for our tissue-engineered repairs. Since the extremely small size of the mouse knee prevents us from directly and accurately measuring murine PT forces, we have chosen to apply these design limits across species based on expected ADLs (). 21% of Normal Failure Load (0.88N).
The 2-week natural healing tissue was functionally inadequate as it did not mimic normal tangent stiffness but required an additional displacement of 0.18mm to achieve 0.88N (p<0.05). By 5 weeks, however, the healing tissue was not significantly different than normal (p>0.05). The 8-week defect healing tissue actually matched normal PT tangent stiffness within this range. 40% of Normal Failure Load (1.65N).
The natural healing tissues at all three time points were inadequate requiring at least an additional 0.123mm of displacement (4% strain) to reach 1.65N. In fact, only 33%, 75%, and 69% of the samples at 2-, 5-, and 8-weeks, respectively, exhibited failure forces equal to or higher than this 40% level. This additional analysis further demonstrates the functional inadequacies of adult natural tendon healing in this murine model.
Multiple studies have looked at natural healing of full-length, central PT tendon defects in rabbits,9
with varying biomechanical outcomes. Differences in surgical procedure, mechanical testing and analysis methods can contribute to these differences but other species- or size-related factors may have an even greater impact. We have attempted to compare biomechanical healing between central patellar tendon injuries in the mouse and rabbit, with average life-spans of approximately 2 years and 6–7 years, respectively. To better compare healing between these two species, we normalized time post-surgery to age at the time of surgery. Although it is apparent when mapping percent of normal force vs. normalized healing time () that the mouse heals better and faster than the rabbit, several confounding factors could affect these outcomes. For instance, while the relative defect size compared to the overall tendon volume is similar in both models, the absolute defect volume in the rabbit model is 180X greater than in the mouse. As type-I collagen structure is likely homologous between species, the rabbit must produce and assemble far more ECM, potentially contributing to its slower and less complete healing response. The increased metabolism of the mouse combined with its lower tissue forces could also benefit defect healing. These comparisons are important as researchers attempt to translate tendon healing and tissue engineered findings to larger preclinical models.
Fig. 6 Comparisons of ultimate load, stiffness, ultimate stress, and modulus vs. time post-surgery normalized to the age at surgery showed improved healing in the mouse model vs previous work done in the rabbit central-third defect model.9 Error bars indicate (more ...)
There were limitations in this study. 1) Using a scalpel blade to create the insertional injury did not ensure that the entire insertion was removed. This limitation could have produced inconsistent spatial healing depending on where the scalpel injured the bone. This could also account for the minimal Col2 expression seen at these insertion injuries. We plan to use a surgical burr in future studies to uniformly disrupt the insertion within the central defect. This method of creating a consistent injury between species is necessary as we try to better mimic the bony trough created in our central-third NZW rabbit PT defect model.9, 12, 19
2) Due to the limited size of the tendon, we could not mark the boundaries of the defect during surgery as we did when creating the rabbit PT defect injury.9, 12, 19
Instead, we relied on the discoloration of the defect region when removing the medial and lateral struts during dissection prior to mechanical testing. Any remaining strut on the test specimen could have led to overestimates of actual mechanical properties. 3) Slipping occurred within the patellar grip during the uniaxial failure tests in a limited number of samples. The slippage typically occurred between 1.5N and 2.5N of load. Therefore, displacement and strain at failure were not reported and stiffnesses were calculated from the linear region prior to slipping. 4) Optical strain measurements were not taken during the biomechanical tests, as tissue markers were difficult to apply to the fragile healing tissue. Therefore, average modulus was reported over the full tissue length. Future studies will utilize ink markers to measure local strains.
Given its genetic power, the murine full-length PT defect injury serves as a potent tool for biological and biomechanical study of natural tendon healing. While its limited size may prevent examination of certain novel tissue engineering treatments, advances made in the murine model could conceivably be translated into larger models where repeatable surgeries and design benchmarks are possible. Developing such analogs is particularly attractive if similar biomechanical and/or biological responses to injury can be identified.
Just as functional tissue engineers have been developing mechanical success criteria, corresponding biological success criteria will be needed as well. Murine injury models allow measurement of such biological success criteria by comparing the expression of tenogenic markers in natural healing tissue to normal adult tendon as well as normal tendon during embryonic and early post-natal development. These comparisons are expected to provide multi-functional design benchmarks for tissue-engineered therapies. Such strategies have the potential to promote tenogenesis and zonal insertion regeneration rather than non-functional scar formation and inferior repair biomechanics.