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Abstract

Purpose:

In the torn rotator cuff muscles, decreased expression of wnt10b prior to elevation of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα) has previously been reported. The purpose of this study is to elucidate the expression profiles of these adipogenesis-related genes after rotator cuff detachment and reattachment in a rabbit model.

Methods:

We investigated gene expression profiles of PPARγ, C/EBPα, and wnt10b in different parts of rabbit supraspinatus (SSP) muscle after tendon detachment (n = 6 for each time point). In addition, we assessed expression of the same genes after SSP reattachment with different intervals from initial detachment (n = 6). Fatty degeneration of the SSP muscle was examined by Oil red-O staining. Gene expression profiles were examined by quantitative real-time polymerase chain reaction.

Results:

After SSP detachment, Oil red-O-positive oil deposits increased after 3 weeks. In the SSP reattachment model, numerous Oil red-O-positive cells were present at 5-week reattachment, following 2- and 3-week detachment. PPARγ and C/EBPα messenger ribonucleic acid expression exhibited a significant increase at 2 and 3 weeks after SSP detachment and remained increased at 5-week reattachment after 2- and 3-week detachment. A decreased expression of wnt10b was observed from 1 week after SSP detachment. Expression of wnt10b was recovered not in the central area of the SSP muscle but in the periphery after reattachment. Adipogenic change was not observed when SSP tendon was reattached after 1-week detachment.

Conclusions:

These results may suggest that once the adipogenic transcription factors, PPARγ and C/EBPα, were elevated, repair surgery after rotator cuff tear could not prevent the emergence of fat in the SSP muscle.

Keywords

C/EBPα fatty degeneration PPARγ rotator cuff wnt10b

Introduction

Rotator cuff tears lead to degeneration of rotator cuff muscles. Degeneration of cuff muscles is often accompanied by deposition of fat, namely fatty degeneration. Severity of fatty degeneration in the rotator cuff muscles correlated with the size and number of tendons ruptured.^1,2 In addition, degrees of fatty degeneration correlated with the time since the beginning of symptoms.² Recent prospective analysis of asymptomatic rotator cuff tears revealed that fatty degeneration was commonly found in degenerative rotator cuff tears with faster enlargement.³ Several lines of evidence indicated that fatty degeneration was one of the prognostic factors of poor recovery after rotator cuff repair.^4
–6 Even though repaired tendon was intact at the time of follow-up, preoperative existence of fatty degeneration was associated with worse outcome.⁷ It is known that fat deposition in the muscle remains unchanged after the repair of rotator cuff tendon. Functional outcome showed improvement after arthroscopic repair of rotator cuff tear with fatty degeneration.⁸ However, fatty degeneration was an irreversible process histologically and radiologically.^9

–12

Transcription factors, peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα), play a critical role in adipogenesis. During adipogenesis, PPARγ and C/EBPα maintain each other’s expression level and activate downstream adipose-specific gene expression.^13,14 A member of wnt gene family, wnt10b, is a secreted signaling factor originally found in human breast cancer.¹⁵ It has been reported that wnt10b keeps preadipocytes undifferentiated and disruption of wnt10b signaling causes transdifferentiation of myoblasts into adipocytes.¹⁶ Muscle-to-fat conversion of muscle satellite cells harvested from Leptin receptor-deficient obese rats also suggested involvement of wnt10b depletion.¹⁷ It is reported that wnt10b inhibits adipogenesis by signals through wnt receptors, Frizzled 1, 2, and/or 5, and co-receptors low-density lipoprotein receptor-related proteins 5 and 6.¹⁸ Expression of wnt10b is upregulated during regenerating process of skeletal muscles possibly due to the inhibition of adipogenic differentiation.¹⁹ Expression levels of wnt10b in the longissimus dorsi muscle of steers showed decreased expression as compared with bulls.²⁰ It may explain that castration of cattle enhances intramuscular fat content of beef through regulation of wnt10b. Itoigawa et al. have examined the gene expression profiles in rotator cuff muscles after experimental resection of tendons.²¹ Elevated expression levels of PPARγ and C/EBPα were confirmed in the supraspinatus (SSP) muscles after rotator cuff resection. Prior to the elevation of these PPARγ and C/EBPα, the expression of wnt10b was decreased in the SSP muscles. Together with the results of an in vitro experiment using cultured myoblasts,^21,22 wnt10b might play a key role in the fatty degeneration of torn rotator cuff muscles. Depletion of wnt10b expression and succeeding elevation of PPARγ and C/EBPα in SSP muscle was suggested as molecular mechanism of fatty degeneration of torn rotator cuff muscles.²¹ Recently, Shah et al. reported that expression of wnt10b in rotator cuff muscles biopsied at the time of repair surgery was negatively correlated with increased tear size.²³ However, the molecular events in the SSP muscle after surgical repair have not been elucidated. An in vitro study exhibited that mechanical stretch enhances the expression of wnt10b and inhibits differentiation of myogenic cells into adipocytes.²² We, therefore, hypothesized that restoration of mechanical stimulation by rotator cuff repair could stimulate the expression of wnt10b and possibly inhibit the elevation of PPARγ and C/EBPα as well as fatty degeneration when repaired after short interval.

In the current study, we first investigated the gene expression profiles of PPARγ, C/EBPα, and wnt10b in different parts of rabbit SSP muscles after tendon detachment. Furthermore, we assessed the same gene expression after rotator cuff repair in different periods from the initial detachment. Fatty degeneration of the SSP muscle was histologically examined by Oil red-O staining.

Methods

Animals

All experimental procedures were approved by the committee of animal experiments at the author’s institution. Seventeen- to nineteen-week-old male Japanese white rabbits were used for this study. All surgical procedures in bilateral shoulders were performed under general anesthesia.

Surgical procedures

Rotator cuff tear and repair models were made based on the previous report from Matsumoto et al.²⁴ In brief, the SSP tendons were exposed by splitting the deltoid muscle. In the rotator cuff tear model, the SSP tendon was transected and detached from its insertion (Figure 1(a)). The proximal stump of the SSP tendon was wrapped with a polyvinylidene fluoride (PVDF) membrane (Durapore7 SVLP, 125 µm thickness, pore size 5 µm, Millipore, Bedford, Massachusetts, USA) to prevent spontaneous reattachment (Figure 1(b)). In the rotator cuff repair model, the SSP tendon, transected and wrapped with PVDF membrane, was reattached to the greater tuberosity at different time points from initial surgery. The PVDF membrane was removed, and a bony trough (2 × 2 × 5 mm³) was made between the articular cartilage of the humeral head and the tuberosity. The tendon stump was pulled into the trough with nonresorbable, monofilament 2-0 nylon (NESCO suture, Alfresa Pharma, Tokyo, Japan) through three drill holes of 1 mm diameter (Figure 1(c)). Experiments for rotator cuff tear and repair were performed in the right shoulder. The left shoulder underwent a sham procedure which included skin incision, deltoid splitting, and closure same as experimental side without cuff detachment or reattachment (control side). After surgery, rabbits were allowed unrestricted activity in the cage.

Figure 1.

(a) Tendon stump of the SSP muscle detached from its insertion. (b) The tendon stump was wrapped with a PVDF membrane to prevent spontaneous reattachment of detached tendon. (c) Five weeks after reattachment of SSP tendon. (d) The SSP muscle of detached side (top) and the SSP muscle of control side (bottom). (e) Schematic drawing of sample harvesting. Central and peripheral areas of distal and middle portion of the SSP muscle were collected. (f) Schematic drawing of study design. (g) Quantification of Oil red-O stained specimen; 12-mm-diameter polypropylene tube was fixed with aqueous mounting medium on the slide. SSP: supraspinatus; PVDF: polyvinylidene fluoride.

Sample harvesting

Rabbits were euthanized with an overdose of sodium pentobarbital. The SSP muscles were excised from the scapula encapsulated by muscle fascia (Figure 1(d)). The weight of the SSP muscles was measured immediately after excision. The most distal 5-mm tendinous part was discarded. The remaining harvested SSP muscles were divided into three portions: distal, middle, and proximal. Five-millimeter cubic muscle tissues sampled from central and peripheral parts of the distal and middle portions were subjected to molecular analysis (Figure 1(e)). Adjacent muscle tissue of the distal portion was used for histological analysis.

Experimental periods

In the rotator cuff tear model, the SSP muscles were harvested at 1, 2, and 3 weeks after surgery (n = 6 for each time point: Figure 1(f)). In the rotator cuff repair model, repair surgery was performed at 1, 2, and 3 weeks after initial surgery. The SSP muscles were harvested 5 weeks after the second surgery (n = 6 for each time point: Figure 1(f)).

Histological evaluation

A small part of the muscle specimen fixed with 10% neutral buffered formaldehyde and embedded in paraffin was sectioned and stained with hematoxylin and eosin. Microscopic images were captured by a digital camera (DP73 Olympus, Tokyo, Japan). The diameter of the muscle fiber was measured using ImageJ software (v.1.48, National Institutes of Health, Maryland, USA). Minimal Feret’s diameter of muscle fiber was considered as the fiber diameter.²⁵

Oil red-O staining and quantification

The central portion of the SSP muscles was diced in 5 mm cubes. The SSP muscle was embedded in OCT compound (Tissue-Tek, Torrance, California, USA) and rapidly frozen in isopentane chilled with liquid nitrogen. Five-micrometer-thick frozen sections were prepared with a cryostat (Bright, UK). The sections were then washed with distilled water and replaced by 60% isopropanol. The sections were stained by the Oil red-O solution (Oil red-O (Sigma, Missouri, USA) in 60% isopropanol) for 30 min and counterstained using hematoxylin. The slides were then thoroughly washed with distilled water. The extent of Oil red-O staining was quantified by Oil red-O elution, which was modified from a previously published method.²⁶ A 10-mm high cylinder was cut out from a 12-mm-diameter round-bottom polypropylene tube using a band saw. This cylinder was placed on the slides surrounding an Oil red-O stained muscle section. The cylinder was fixed in an aqueous mounting medium circumferentially applied for waterproofing. Oil red-O dye was extracted from three sections with 100 µL of 100% isopropyl alcohol placed in the cylinder (Figure 1(g)). Furthermore, its absorbance was measured by a spectrophotometer (Infinite M200, Tecan, Switzerland) at a wavelength of 510 nm.

Quantitative real-time polymerase chain reaction

The SSP muscles were aseptically harvested at each time point and homogenized using POLYTRON (Kineatics, Lucern, Switzerland). Total ribonucleic acid (RNA) was extracted by TRIZOL reagent (Invitrogen, Carlsbad, California, USA) and cleaned using RNeasy Mini Kit (Qiagen, West Sussex, UK) according to the manufacturer’s protocol. First-strand complementary deoxyribonucleic acid (cDNA) was synthesized using high-capacity cDNA archive kit (Applied Biosystems, Carlsbad, California, USA). Quantitative real-time polymerase chain reaction (RT-PCR) was performed using ABI StepOnePlus, Power SYBR Green PCR Master Mix (Applied Biosystems) and 500 nM of each primer, routinely in duplicate. The primers were designed based on the sequences in the GenBank database. The primer pairs used for this study include β-actin: L, 5′-tgggagtgggttgaggtg-3′ and R, 5′-ttattgaactggtctcgtcagtg-3′; wnt10b: L, 5′-ggcaagctggtgagctgt-3′ and R, 5′-gacctgagccgatcctgtt-3′; PPARγ: L, 5′-cccatcgaggacatccag-3′ and R, 5′-ggggtggttcagcttgag-3′; and C/EBPα: L, 5′-ggcagactggagcctctgta-3′ and R, 5′-ctgcttcgcttcgtcctc-3′.

The fractional cycle number at which fluorescence passes the threshold (Ct values) was used for quantification using a comparative Ct method. Sample values were normalized to the threshold value for β-actin: ΔCt = Ct (experiments) − Ct (β-actin). The Ct value of control was used as a reference. ΔΔCt = ΔCt (experiment) − ΔCt (control). The fold change in messenger RNA (mRNA) expression was calculated by the formula 2^−ΔΔCt.

Statistics

Paired student’s t-test was used to determine significant differences between experimental and sham-operated shoulders. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used for comparison between experimental shoulders at different times. Results are presented as mean values and the standard deviation. A value of p < 0.05 was considered statistically significant.

Results

Muscle weight and fiber diameter of the SSP muscles

To validate the effects of cuff tear on muscle volume and its properties, wet weight and fiber diameter of the SSP muscles were measured after experiments. Wet weight of the SSP muscles was significantly decreased by detachment of the tendon. The rate of reduction after detachment of the tendon was time-dependently increased. In addition, wet weight of the SSP muscles was decreased 5 weeks after reattachment surgery. The diameter of muscle fibers was decreased 2 and 3 weeks after detachment of SSP tendons. After reattachment surgery, the diameter of the muscle fibers was decreased compared with the controls in each time period. Infiltration of small mesenchymal-like cells was observed adjacent to the intramuscular tendon by histological examination (Figure 2).

Figure 2.

(a to e) Axial section of the SSP muscle. At 1 week (1 week) after detachment of the SSP tendon (b) and 5-week reattachment following 1-week detachment (d: 1 + 5 weeks), infiltration of small mesenchymal cells was observed adjacent to the intramuscular tendon. At 3 weeks (3 weeks) after detachment of SSP tendon (c) and 5-week reattachment, following 3-week detachment (e: 1 + 3 weeks), adipose tissue was observed adjacent to the intramuscular tendon. (f) Wet weight of the SSP muscle. (g) Mean of muscle fiber diameters (n = 6). *p < 0.05; **p < 0.01. SSP: supraspinatus.

Fatty degeneration of the SSP muscle in the rotator cuff tear model

In the rotator cuff tear model, the Oil red-O staining of frozen section showed positively stained adipose cells especially at 3 weeks after detachment. The quantification of oil deposit exhibited significant increase at 3 weeks after detachment (Figure 3). In the gene expression profiles of the SSP muscles examined by RT-PCR at 1, 2, and 3 weeks after detachment, a decreased expression of wnt10b in the cuff tear side was observed from 1 to 3 weeks in both central and peripheral areas of distal portions. PPARγ mRNA expression exhibited significant increase at 2 and 3 weeks after detachment in the peripheral and central areas of the distal portion and at 3 weeks in central area of the middle portion. PPARγ mRNA expression at 2 and 3 weeks after detachment showed significant increase when compared to 1 week after detachment in both central and peripheral areas in distal portion. C/EBPα mRNA expression showed significant increase at 2 weeks after detachment in the central and peripheral area and 3 weeks in central area of the distal portion. C/EBPα mRNA expression at 2 and 3 weeks after detachment showed significant increase when compared to 1 week after detachment in central area of distal portion. There was no significant difference in wnt10b and C/EBPα mRNA expression in the middle portion (Figure 4).

Figure 3.

(a) Oil red-O staining of frozen section. Positive Oil red-O staining was clearly observed at 3 weeks after detachment of the SSP tendon (3 weeks). (b) Quantification of lipid measured by elution of Oil red-O staining. Significant difference between experimental and sham-operated shoulders was confirmed at 3 weeks after detachment. Significant increase was found between 2 and 3 weeks after detachment by one-way ANOVA (n = 6). *p < 0.05. ANOVA: analysis of variance; SSP: supraspinatus.

Figure 4.

Quantitative RT-PCR examination of (a) wnt10b, (b) PPARγ, and (c) C/EBPα gene expression. The middle portion of the SSP muscles did not show significant change. In the distal portion of the SSP muscles, the expression of wnt10b was decreased at each time point of observation. PPARγ and C/EBPα were increased at 2 and 3 weeks after detachment when comparing to sham-operated shoulders. Significant difference between different time points was also shown (n = 6). *p < 0.05; **p < 0.01. RT-PCR: real-time polymerase chain reaction; PPARγ: peroxisome proliferator-activated receptor γ; C/EPBα: CCAAT/enhancer-binding protein α; SSP: supraspinatus.

Fatty degeneration of the SSP muscle in the rotator cuff repair model

In the rotator cuff repair model, numerous adipose cells were Oil red-O positive on frozen sections from samples at 5-week reattachment after 2- and 3-week detachment. The quantification of oil deposits exhibited significant increase in lipids at 5-week reattachment after 2- and 3-week detachment when comparing to sham-operated shoulders. When comparing between different time points, oil deposit was significantly increased in 5-week reattachment after 2- and 3-week detachment than 5-week reattachment after 1-week detachment (Figure 5). The gene expression profiles in the distal portion of the SSP muscles were examined by RT-PCR. Depletion of wnt10b was observed in the central area at 5-week reattachment after 1-, 2-, and 3-week detachment. Elevation of PPARγ and C/EBPα mRNA expression in the detached muscles was observed in both central and peripheral areas at 5-week reattachment after 2- and 3-week detachment. Comparison between different time points exhibited significant increase of PPARγ and C/EBPα mRNA expression in peripheral area between 1- and 2-week detachment followed by 5-week reattachment (Figure 6).

Figure 5.

(a) Oil red-O staining of frozen section. Remarkable Oil red-O staining was observed at 2- and 3-week detachment, followed by 5-week reattachment of the SSP tendon. (b) Quantification of lipid measured by elution of Oil red-O staining. A significant increase was confirmed at 2- and 3-week detachment, followed by 5-week reattachment. Significant increase between different time points by one-way ANOVA was also shown (n = 6). **p < 0.01. ANOVA: analysis of variance; SSP: supraspinatus.

Figure 6.

Quantitative RT-PCR examination of (a) wnt10b, (b) PPARγ, and (c) C/EBPα gene expression. In the central area of distal portion of rabbit SSP muscle, the expression of wnt10b was decreased at each time point of observation. PPARγ and C/EBPα were increased at 2- and 3-week detachment, followed by 5-week reattachment. Significant difference between different time points was also shown (n = 6). *p < 0.05; **p < 0.01. RT-PCR: real-time polymerase chain reaction; PPARγ: peroxisome proliferator-activated receptor γ; C/EPBα: CCAAT/enhancer-binding protein α; SSP: supraspinatus.

Discussion

Our investigation on rabbit SSP muscles after rotator cuff detachment demonstrated an emergence of oil deposits in the muscle and elevation of the adipogenic transcription factors, PPARγ and C/EBPα. The expression of wnt10b showed significant depletion prior to the change of histology and adipogenic transcription factors. These results from the rabbit experimental model were consistent with the results from a rat model previously reported by Itoigawa et al.²¹ According to our findings in tendon repair model, numerous oil deposits were observed in the SSP muscles at 5 weeks after repair, following detachment for 2 and 3 weeks. In addition, the expression of PPARγ and C/EBPα was significantly increased in these samples. The expression of wnt10b remained decreased 5 weeks after repair in the central area of the distal portion of the SSP muscles. Significant fatty degeneration was undetectable if the tendon was repaired after 1-week detachment when adipogenic transcription factors had not yet elevated. These results suggest that elevation of adipogenic transcription factors indicates the fate of fatty degeneration in the SSP muscles. Shah et al.²³ investigated gene expression profiles of adipogenic transcription factors and wnt10b in torn human rotator cuff tears, which are consistent with our results. Frey et al.²⁷ examined adipogenic gene expression in rotator cuff muscle of the sheep after tendon tear. PPARγ and C/EBPα expression started increasing at 12 and 13 weeks after the tear, respectively. Larger animals may exhibit delayed responses of adipogenic transcription factors after rotator cuff tear. In other words, there seems to be a time threshold of tendon detachment for irreversibility of fatty degeneration, which may or may not be specific to animal species. Further analyses on gene expression profiles of PPARγ, C/EBPα, and wnt10b in human torn rotator cuff muscles may elucidate appropriate time for surgical rotator cuff repair.

Rabbits were used as experimental model in this study. To date, several animal models have been developed to investigate the pathophysiology of rotator cuff tears.^28,29 Kovacevic et al.³⁰ reported the rat surgical rotator cuff repair model. In most of reported surgical repair models, rats underwent acute repair of rotator cuff tendon. In animal models, it is well known that detached rotator cuff tendons are spontaneously reattached. Reattachment surgery models after certain cuff tear period could have a possibility of loss of discontinuity of rotator cuff during waiting period. Matsumoto et al.²⁴ wrapped the detached rotator cuff tendon of rabbits with PVDF membrane to prevent spontaneous reattachment. Because of small size, the rat models with transection, prevention of spontaneous reattachment by wrapping with membrane and delayed repair of rotator cuff tendon are technically demanding and prone to low reproducibility. Rabbits provided an excellent model for histological and biomechanical examination^28,29; thus, several researchers have utilized rabbit models to study events after rotator cuff repair.^24,31,32

Rotator cuff tears have a substantial impact on cuff muscles. In addition, we examined muscle weight of the SSP muscles after detachment and reattachment. Muscle weight of the SSP muscles failed to indicate recovery 5 weeks after reattachment. The area of cuff muscles examined by computed tomography (CT) in a sheep cuff tear model³³ and the volume of SSP in the rabbit tear model²⁹ also indicated prolonged atrophy even after reattachment of SSP tendons. Although the observation period after reattachment was shorter in our experiment than in these previous studies, weight of the SSP muscle showed similar tendencies.

Previous studies indicated that fatty degeneration was mostly located in the musculotendinous junction.²¹ In the rat model, more oil deposits were observed in the distal portion of the SSP muscle than in the middle portion.²¹ In the current rabbit model study, we examined the localization of the fatty degeneration in detail. The central area of the distal SSP muscles contains intramuscular tendons and surrounding muscle tissues, although the peripheral area almost completely consists of muscles. Our histological findings showed emergence of oil deposits mainly in central area of the distal SSP muscles. The reason why emergence of adipocytes was localized to the central area of the distal SSP muscles remains unknown. We considered that adipocytes in muscle tissue had risen from stem cells that reside in the muscle. A study using electron microscope exhibited that satellite cells are uniformly distributed throughout the whole muscle.³⁴ Davies et al.³⁵ reported transient activation of muscle stem cell in the murine rotator cuff muscles after tendon transection. They, however, did not examine the distribution of stem cells in the muscle. Injury of muscle fibers which cause activation of muscle stem cells was mostly observed in distal portion of rotator cuff muscles after reattachment surgery.³⁶ Further study will be required to reveal the distribution of adipocyte-inducing signals and stem cell source in the rotator cuff muscles with torn tendon.

The expression levels of wnt10b remained decreased after reattachment surgery in the central area of the distal SSP muscles. Expression of wnt10b in the central area was higher in 1-week detachment, followed by 5-week reattachment compared with 2- and 3-week detachments. Oil red-O staining and adipogenic markers were not significantly changed in 1-week detachment, followed by 5-week reattachment. There may be a threshold in the wnt10b depletion level and duration, which initiates the successive change of adipogenic markers. Peripheral areas of the SSP muscles after reattachment did not exhibit significant change in wnt10b expression level compared with the control side. We examined only mRNA expression of wnt10b, which is secreted as a signal protein. Although the spatial effectiveness of the wnt10b signal is not well known, the total amount of wnt10b protein expression in the muscle may be recovered by the repair of the SSP muscle tendon in samples from 1-week detachment, followed by 5-week reattachment.

The extent of fatty degeneration was quantified by elution of Oil red-O staining of the frozen sections. To date, this method was applied for the quantification of adipogenesis on the culture dish.²⁶ To the best of our knowledge, this is the first study to utilize this method for quantification of adipogenesis in histological sections. We standardized the volume of sections by dissecting the same cubic size of muscle and using fixed-thickness frozen section. CT images have been used for the measurement of fatty degeneration of experimental animal models. However, our method could be easily measured with a spectrophotometer, although there is a possibility of measurement error as that with any other method. Howald et al.³⁷ described two types of lipid accumulations in the muscle, intramyocellular lipid (IMCL) and extramyocellular lipid (EMCL). The lipid droplets of IMCL are located in close contact with the mitochondria, which resides between myofibrils in the myotubes. Free fatty acid in IMCL is readily available as the energy source of muscle contraction. IMCL is quite tiny droplets which require an electron microscope to detect in non-pathologic condition. Conversely, EMCL is a lipid droplet in the adipocytes, which is characterized by a large lipid droplet occupying the most of cytoplasm. The rotator cuff tear possibly increases IMCL in the cuff muscle as well as EMCL. In addition, lipid-laden macrophages³⁸ and non-cell-based lipid accumulation can be found in the muscle with rotator cuff tears. Elution with Oil red-O probably evaluated all those types of fatty accumulation in the rotator cuff muscles.

Muscle fiber diameters were decreased in 1-week detachment followed by 5 week-reattachment compared with 1-week detachment. At the time of reattachment surgery, the stump of the detached SSP tendon was pulled into the trough made between humeral head cartilage and greater tuberosity. This procedure possibly elongated the length of the SSP muscle fibers and increased its muscular tension. Pennation angle of rotator cuff muscle fibers thought to be increased after rotator cuff tear by retraction³⁹ and could be decreased by elongation of cuff muscles. Decreased pennation angle of fiber muscle after the reattachment surgery may be responsible for the decreased muscle fiber diameter. Gene expression of wnt10b in central and peripheral areas of the SSP muscle showed different response after reattachment. The wnt10b expression was recovered in peripheral area, whereas it remained depleted in central area. A previous in vitro study suggested the relationship between wnt10b expression and mechanical stimulation.²² Elongation of SSP by reattachment might result in the difference of stress distribution and mechanical loading environment in SSP muscles. Correlation between tear size and repair tension, which affects healing of repair site, has been reported.⁴⁰ However, detailed distribution of mechanical stress in the muscle after tendon repair remains unelucidated. Muscle fiber tension could be associated with size of tear and adaptation of muscle sarcomere length after injury.⁴¹

The severity of the full-thickness rotator cuff tears was classified by the size and number of involved tendons: small, medium, large, and massive.⁴² In large and massive tears, the SSP muscle and tendon completely lost connection with the humeral head. Experimental animal cuff tear models, including our rabbit model, mimicked a large tear. From the results in the current study, the progression of fatty degeneration of the SSP muscle could not be ceased by surgical repair of the rotator cuff tendon once the adipogenic marker had been changed. It may be recommended that surgical repair of the torn rotator cuff tendon should be performed as soon as possible to avoid fatty degeneration of cuff muscles.

There are several limitations to our study. We only examined gene expression of wnt10b. Protein expression of wnt10b was not examined in this study. The length of observation after surgical repair was only 5 weeks after reattachment surgery. Trudel et al.⁴³ observed fatty degeneration of the SSP tendon 12 weeks after reattachment surgery using a rabbit model. Even after 12 weeks, there was distinct fatty degeneration. In our preliminary experiment, rotator cuff repair model with 3- and 12-week reattachment after 3-week detachment showed similar extent of fatty degeneration in the SSP muscle. It is less likely that prolonged periods of observation after reattachment surgery will show decreased fatty degeneration. In clinical settings, rehabilitation has importance in the results of cuff repair surgery,^44

–47 but it is very difficult to evaluate the effect of rehabilitation after shoulder surgery using animal models. In addition, rotator cuff tears often occur in the degenerated tendon of elderly people. Furthermore, a sharply cut tendon stump in the young rabbit model may not be representative of that in clinical cases.

Conclusion

In conclusion, once the adipogenic transcription factors, PPARγ and C/EBPα, were elevated, repair surgery after rotator cuff tear could not prevent the emergence of fat in the SSP muscle. The expression of wnt10b decreased prior to the alteration of PPARγ and C/EBPα and recovered by tendon repair in the peripheral area of the SSP muscle.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Koshi N Kishimoto

References

Barry

Cheung

Lansdown

et al. The relationship between tear severity, fatty infiltration, and muscle atrophy in the supraspinatus. J Shoulder Elbow Surg 2013; 22: 18–25.

Melis

Nemoz

Walch

Muscle fatty infiltration in rotator cuff tears: descriptive analysis of 1688 cases. Orthop Traumatol Surg Res 2009; 95: 319–324.

Herbert-Davis

Teefey

Steger-May

et al. Progression of fatty muscle degeneration in atraumatic rotator cull tears. J Bone Joint Surg Am 2017; 99: 832–839.

Fermont

AJM

Wolterbeek

Wessel

et al. Prognostic factors for successful recovery after arthroscopic rotator cuff repair: a systematic literature review. J Orthop Sports Phys Ther 2014; 44(3): 153–163.

McElvany

McGoldrick

Gee

et al. Rotator cuff repair: published evidence on factors associated with repair integrity and clinical outcome. Am J Sports Med 2014; 43(2): 491–500.

Mall

Tanaka

Choi

et al. Factors affecting rotator cuff healing. J Bone Joint Surg Am 2014; 96(9): 778–788.

Ohzono

Gotoh

Nakamura

et al. Effect of preoperative fatty degeneration of the rotator cuff muscles on the clinical outcome of patients with intact tendons after arthroscopic rotator cuff repair of large/massive cuff tear. Am J Sports Med 2017; 45(13): 2975–2981.

Burkhart

Barth

Richards

et al. Arthroscopic repair of massive rotator cuff tears with stage 3 and 4 fatty degeneration. Arthroscopy 2007; 23(4): 347–354.

Goutallier

Postel

Bernageau

et al. Fatty muscle degeneration in cuff ruptures: pre- and postoperative evaluation by CT scan. Clin Orthop Relat Res 1994; 304: 78–83.

10.

Fuchs

Gilbart

Hodler

et al. Clinical and structural results of open repair of an isolated one-tendon tear of the rotator cuff. J Bone Joint Surg Am 2006; 88(2): 309–316.

11.

Jost

Zumstein

Pfirrmann

et al. Long-term outcome after structural failure of rotator cuff repairs. J Bone Joint Surg Am 2006; 88(3): 472–479.

12.

Liem

Lichtenberg

Magosch

et al. Magnetic resonance imaging of arthroscopic supraspinatus tendon repair. J Bone Joint Surg Am 2007; 89(8): 1770–1776.

13.

Shao

Lazar

. Peroxisome proliferator activated receptor γ, CCAAT/enhancer-binding protein α, and cell cycle status regulate the commitment to adipocyte differentiation. J Biol Chem 1997; 272: 21473–21478.

14.

Rosen

Hsu

Wang

et al. C/EBPα induced adipogenesis through PPARγ: a unified pathway. Genes Dev 2002; 16: 22–26.

15.

Bui

Rankin

Smith

et al. A novel human Wnt gene, WNT10B, maps to 12q13 and is expressed in human breast carcinomas. Oncogene 1997; 14: 1249–1253.

16.

Ross

Hemati

Longo

et al. Inhibition of adipogenesis by wnt signaling. Science 2000; 289: 950–952.

17.

Scarda

Franzin

Milan

et al. Increased adipogenic conversion of muscle satellite cells in obese Zucker rats. Int J Obes 2010; 34: 1319–1327.

18.

Bennett

Ross

Longo

et al. Regulation of Wnt signaling during adipogenesis. J Biol Chem 2002; 277(34): 30998–31004.

19.

Wagatsuma

. Adipogenic potential can be activated during muscle regeneration. Mol Cell Biochem 2007; 304(1–2): 25–33.

20.

Jeong

Kim

Nguyen

et al. Wnt/β-catenin signaling and adipogenic genes are associated with intramuscular fat content in the longissimus dorsi muscle of Korean cattle. Anim Genet 2013; 44(6): 627–635.

21.

Itoigawa

Kishimoto

Sano

et al. Molecular mechanism of fatty degeneration in rotator cuff muscle with tendon rupture. J Orthop Res 2011; 29(6): 861–866.

22.

Akimoto

Ushida

Miyake

et al. Mechanical stretch inhibits myoblast-to-adipocyte differentiation through Wnt signaling. Biochem Biophys Res Commun 2005; 329: 381–385.

23.

Shah

Kormpakis

Cavinatto

et al. Rotator cuff muscle degeneration and tear severity related to myogenic, adipogenic, and atrophy genes in human muscle. J Orthop Res 2017; 35(12): 2808–2814.

24.

Matsumoto

Uhthoff

Trudel

et al. Delayed tendon reattachment does not reverse atrophy and fat accumulation of the supraspinatus—an experimental study in rabbits. J Orthop Res 2002; 20: 357–363.

25.

Briguet

Courdier-Fruh

Foster

et al. Histological parameters for the quantitative assessment of muscular dystrophy in the mdx-mouse. Neuromuscul Disord 2004; 14: 675–682.

26.

Ramírez-Zacarías

Castro-Muñozledo

Kuri-Harcuch

. Quantification of adipose conversion and triglycerides by staining intracytoplasmic lipids with Oil red O. Histochemistry 1992; 97(6): 493–497.

27.

Frey

Regenfelder

Sussmann

et al. Adipogenic and myogenic gene expression in rotator cuff muscle of the sheep after tendon tear. J Orthop Res 2009; 27(4): 504–509.

28.

Soslowsky

Carpenter

DeBano

et al. Development and use of animal model for investigations on rotator cuff disease. J Shoulder Elbow Surg 1996; 5: 383–392.

29.

Edelstein

Thomas

Soslowsky

. Rotator cuff tears: what have we learned from animal models? J Musculoskelet Neuronal Interact 2011; 11(2): 150–162.

30.

Kovacevic

Gulotta

Ying

et al. rhPDGF-BB promotes early healing in a rat rotator cuff repair model. Clin Orthop Relat Res 2015; 473(5): 1644–1654.

31.

Uhthoff

Matsumoto

Trudel

et al. Early reattachment does not reverse atrophy and fat accumulation of the supraspinatus—an experimental study in rabbits. J Orthop Res 2003; 21: 386–392.

32.

Gupta

Lee

. Contributions of the different rabbit models to our understanding of rotator cuff pathology. J Shoulder Elbow Surg 2007; 16: 149–157.

33.

Gerber

Meyer

Schneeberger

et al. Effect of tendon release and delayed repair on the structure of the muscles of the rotator cuff: an experimental study in sheep. J Bone Joint Surg Am 2004; 86A(9): 1973–1982.

34.

Snow

. Satellite cell distribution within the soleus muscle of the adult mouse. Anat Rec 1981; 201(3): 463–469.

35.

Davies

Garcia

Tamaki

et al. Muscle stem cell activation in a mouse model of rotator cuff injury. J Orthop Res 2017; 36(5): 1370–1376.

36.

Davis

Stafford

Jergenson

et al. Muscle fibers are injured at the time of acute and chronic rotator cuff repair. Clin Orthop Relat Res 2015; 473: 226–232.

37.

Howald

Boesch

Kreis

et al. Content of intramyocellular lipids derived by electron microscopy, biochemical assays, and ¹H-MR spectroscopy. J Appl Physiol 2002; 92(6): 2264–2272.

38.

Mendias

Roche

Harning

et al. Reduced muscle fiber force production and disrupted myofibril architecture in patients with chronic rotator cuff tears. J Shoulder Elbow Surg 2015; 24(1): 111–119.

39.

Meyer

Hoppeler

von Rechenberg

et al. A pathomechanical concept explains muscle loss and fatty muscular changes following surgical tendon release. J Orthop Res 2004; 22(5): 1004–1007.

40.

Kim

Jang

Choi

et al. Evaluation of repair tension in arthroscopic rotator cuff repair. Am J Sports Med 2016; 44(11): 2807–2812.

41.

Gibbons

Sato

Bachasson

et al. Muscle architectural changes after massive human rotator cuff tear. J Orthop Res 2016; 34(12): 2089–2095.

42.

Snyder

Pachelli

Del

et al. Partial thickness rotator cuff tears: results of arthroscopic treatment. Arthroscopy 1991; 7(1): 1–7.

43.

Trudel

Ryan

Rakhra

et al. Muscle tissue atrophy, extramuscular and intramuscular fat accumulation, and fat gradient after delayed repair of the supraspinatus tendon: a comparative study in the rabbit. J Orthop Res 2012; 30: 781–786.

44.

Keener

. Revision rotator cuff repair. Clin Sports Med 2012; 31(4): 713–725.

45.

Baumgarten

Vidal

Wright

. Rotator cuff repair rehabilitation: a level I and II systematic review. Sports Hearth 2009; 1(2): 125–130.

46.

Koo

Burkhart

. Rehabilitation following arthroscopic rotator cuff repair. Clin Sports Med 2010; 29(2): 203–211.

47.

Blislin

Field

Savoie

. Complications after arthroscopic rotator cuff repair. Arthroscopy 2007; 23(2): 124–128.

Fatty degeneration and wnt10b expression in the supraspinatus muscle after surgical repair of torn rotator cuff tendon

Abstract

Purpose:

Methods:

Results:

Conclusions:

Keywords

Introduction

Methods

Animals

Surgical procedures

Sample harvesting

Experimental periods

Histological evaluation

Oil red-O staining and quantification

Quantitative real-time polymerase chain reaction

Statistics

Results

Muscle weight and fiber diameter of the SSP muscles

Fatty degeneration of the SSP muscle in the rotator cuff tear model

Fatty degeneration of the SSP muscle in the rotator cuff repair model

Discussion

Conclusion

Footnotes

Declaration of conflicting interests

Funding

ORCID iD

References