The impact of mild hypercholesterolemia on injury repair in the rat patellar tendon

Hypercholesterolemia is associated with tendon pathology and injury prevalence. Lipids can accumulate in the tendon's extracellular spaces, which may disrupt its hierarchical structure and the tenocytes physicochemical environment. We hypothesized that the tendon's ability to repair after injury would be attenuated with elevated cholesterol levels, leading to inferior mechanical properties. Fifty wild‐type (sSD) and 50 apolipoprotein E knock‐out rats (ApoE−/−) were given a unilateral patellar tendon (PT) injury at 12 weeks old; the uninjured limb served as a control. Animals were euthanized at 3‐, 14,‐ or 42‐days postinjury and PT healing was investigated. ApoE−/− serum cholesterol was double that of SD rats (mean: 2.12 vs. 0.99 mg/mL, p < 0.001) and cholesterol level was related to the expression of several genes after injury; notably rats with higher cholesterol demonstrated a blunted inflammatory response. There was little physical evidence of tendon lipid content or differences in injury repair between groups, therefore we were not surprised that tendon mechanical or material properties did not differ between strains. The young age and the mild phenotype of our ApoE−/− rats might explain these findings. Hydroxyproline content was positively related to total blood cholesterol, but this result did not translate to observable biomechanical differences, perhaps due to the narrow range of cholesterol levels observed. Tendon inflammatory and healing activity is modulated at the mRNA level even with a mild hypercholesterolemia. These important initial impacts need to be investigated as they may contribute to the known consequences of cholesterol on tendons in humans.


| INTRODUCTION
Hypercholesterolemia is a condition characterized by elevated blood cholesterol levels. Once a distinct feature of high-income western countries, acquired high cholesterol is rising in low-and middleincome countries with changes in diet. 1 Hypercholesterolemia can also be genetically inherited (familial) and as prevalent as 1/100. 2 Excess blood cholesterol is a major risk factor for developing cardiovascular disease due to its role in atherosclerosis and is associated with other preventable, age-related diseases (e.g., dementia 3 ) as well as tendon ruptures 4,5 and tendinopathies. 6 Xanthomas-lesions containing lipid-laden macrophages (or "foam cells")-are a physical manifestation of hypercholesterolemia commonly found under the skin and around superficial tendons. 7 Lipid accumulation among collagen fibers and fascicles may disrupt collagen organization within tendon and negatively affect its mechanical properties. 8,9 There is evidence of this type of structure-function relationship in tendinosis 10 and tendinopathy, 11 where expansion of the non-collagenous matrix from elevated proteoglycan content results in reduced stiffness and elastic storage potential, and increased hysteresis. Subclinical xanthomas are found in degenerative tendons, particularly with advancing age. 12 The pathophysiological mechanisms underpinning cholesterol deposition in tendons are not well characterized but are hypothesized to be similar to atherosclerosis. 13 Despite the prevalence of acute and chronic tendon injuries, there has been little research into the mechanisms by which cholesterol weakens tendon, hence their underlying etiology often goes unexplained. 14 Recently, Steplewski et al. 15 discovered that high cholesterol weakens tendons via an unknown mechanism that causes physical damage to collagen fibrils. Hypercholesterolemia may impair tendon healing after an acute injury, 16,17 perhaps from lipid infiltration changing the mechanosensitive environment of tendon cell populations.
Tendon cholesterol may therefore reduce an already slow and poor healing response to injury and subsequently increase the risk of progressive pathology.
We aim to ascertain the influence of hypercholesterolemia on tendon injury and repair using an established rat knock-out model to mimic familial hypercholesterolemia. This model results in defects in apolipoprotein E (ApoE)-a critical protein used for transporting lipoproteins, fat-soluble vitamins, and cholesterol around the body-and brings about mild-moderate hypercholesterolemia without feeding a highfat diet. 18 We will compare cell and tissue mechanobiology in this KO model with wild-type controls to explore the hypotheses that tendons exposed to chronically elevated levels of serum cholesterol will exhibit (a) greater localized levels of cholesterol accumulation, (b) abnormal mechanical properties, and (c) a hampered repair response to injury.
The patellar tendon (PT) was chosen for investigation due to its loadingbearing function, superficial location, and established injury model. 19 It also has a simple form for biomechanical testing and has previously shown evidence of lipid accumulation. 8,20 The information obtained from our studies is of relevance because tendon disorders and injuries are widespread, 21 cause significant pain and disability, and are notoriously complex to fully rehabilitate. Further, an association between cholesterol levels and tendon health is a particular concern, in light of the vicious cycle in which poor mobility and pain 22,23 restricts exercise opportunities for patients, furthering the risk of elevated cholesterol. 24 Our findings will help to substantiate the mechanisms linking cholesterol levels and tendon injury risk, and the consequences of tendon cholesterol on tendon healing.

| METHODS
Animal breeding and experimental procedures were approved by the local Animal Care Committee at the University of British Columbia (protocols #A17-0033 and A16-0256, respectively), and were carried out in accordance with the principles and standards of the Canadian Council on Animal Care. The ARRIVE 2.0 checklist was consulted for transparent and accurate reporting. 25

| Experimental animals
We used young ApoE −/− rats (SD-ApoE tm1sage ; Horizon Discovery), which demonstrate significantly elevated blood lipid profiles without feeding a high fat diet, to examine the effect of high cholesterol on tendon injury and repair processes. Whilst severity of hypercholesterolemia can be increased with feeding a high-fat diet, we wanted to avoid any comorbidities associated with it. Moreover, there is an associated increase in unexpected mortality and significantly reduced lifespan in these animals and so is not recommended. 18 Sprague−Dawley wild-type rats (SD; Charles River Laboratories) were used as control animals. Breeding pairs were cohoused when animals were 24 and 14 weeks, respectively. Rat husbandry was carried out by certified animal laboratory technicians.
Rats were maintained on a 24 h light/dark cycle between 21°C and 24°C. All rats were fed regular chow (5% fat; PicoLab Rodent Diet 20: 5053; LabDiet) for the study duration. Cages contained nesting material and environmental enrichment (plastic tubes, chew toys).
Litters were weaned and sexed at 21 days old and sorted into group housing (2−3 per cage) located in a room separate from the breeders.
Allocation to a postinjury group was completed on a cage-by-cage basis to help balance the male/female ratio at each time-point, and based on surgical suite and researcher scheduling logistics.
We investigated different aspects of tendon healing after injury at 3,-14,-and 42-days postinjury. These timepoints were chosen to target inflammatory, repair and remodeling processes, respectively.
We determined that 20 animals per strain (tissue histology, n = 6; gene expression, n = 4; biomechanics, n = 10) would allow a reasonable investigation of each timepoint. However, only 50 ApoE −/− rats were successfully bred in total (50 SD rats were bred to match) and we omitted the 3-day postinjury timepoint from biomechanical testing, it being the least relevant to our hypotheses. All animals were used in this study; no inclusion or exclusion criteria were established.

| Surgical protocol
All rats underwent a unilateral PT injury surgery at 12 weeks of age; the uninjured limb served as a control. Following UBC standard operating procedures, rats were anesthetized with a mixture of isoflurane (2%−3%) and oxygen (100%) delivered via nose cone, and injected with warmed fluids (sterile lactated ringer's solution) and a preemptive dose of buprenorphine subcutaneously. Animals' ears were notched for identification, the incision area was prepared for surgery and a local analgesic delivered (by R. M.) to the incision site (Bupivacaine 0.25%, 4−8 mg/kg subcutaneous). Rats were kept warm during the procedure via a warming pad. Surgery was conducted aseptically; a 1 cm long incision was made on the medial side of the right knee and lateral displacement of the skin exposed the PT. An iris spatula was inserted behind the PT to provide a backing for a full thickness partial transection to the center of the PT with a 0.75 mm diameter biopsy punch. Skin wounds were closed with subcuticular sutures and surgical glue.
Animals resumed full mobility on regaining consciousness and were allowed to continue normal cage activity until they were killed. Rats were monitored postsurgically and additional doses of analgesic given where necessary. >4% of animals required follow-up sutures after removing their original sutures; these animals were temporarily fitted with an Elizabethan collar. There were no additional complications postsurgery.

| Tissue harvesting
Rats were anaesthetized with a mixture of isoflurane (2%−3%) and oxygen (100%) delivered via nose cone. 0.5 mL of blood was collected in a lithium heparin tube via cardiac puncture just before euthanization with carbon dioxide, with death assured by cervical dislocation. Tendon tissues were harvested immediately thereafter.

| Blood lipids
Immediately after collection, blood samples were centrifuged for 10 min at 2000g to separate plasma, and plasma samples stored at −80°C. Total blood serum cholesterol (free cholesterol and cholesteryl esters) was assessed with a fluorometric cholesterol assay (Invitrogen; cat. no. A12216).

| qPCR
PT tissue was dissected from between the tibial tuberosity and patella with a sterile blade, taking care to remove the surrounding connective tissues and posterior fat pad, and stored short-term at −80°C. For RNA extraction, tendons were powdered in a tissue mill  (Table 1).

| Histology
PT tissue was harvested as described for qPCR and embedded in OCT with the orientation noted. Samples were flash frozen on a liquid nitrogen-cooled metal block and kept at −80°C until required.

| Biomechanics
The tibia-PT-patella complex of both legs were harvested, wrapped in  Figure 2) with some overlap between groups. There were no sex differences in TC within each strain and no obvious macroscopic differences in tissue appearance between groups.

| Histology
There were no apparent histological differences in the PT of ApoE −/− versus SD rats, either in injured or uninjured tendons. Areas of injury were easily identified by increased cellularity, disrupted fibril alignment, and deposition of ground substance (Figure 3). The injury site became less obvious with timepoint progression although there were notable histological differences between injuries. Close inspection of the histology indicated that these differences likely arose from the surgical procedure. In some instances, the biopsy punch fully removed the tissue (hereafter termed "large injury") whilst in others, the tissue remained in place afterwards (hereafter termed "small injury"). An example is shown in Figure 3.

| qPCR
As expected, inflammatory markers COX2 and IL1 were more highly

| Hydroxyproline content
Total hydroxyproline content did not differ significantly between comparable limbs of SD and ApoE −/− rats, or between the uninjured versus injured limbs of each group ( Figure 6A). However, hydroxyproline content was significantly, positively related to total serum cholesterol ( Figure 6B, p = 0.011).

| DISCUSSION
In this study, we investigated the impact of elevated cholesterol levels on tendon healing after an acute injury. We found injury repair at the mRNA level was related to TC level rather than differing between the ApoE −/− rat model and control animals. We did not find differences between rat strains in histology or biomechanical properties. Hydroxyproline content was significantly associated with cholesterol level but did not differ between strains. Our results indicate that mild hypercholesterolemia-rather than rat strain-may impact healing processes but does not significantly impact tendon strength.
The ApoE −/− rats in this study showed elevated blood cholesterol levels compared to SD control rats, but only to about half of that predicted. 18 This resulted in some overlap in cholesterol levels between the ApoE −/− the SD group. A high fat diet would have enhanced the lipid profiles of these animals in the present study 31,32 but at the expense of higher mortality rates and reduced lifespan, 18,32 hence it is not recommended by the supplier.
The relative absence of lipid staining in our histological sections suggests that physical accumulation of lipids in the PT is minimal in ApoE −/− rats, confirming the mild phenotype. Due to these initial findings we found we could not test the hypothesis that tendons  (14, 42 days). No differences between groups or timepoints were found in any variable examined. PT, patellar tendon.
We examined immune cell populations in injured and non-injured tendon tissue due to reports of macrophage polarization in injury repair, atherosclerotic plaque, and xanthoma development. 35,36 CD11b is considered a total macrophage marker 37 and CD206 is, useful for differentiating the pro-healing M2 macrophages. 38 While both markers were identified with IHC, the lack of consistency across timepoints and strains, coupled with unintentionally creating two injury types, makes it impossible to draw conclusions from this finding. Other histochemical methods gave us a sense of total immune cell numbers (which were surprisingly low, even in large injuries) and locations, but examining immune cells in tendons with confirmed intratendinous lipids should be done in future to better gauge their associations with cholesterol levels.
We hypothesized that intratendinous lipid accumulation would alter the tenocyte's local environment and negatively affect the ApoE −/− cellular repair profile, but found no difference in the magnitude or time-course of gene expression compared to SD rats ( Figure 4A). This is likely due to low statistical power and significant within-group data variability. When combining groups by timepoint (which increased our statistical power) we found the expression of inflammatory markers COX-2 and IL1 were significantly correlated with cholesterol levels in early injury repair, such that the inflammatory response was reduced as cholesterol increased ( Figure 4B). Acute inflammation is essential for tendon healing thus a diminished inflammatory response with elevated cholesterol might translate to impaired tissue healing. 39 Although we did not detect impaired healing via histological or biomechanical investigations, the small injury size relative to tendon size may have been insufficient to warrant detectable phenotypic differences resulting from the observed alterations in gene expression.
LOX plays a significant role in tissue repair and remodeling processes, namely by catalyzing the cross-linking of ECM components for tissue stability. 40 Both LDL 41 and oxLDL 42  There are several factors that should be considered when explaining our biomechanical findings. The biopsy punch injury is an accepted and reproducible model for studying basic mechanisms of tendon repair, 19 however the 0.75 mm punch size in relation to the PT width of our rats was perhaps too small to affect tendon biomechanics. Moreover, discovering that the biopsy had not always removed tissue and so created two distinct injury types may have made it more difficult to identify differences between strains.
However, variability seems to be comparable between injured and non-injured tendons, indicating that the different injuries might not cause a higher variation. Other injury/repair models such as a window defect, partial or full transection [45][46][47] may have delivered a more pronounced affect on tendon biomechanics. However, our chosen method did not require suture repair, which might add to or alter the normal biological processes at the injury site (e.g., increased inflammation), nor did it result in unloading from animals avoiding weightbearing on the injured limb. This is particularly important as mechanical loading has been shown to accelerate tendon healing and recovery. 48 We also had a lower-than-expected sample size for properties compared to wild-type controls. 8,49 Young ApoE −/− rats fed a regular diet produced a mild hypercholesterolemia model in our study. Older animals with higher plasma cholesterol 50 and/or a longer exposure to high cholesterol levels 49 may have provided a more moderate hypercholesterolemia model. Certainly other hyperlipidemic models demonstrate moderate or severe phenotypes and evidence of tendon lipid deposits to better reflect FH in humans. 34,51,52 With the availability of genome editing techniques, models of hyperlipidemia for generalizing research results from animals to humans 53 should be taken advantage of in future research.

| CONCLUSION
We found elevated blood cholesterol levels were related to the expression of injury repair genes and may negatively affect tendon repair after injury. Although there was little evidence of lipid accumulation at the tissue level (perhaps due to our young rat cohorts) there was a significant relationship between total blood cholesterol and PT hydroxyproline content. Our results substantiate the link between cholesterol level and tendon injury risk and indicate that even mildly elevated cholesterol levels can modulate tendon inflammatory and healing activity. These impacts need further investigation as they may contribute to the known consequences of cholesterol on tendons in humans.

AUTHOR CONTRIBUTIONS
Conceptualization: Charlie M. Waugh, Alexander Scott, and Hazel R.