Cellular and morphological changes with EAA supplementation before and after total knee arthroplasty

Abstract

Investigate the underlying cellular basis of muscle atrophy (Placebo) and atrophy reduction (essential amino acid supplementation, EAAs) in total knee arthroplasty (TKA) patients by examining satellite cells and other key histological markers of inflammation, recovery, and fibrosis. Forty-one subjects (53-76 yr) scheduled for TKA were randomized into two groups, ingesting 20 g of EAAs or placebo, twice-daily, for 7 days before TKA and for 6 wk after surgery. A first set of muscle biopsies was obtained from both legs before surgery in the operating room, and patients were randomly assigned and equally allocated to have two additional biopsies at either 1 or 2 wk after surgery. Biopsies were processed for gene expression and immunohistochemistry. Satellite cells were significantly higher in patients ingesting 20 g of essential amino acids twice daily for the 7 days leading up to surgery compared with Placebo (operative leg P = 0.03 for satellite cells/fiber and P = 0.05 for satellite cell proportions for Type I-associated cells and P = 0.05 for satellite cells/fiber for Type II-associated cells.) Myogenic regulatory factor gene expression was different between groups, with the Placebo Group having elevated MyoD expression at 1 wk and EAAs having elevated myogenin expression at 1 wk. M1 macrophages were more prevalent in Placebo than the EAAs Group. IL-6 and TNF-α transcripts were elevated postsurgery in both groups; however, TNF-α declined by 2 wk in the EAAs Group. EAAs starting 7 days before surgery increased satellite cells on the day of surgery and promoted a more favorable inflammatory environment postsurgery.NEW & NOTEWORTHY Clinical studies by our group indicate that the majority of muscle atrophy after total knee arthroplasty (TKA) in older adults occurs rapidly, within the first 2 wks. We have also shown that essential amino acid supplementation (EAAs) before and after TKA mitigates muscle atrophy; however, the mechanisms are unknown. These results suggest that satellite cell numbers are elevated with EAA ingestion before surgery, and after surgery, EAA ingestion positively influences markers of inflammation. Combined, these data may help inform further studies designed to address the accelerated sarcopenia that occurs in older adults after major surgery.

Keywords: M1 and M2 macrophage; atrophy; clinical trial; muscle stem cell; sarcopenia.

Conflict of interest statement

No conflicts of interest, financial or otherwise, are declared by the authors.

Figures

Fig. 1.

CONSORT diagram. CONSORT flow diagram showing the numbers of patients who were randomly assigned to each treatment group, who were excluded or withdrew from the study, and who were included in the analysis.

Fig. 2.

Study design. Bilateral biopsies were obtained in the operating room, before total knee arthroplasty (TKA) surgery began, after 7 days of twice-daily ingestion of 20 g of essential amino acid supplement (EAA) or placebo, and at 1 and 2 wk post-TKA (random allocation). Supplements were not taken on the day of surgery, but continued the next day through the end of the trial at 6 wk post-TKA. The rationale for post-TKA biopsy time points was to determine the cell level changes occurring at week 1 and week 2 after TKA, when MRI data from our previously published clinical trial showed that 80% or more of the total muscle atrophy measured at week 6 after TKA. Additionally, the parent study included analysis of functional outcomes, blood analysis, and MRI analysis for 6 wk following surgery, but the results of these measures have been or will be reported in other articles.

Fig. 3.

Group activity levels. The histogram shows change in daily calories expended in physical activity for individuals in the EAA and Placebo Groups from presurgery to 2- and 6 wk postsurgery. The histogram presents mean values by group at the three time points.

Fig. 4.

Satellite cell content in vastus lateralis tissue of study patients. Representative images from vastus lateralis biopsy sections of EAA (A–C) and Placebo (D–F) Group subjects. Scale bars indicate 150 µm. A and D: satellite cells labeled with anti-Pax7+ (green), fiber types labeled with anti-MyHC Type I (red), and nuclei labeled with DAPI (blue). B and E: Pax7+ cells alone. C and F: expanded view of the lower right corner of the adjacent images with Pax7+ (green), the extracellular matrix surrounding each muscle cell labeled with anti-laminin (blue), and MyHC1 (red). Arrows indicate Pax7+ cells associated with Type I fibers. Arrowheads show Pax7+ cells associated with Type II fibers. G: histograms of satellite cells/fiber in the EAA versus Placebo groups at presurgery versus combined 1- and 2 wk postsurgery in the operative and nonoperative legs. Histograms show satellite cells per Type I fiber in the operative leg (H) and nonoperative leg (I) presurgery and at 1 and 2 wk postsurgery. Histograms show satellite cells per Type II fiber in the operative leg (J) and nonoperative leg (K) leg at baseline and at 1 and 2 wk. L: histograms of satellite cell proportions in the EAA versus Placebo groups presurgery versus combined 1 and 2 wk postsurgery in the operative and nonoperative legs. Histograms show satellite cell proportions per Type I fibers in the operative leg (M) and the nonoperative leg (N) presurgery and at 1 and 2 wk postsurgery. Histograms show satellite proportions per Type II fiber in the operative leg (O) and nonoperative leg (P) presurgery and at 1 and 2 wk postsurgery. *Significance (P) values represent the treatment group main effect in separate analyses of (co)variance. Presurgery values were covaried in models evaluating postsurgery treatment group differences. Values denoted with a # symbol trended toward significance (had a P value between 0.051 and 0.09).

Fig. 5.

Macrophage content in vastus lateralis tissue of study patients. Representative images of tissue sections from EAA (A–C) and Placebo (D–F) study subjects. Scale bar indicates 150 µm. In A and D, M1 macrophages are labeled with anti-cd86 (green). M2c macrophages are labeled with anti-cd163 (blue). The extracellular matrix is labeled with anti-laminin2 (red). In B and E, cd86-positive staining is shown. In C and F, cd163 is shown. G: histogram showing the changes in M1 area normalized to the area of the extracellular matrix marked with laminin. Black bars indicate average area of the baseline time point, and white bars indicate the combined average of the 1- and 2-wk time points. H: histogram showing the changes in M1 area normalized to the area of the extracellular matrix marked with laminin. Black bars indicate the average area of the baseline time point, and white bars indicate the combined average of the 1- and 2-wk time points. I: ratio of M1 to M2c. J: representative image used for assessment of fibrotic tissue, with TE7 (red), desmin (blue), and laminin (green). K: histogram showing measurements of TE7 area in the various experimental conditions. *Significance (P) values represent the treatment group main effect in separate analyses of (co)variance. Presurgery values were covaried in models evaluating postsurgery treatment group differences. Values denoted with a # symbol trended toward significance (had a P value between 0.051 and 0.099).

Fig. 6.

NCAM+ fibers, TUNEL and central nuclei assessment in vastus lateralis biopsies of study subjects. A: representative image of NCAM+ fibers (green; positive fibers are marked with an *). The image is costained with laminin (red) and DAPI (blue). Scale bar is 75 µm. B: histogram showing NCAM+ fibers in the various experimental conditions. C: representative image used for determining central nuclei number. Nuclei are labeled with DAPI (red) and laminin (green) marks the extracellular matrix. Solid triangles indicate central myonuclei. Scale bar is 150 µm. D: histogram showing central nuclei distribution in the various experimental conditions. E: TUNEL-positive cell (green) counterstained with DAPI (blue). Scale bar is 150 µm. F: histogram showing the distribution of TUNEL cells across the experimental conditions. *Significance (P) values represent the treatment group main effect in separate analyses of (co)variance. Presurgery values were covaried in models evaluating postsurgery treatment group differences.

Fig. 7.

Capillary numbers per fiber in vastus lateralis biopsies of study subjects. A: representative image of capillaries labeled with Ulex europeaus agglutinin (green), smooth muscle actin (red), and laminin (blue). Scale bar is 300 µm. B: histogram showing the relative numbers of capillaries per fiber across the experimental conditions. C: histogram summarizing the capillary-to-fiber perimeter exchange (CFPE) index across the experimental conditions. *Significance (P) values represent the treatment group main effect in separate analyses of (co)variance. Presurgery values were covaried in models evaluating postsurgery treatment group differences.