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Abstract

The purpose of this study was to identify strength gains after an anterior cruciate ligament injury and surgery and during inflammatory challenge in participants with disparate vitamin D levels. Plasma samples were obtained from those who had not previously experienced an anterior cruciate ligament injury and from injured patients 2 weeks before and 3 months after anterior cruciate ligament surgery. Plasma 25-hydroxyvitamin D and cytokine concentrations were measured in each blood sample. Single-leg peak isometric forces were measured 2 weeks presurgery and 3 months postsurgery. Compared with noninjured participants, inflammatory cytokines were elevated prior to and following anterior cruciate ligament reconstruction. During this inflammatory challenge, the peak isometric force increases after surgery were significantly lower in those with plasma 25-hydroxyvitamin D concentrations <30 ng/mL compared with those with concentrations ≥30 ng/mL. The authors conclude that low vitamin D appears to hinder strength recovery after anterior cruciate ligament surgery and during inflammatory insult.

Keywords

vitamin D inflammatory cytokines muscular strength anterior cruciate ligament 25-hydroxyvitamin D

Introduction

Muscular (quadriceps) weakness is an immediate impairment that follows an anterior cruciate ligament injury and surgery. Muscular weakness can persist years after an anterior cruciate ligament injury and/or surgery and can be a predisposing factor for degenerative joint disease and subsequent surgeries later in life. Recently, the prevalence of low vitamin D (ie, serum 25-hydroxyvitamin D < 30 ng/mL) has been shown in patients scheduled to undergo anterior cruciate ligament reconstruction.¹ In patients with osteomalacia and chronic renal failure, low vitamin D is associated with muscular weakness.^2,3 Furthermore, low vitamin D increased the risk of muscular weakness,^4–6 whereas conversely, a higher vitamin D status related to better lower-extremity function^7–9 and strength in older people.^10,11 Despite the association with diverse pathophysiological and nonpathophysiological conditions in humans, it is unknown if vitamin D relates to muscular strength after an anterior cruciate ligament injury and surgery.

Similar to anterior cruciate ligament surgery patients, patients scheduled to undergo total hip or knee arthroplasty have also been shown to have low vitamin D levels.¹ Moreover, Reid et al¹² recently demonstrated a decrease in vitamin D (serum 25-hydroxyvitamin D) following knee arthroplasty, which was attributed to inflammatory insult.¹² The inflammatory response to trauma or surgery is essential for the muscle degeneration/regeneration biological progression, but an excessive or prolonged proinflammatory cytokine response causes muscle damage and perpetuates muscular weakness.¹³

Inflammatory cytokines initiate and propagate physiological events and signaling pathways that regulate skeletal muscle function. Following an anterior cruciate ligament injury^14–19 and surgery,^20,21 proinflammatory and anti-inflammatory cytokines increase in the knee, indicating a localized inflammatory response. We previously demonstrated a transient increase in circulating proinflammatory and anti-inflammatory cytokines following an anterior cruciate ligament injury and surgery.^22,23 However, circulating cytokine results were obtained for anterior cruciate ligament–injured patients across time (ie, from before to after anterior cruciate ligament surgery) without comparisons with noninjured participants. Determining the circulating cytokine difference between injured and noninjured participants would identify if a systemic inflammatory response exists following an anterior cruciate ligament injury. This is an important gap to fill in our scientific and medical knowledge because circulating cytokines could have substantial systemic and rehabilitative consequences, such as regulating vitamin D status and muscular strength.

Therefore, the purpose of this study was to identify strength gains after an anterior cruciate ligament injury and surgery and during inflammatory challenge in those with disparate vitamin D levels. We hypothesized that muscular strength improvements following an anterior cruciate ligament injury and surgery are impaired in those with a lower vitamin D status.

Methods

We performed a retrospective analysis of plasma samples and peak isometric force data collected from a previous clinical study conducted in 18 male (Caucasian, light skin pigmentation) patients who underwent anterior cruciate ligament surgery.^22,24,25 Besides our antioxidant (400 IU of vitamin E [50% RRR-α-tocopheryl acetate and 50% RRR-α-tocopherol] and 1000 mg of vitamin C) and matching placebo interventions, which were taken starting ~2 weeks prior to and concluded 3 months after anterior cruciate ligament surgery,^22,24,25 participants were not consuming any other supplements consistently on enrollment (or prior to injury) and refrained from other supplements during study participation.

Additional plasma samples were obtained from 11 participants who had not previously experienced an anterior cruciate ligament injury and matched for gender, age, and skin pigmentation to the anterior cruciate ligament–injured participants, who were not taking any dietary supplements. The institutional review boards at Intermountain Healthcare (Salt Lake City, UT) and Oregon State University (Corvallis, OR) approved this study. Participants were informed of and consented to the experimental protocol and procedures. Participant inclusion and exclusion criteria, surgical procedures, oral and intravenous medications, and physical therapy have been reported previously for those who underwent anterior cruciate ligament reconstructive surgery.^22,24

Study Protocol

Blood draws and sample handling

For those with an anterior cruciate ligament injury, fasting blood samples were obtained ~2 weeks prior to and 3 months after anterior cruciate ligament surgery. All plasma samples were collected between May 2007 and August 2008 in Salt Lake City, UT (latitude ≈40° N). A single plasma sample was collected from noninjured participants between December 2009 and April 2010 in Salt Lake City, UT. Samples were collected from the antecubital vein into one 4.0-cc purple-top Vacutainer tube (1 mg/mL EDTA). Plasma was separated by centrifugation (Fisher Scientific Centrific, Model 228, Pittsburgh, PA) at 1380g for 15 minutes within 20 minutes of sample collection. Samples were immediately flash frozen in liquid nitrogen and stored at −80° C until the day of analysis.

Single-leg strength testing

Single-leg strength testing was performed 2 weeks before and 3 months after anterior cruciate ligament surgery and immediately after the blood draw procedure. Results comparing the noninjured and injured limbs are reported elsewhere.²⁴ Here, we report the peak isometric forces for the injured and noninjured limbs at 2 weeks before and 3 months after anterior cruciate ligament surgery in relation to plasma 25-hydroxyvitamin D concentrations. Single-leg strength testing was not performed in those participants with no anterior cruciate ligament injury.

Nonsteroidal anti-inflammatory drugs

Participants were asked to refrain from using any nonsteroidal anti-inflammatory drugs throughout the duration of the study.

Anterior cruciate ligament reconstructive surgery

Reconstructive, anesthesia, postoperative medication, and physical rehabilitation procedures have been described elsewhere.^22,24 Briefly, regarding anterior cruciate ligament surgical procedures, the majority of the ruptured anterior cruciate ligaments were arthroscopically repaired with a double-bundle anatomic auto (semitendinosus)/allo (semitendinosus) graft. One particpant had a bone-tendon bone autograft that was repaired arthroscopically as well.

Analytical Procedures

Plasma 25-hydroxyvitamin D total concentrations

Plasma 25-hydroxyvitamin D total (sum of 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃) concentrations (ng/mL) were measured using a chemiluminescent assay at ARUP Laboratories (Salt Lake City, UT). The average of the plasma 25-hydroxyvitamin D concentrations 2 weeks before and 3 months after surgery was used to identify each participant’s vitamin D status (< 30 or ≥30 ng/mL). We chose 30 ng/mL as our cutoff criteria because Bogunovic et al¹ recently found that approximately 50% of participants scheduled for anterior cruciate ligament surgery had serum 25-hydroxyvitamin D concentrations below 30 ng/mL.

Plasma cytokine concentrations

A multiplex microsphere-bead array was used to measure a number of circulating inflammatory cytokines (pg/mL) as described previously.^26,27 Briefly, cytokines were quantitated using a multiplexed sandwich capture assay developed in the ARUP Institute for Clinical and Experimental Pathology (University of Utah, Salt Lake City, UT) using the Luminex Multi-Analyte Profiling system (Luminex, Austin, TX).²⁶ Procedural precision of this multiplex-cytokine bead assay has been reported previously.^26,27

Single-leg peak isometric forces

Single-leg peak isometric force procedures have been published previously.²⁴ Briefly, single-leg peak isometric force testing was performed on a custom, horizontal plyo-press (Athletic Republic, Park City, UT). After a self-selected warm-up, participants were positioned on the plyo-press to elicit knee and hip joint knee flexion angles of 90°. Hip and knee extension isometric contractions were accomplished by overloading the weight stack resistance (~2260 N). Each participant performed 3 submaximal contractions followed by 3 maximal single-leg isometric contractions. Leg selection at the start of each testing session was randomized and subsequently followed by an alternating leg sequence of contractions. Each isometric contraction was 3 s in duration and separated by at least 1 minute of rest. Output data were measured from signals obtained from a mounted force plate (Advanced Mechanical Technology, Watertown, MA). All data were sampled at 200 Hz with a low-pass filter at 10 Hz. Peak isometric forces were defined as the highest resultant force produced during the 3 s test for each leg separately and expressed in absolute (N) and relative (N/kg) terms.

Statistical Analyses

The statistical analysis of plasma 25-hydroxyvitamin D and cytokine data for noninjured and anterior cruciate ligament–injured participants was performed using a one way analysis of variance followed by a Tukey Honestly Significant Difference test to test multiple pairwise comparisons. If data were not normally distributed, a Kruskal-Wallis one way analysis of variance on ranks was performed followed by a Dunn’s Method to test multiple pairwise comparisons. Separate t tests were performed to identify differences in age, height, body mass, and body mass index between anterior cruciate ligament–injured and noninjured participants.

For the anterior cruciate ligament–injured participants, the statistical significance of plasma 25-hydroxyvitamin D and cytokine concentrations and single-leg (injured and noninjured) peak isometric forces were assessed with a two way (vitamin D status and time) analysis of variance followed by a Tukey Honestly Significant Difference test for multiple pairwise comparisons. To achieve normality, rank transformations were performed on cytokine data. Transformations were checked for normality with a Kolmogorov-Smirnov test. Separate t tests were performed to identify differences in age, height, body mass, body mass index, and the change in peak isometric forces in the injured limb from 2 weeks before to 3 months after surgery between those with plasma 25-hydroxyvitamin D concentrations <30 and ≥30 ng/mL. A Mann-Whitney rank sum test was performed if participant characteristic data were not normally distributed.

A multiple linear regression analysis was used to identify which independent variable (age, height, body mass, body mass index, and season of data collection) or combination of independent variables predicted plasma 25-hydroxyvitamin D concentrations. A separate multiple linear regression analysis was used to identify which variable (age, height, body mass, body mass index, season of data collection, and plasma 25-hydroxyvitamin D concentrations) or combination of variables predicted the strength of the injured limb. To identify differences in plasma 25-hydroxyvitamin D concentrations across seasons, a one way analysis of variance was performed on the 4 seasons (winter, spring, summer, and autumn) of data collection and plasma 25-hydroxyvitamin D concentrations. All statistical analyses were performed with SysStat Software (SigmaPlot 10.0, SigmaStat 3.5, Chicago, IL). Statistical significance was set at P < .05.

Results

Plasma samples from anterior cruciate ligament–injured participants were collected from a previous vitamin E and C supplementation study.^22–25 To identify if vitamin E and C supplementation was influential on vitamin D status, we performed a t test to evaluate the effect of supplementation on plasma 25-hydroxyvitamin D concentrations. Plasma 25-hydroxyvitamin D concentrations were not significantly (P > .05) different between those treated with or without vitamins E and C (data not shown). Additionally, plasma samples were collected year-round, and therefore, to account for seasonal variability in vitamin D status, a one way analysis of variance was performed on the data collected for the 4 seasons and plasma 25-hydroxyvitamin D concentrations. Plasma 25-hydroxyvitamin D concentrations were not significantly (P > .05) different between seasons of data collection (data not shown). Based on these results, plasma 25-hydroxyvitamin D concentrations from the anterior cruciate ligament–injured participants were pooled and analyzed irrespective of the previous vitamin E and C intervention and season of sample collection.

A multiple linear regression analysis was performed to identify which independent variable or combination of independent variables predicted plasma 25-hydroxyvitamin D concentrations. None of the investigated variables predicted plasma 25-hydroxyvitamin D concentrations when combining the data from the injured and noninjured anterior cruciate ligament participants.

Participant Characteristics

Participant characteristics were similar between injured and noninjured participants. Specifically, age, height, body mass, and body mass index were comparable between noninjured (n = 11) and injured (n = 18) participants (Table 1).

Table 1.

Characteristics of Injured and Noninjured Participants^a

		Injured
	Noninjured	2 Weeks Presurgery	3 Months Postsurgery
Age (years)	31 ± 1	32 ± 2	32 ± 2
Height (cm)	181 ± 2	175 ± 2	175 ± 2
Body mass (kg)	89.4 ± 3.4	85.4 ± 3.2	85.6 ± 3.3
Body mass index (kg/m²)	27.3 ± 1.1	28.1 ± 1.2	28.2 ± 1.3

^a Participant characteristics were not significantly different between groups. Data are presented as mean ± standard error of the mean.

The average of the plasma 25-hydroxyvitamin D concentration 2 weeks before and 3 months postsurgery were used to establish vitamin D status in the anterior cruciate ligament–injured participants. Based on this average concentration, participants were separated into 1 of 2 groups (n = 9 per group): plasma 25-hydroxyvitamin D concentrations < 30 ng/mL (group 1) or ≥30 ng/mL (group 2). Age, height, body mass, and body mass index were not significantly different between anterior cruciate ligament–injured participants in the 2 groups (Table 2).

Table 2.

Injured Participant Characteristics Relative to Vitamin D Status^a

Plasma 25(OH) D	<30 ng/mL	≥30 ng/mL
n	9	9
Age (years)	35 ± 2	29 ± 3
Height (cm)	177 ± 3	172 ± 4
Body mass (kg)
2 Weeks before ACL surgery	90.8 ± 4.0	79.8 ± 4.3
3 Months after ACL surgery	90.2 ± 4.2	81.0 ± 4.8
Body mass index (kg/m²)
2 Weeks before ACL surgery	28.9 ± 1.4	27.2 ± 2.1
3 Months after ACL surgery	28.7 ± 1.4	27.6 ± 2.2
ACL surgical procedure
Double-bundle	8	9
BTB	1	0
Additional surgical procedures (n)
Lateral meniscus repair	0	1
Medial meniscus repair	0	4
Partial medial meniscectomy	3	0
Partial lateral meniscectomy	1	1

Abbreviations: 25(OH)D, 25-hydroxyvitamin D; ACL, anterior cruciate ligament; BTB, bone-tendon-bone autograft.

^a Participant characteristics were not significantly different between groups. Data are presented as mean ± standard error of the mean.

For the majority of participants in both vitamin D status groups, ruptured anterior cruciate ligaments were arthroscopically repaired with a double-bundle anatomic auto/allograft. One participant with plasma 25-hydroxyvitamin D concentrations <30 ng/mL had a bone-tendon-bone autograft, which was also repaired arthroscopically. Regarding additional surgical procedures, 5 participants had meniscus repair, and 1 participant had a partial lateral meniscectomy in the group with plasma 25-hydroxyvitamin D concentrations ≥30 ng/mL (Table 2). In the group with plasma 25-hydroxyvitamin D concentrations <30 ng/mL, a partial meniscectomy was performed in 4 participants (Table 2).

Plasma 25-Hydroxyvitamin D Concentrations

In the noninjured group, 73% of the participants had plasma 25-hydroxyvitamin D concentrations less than 30 ng/mL. With regard to the anterior cruciate ligament–injured group, plasma 25-hydroxyvitamin D concentrations were less than 30 ng/mL in 56% of the participants at 2 weeks presurgery and in 50% of the participants at 3 months postsurgery. Plasma 25-hydroxyvitamin D concentrations were not significantly different between the noninjured and injured participants (Table 3). As would be expected following vitamin D status delineation, mean plasma 25-hydroxyvitamin D concentrations were significantly (P < .05) lower in those with concentrations <30 ng/mL (23.4 ± 3.2 and 22.7 ± 1.4 ng/mL at 2 weeks presurgery and 3 months postsurgery, respectively) compared with those with ≥30 ng/mL (37.6 ± 4.0 and 43.1 ± 4.4 ng/mL at 2 weeks presurgery and 3 months postsurgery, respectively).

Table 3.

Plasma 25(OH)D (ng/mL) and Cytokine (pg/mL) Concentrations^a

	Noninjured	Injured
	Noninjured	2 Weeks Before ACL Surgery	3 Months After ACL Surgery
25(OH)D	27.0 ± 2.8	30.5 ± 3.0	32.9 ± 3.3
IFN-γ	8.13 ± 5.56	12.9 ± 3.3	9.44 ± 2.67
IL-4	3.02 ± 1.67	3.20 ± 1.03	2.88 ± 0.66
IL-1β	32.4 ± 19.8	19.4 ± 5.9	29.0 ± 7.5
TNF-α	5.51 ± 3.49	5.89 ± 1.38	6.11 ± 1.34

Abbreviations: ACL, anterior cruciate ligament; 25(OH)D, 25-hydroxyvitamin D; IFN, interferon; IL, interleukin.

^a Plasma 25(OH) D, IFN-γ, IL-4 and IL-1β were not significantly different between noninjured and injured participants. Data are presented as mean ± standard error of the mean.

Cytokine Concentrations

We previously demonstrated an increase in circulating inflammatory cytokines within anterior cruciate ligament–injured participants prior to and following surgery.^22,23 However, it is unknown if cytokines increase in the circulation in these patients when compared with noninjured individuals. To address this gap in our knowledge, we compared plasma cytokine concentrations in noninjured and injured participants. Plasma interferon-γ, interleukin-4, interleukin-1β, and tumor necrosis factor-α concentrations were not significantly different between injured and noninjured participants (Table 3). In contrast, plasma interleukin-2, interleukin-2 receptor α, interleukin-6, interleukin-12, interleukin-5, interleukin-10, and interleukin-13 (Figures 1A-1G ) concentrations were significantly (P < .05) greater in injured compared with noninjured participants. Although detected in injured participants, circulating interleukin-8 was not detected in the noninjured participants (Figure 1H). Thus, these data identify a systemic cytokine response following an anterior cruciate ligament injury that does not subside even 3 months after surgery.

Figure 1.

Plasma cytokine concentrations: plasma interleukin (IL)-2 (A), IL-2 receptor α (rα; B), IL-6 (C), IL-12 (D), IL-5 (E), IL-10 (F) and IL-13 (G) concentrations (pg/mL) were significantly (*P < .05) greater prior to (2 weeks [wks; pre]) and following (3 months [mos; post]) anterior cruciate ligament (ACL) surgery compared with those in noninjured participants. Plasma IL-8 (H) concentrations were not detected (ND) in the noninjured participants. Data are presented as mean ± standard error of the mean

It is unknown if inflammatory cytokines vary depending on plasma 25-hydroxyvitamin D concentrations following an anterior cruciate ligament injury and surgery. For the most part, inflammatory cytokines were not significantly different between participants with disparate plasma 25-hydroxyvitamin D concentrations (supplemental Table). However, plasma interferon-γ concentrations were significantly (P < .05) greater in participants with plasma 25-hydroxyvitamin D ≥30 ng/mL than in those with concentrations <30 ng/mL (Figure 2). Nonetheless, these data collectively imply that circulating cytokines following an anterior cruciate ligament injury and surgery were not significantly different between participants with disparate circulating 25-hydroxyvitamin D concentrations.

Figure 2.

Plasma interferon-γ concentrations. Plasma interferon (IFN)-γ concentrations (pg/mL) were significantly (main effect of vitamin D status, P < .05, bar) greater in participants with plasma 25-hydroxyvitamin D (25(OH)D) concentrations ≥30 ng/mL (gray bar) than those with <30 ng/mL (black bar) at 2 weeks before (wks; pre) and 3 months after (mos; post) surgery. Data are presented as mean ± standard error of the mean

Single-Leg Forces

For the participants in this study, we reported previously that the injured limb was significantly weaker than the noninjured limb prior to and following anterior cruciate ligament surgery.²⁴ In the present investigation, we sought to examine the strength of each limb separately relative to time (2 weeks presurgery and 3 months postsurgery) and plasma 25-hydroxyvitamin D concentrations (<30 and ≥30 ng/mL). Because of the variability in body mass (kg) between vitamin D status groups (Table 2), we report single-leg peak isometric forces in both absolute (N; Figure 3A) and relative (N/kg; Figure 3B) terms.

Figure 3.

Single-leg peak isometric forces prior to and following anterior cruciate ligament surgery in participants with plasma 25-hydroxyvitamin D concentrations <30 and ≥30 ng/mL. Single-peak isometric forces (A, N; B, N/kg) in the injured (INJ) limb were significantly (Vitamin D Status × Time interaction; P < .05, bar) greater at 3 months after (mos; post) compared with 2 weeks before (wks; pre) anterior cruciate ligament (ACL) surgery in participants with plasma 25-hydroxyvitamin D (25(OH)D) concentrations ≥30 ng/mL. Data are presented as mean ± standard error of the mean

Peak isometric forces in the noninjured limb were not significantly different between groups or across time (Figures 3A and 3B). Similarly, peak isometric forces in the injured limb were not significantly different between groups (Figures 3A and 3B). However, the peak isometric forces of the injured limbs were significantly (Vitamin D Status × Time interaction, P < .05) greater at 3 months postsurgery compared with 2 weeks presurgery in participants with plasma 25-hydroxyvitamin D concentrations ≥30 ng/mL (Figures 3A and 3B). This latter observation was not found in participants with plasma 25-hydroxyvitamin D concentrations <30 ng/mL (Figures 3A and 3B).

To examine strength recovery, we analyzed the change in peak isometric forces from presurgery to postsurgery in the injured limb relative to the plasma 25-hydroxyvitamin D concentration groups. Strikingly, peak isometric force increases from 2 weeks presurgery to 3 months postsurgery in the injured limb were significantly (P < .05) greater (by nearly 6-fold) in participants with plasma 25-hydroxyvitamin D concentrations ≥30 ng/mL compared with those with <30 ng/mL (Figure 4).

Figure 4.

Percentage change (from 2 weeks before [wks; pre] and 3 months after [mos; post] surgery) in peak isometric forces in the injured limb. The increases from 2 weeks before (wks; pre) and 3 months after (mos; post) anterior cruciate ligament (ACL) surgery were significantly (P < .05, bar) greater in participants with plasma 25-hydroxyvitamin D (25(OH)D) concentrations ≥30 ng/mL when compared with those with concentrations <30 ng/mL. Data are presented as mean ± standard error of the mean

A multiple linear regression analysis was performed to identify which independent variable or combination of independent variables predicted peak isometric forces in the injured limb. Although the best predictor was plasma 25-hydroxyvitamin D concentrations (albeit nonsignificantly, P > .05), none of the examined variables predicted the peak isometric forces in the injured limb following anterior cruciate ligament surgery.

Discussion

The novelty of this study—which is also consistent with our hypothesis—was the finding that strength gains after an anterior cruciate ligament injury and surgery were lower in participants with plasma 25-hydroxyvitamin D concentrations less than 30 ng/mL compared with those with concentrations ≥30 ng/mL (Figures 3 and 4). These results are of clinical importance because muscular weakness continues to challenge the physical rehabilitation from an anterior cruciate ligament injury and surgery, and monitoring for low vitamin D could provide a safe, complementary, and easily treatable therapeutic approach intended to ameliorate strength deficits.

Although plasma (or serum) 25-hydroxyvitamin D concentration is the best indicator for vitamin D status, the hormonal form of vitamin D (1α, 25-dihyroxyvitamin D₃) is responsible for modulating muscle function by initiating genomic and nongenomic events in skeletal muscle on receptor binding. On binding to the nuclear vitamin D receptors in skeletal muscle, vitamin D induces gene transcription and protein synthesis,²⁸ which influences muscle cell proliferation and differentiation,^29,30 calcium uptake,³¹ and phosphate transport across the sarcolemma.^32,33 The nongenomic responses in skeletal muscle include rapid calcium uptake³⁴ and the activation of mitogen-activated protein kinase signaling pathways.^35,36 It is plausible that vitamin D modulates the genomic and nongenomic signaling pathways in skeletal muscle and improves strength recovery after anterior cruciate ligament reconstruction. Considering recent evidence refuting the strong presence of vitamin D receptors in skeletal muscle,³⁷ it is unclear if vitamin D exerts a direct effect on skeletal muscle or alters indirect systemic mediators of skeletal muscle function, such as inflammatory cytokines.

In the present report, we provide the first evidence of a systemic cytokine challenge in injured compared with noninjured participants prior to and following surgery. Furthermore, the cytokine response to injury and surgery was not significantly different between participants with disparate vitamin D levels, with the exception of interferon-γ. This latter finding is an interesting observation because it contrasts previous reports indicating that vitamin D inhibits interferon-γ production.^38–43 Considering these discrepancies when interpreting the present results, it is likely that other inducers are responsible for the interferon-γ increase, such as interleukin-2 or reactive oxygen species.^44–46 However, interleukin-2 (Figure 1A) or 8-isoprostane prostaglandin F_2α (a marker of oxidative stress-induced lipid peroxidation; data not shown)²⁵ concentrations were not significantly different between vitamin D groups. Alternatively, more meniscus injuries that required repair, as opposed to meniscectomies, in participants with plasma 25-hydroxyvitamin D concentrations ≥30 ng/mL could account for the interferon-γ increase prior to and following anterior cruciate ligament reconstruction. Although the mechanism for the interferon-γ increase following an anterior cruciate ligament injury and surgery awaits future resolve, our results indicate lower strength gains in participants with (1) fewer meniscus repair procedures performed during anterior cruciate ligament surgery and (2) lower plasma 25-hydroxyvitamin D and interferon-γ concentrations.

Interferon-γ is synthesized by T lymphocytes and natural killer cells in response to inflammatory stimuli. Among other proinflammatory cytokines, interferon-γ is capable of impairing skeletal muscle recovery by increasing nitric oxide production from inducible nitric oxide synthase.⁴⁷ Contrasting evidence indicates that interferon-γ accentuates muscle regeneration by impairing fibrosis⁴⁸ and increasing myosin heavy chain content.⁴⁹ Furthermore, in experimental mice, contractile properties of skeletal muscle that had been lacerated improved following interferon-γ administration.⁴⁸ Based on these findings, interferon-γ could be responsible for improving strength recovery following trauma or surgery and independent from vitamin D. Studies investigating the direct, indirect, and synergistic influences of vitamin D and/or interferon-γ on strength recovery following an anterior cruciate ligament injury and surgery are warranted to resolve this issue.

There are a couple of limitations of this study that require attention. First, circulating calcium, parathyroid hormone, and 1,25-dihyroxyvitamin D concentrations were not measured. Second, it is plausible that participants with a better vitamin D status were more physically active and lived healthier lifestyles prior to injury, which could be influential on strength recovery after surgery. Third, vitamin D status was demarcated to a single plasma 25-hydroxyvitamin D concentration. Future studies are encouraged to investigate how strength recovery varies between participants grouped to the new vitamin D status criteria provided by the United States Institute of Medicine.^50,51 Finally, participants with plasma 25-hydroxyvitamin D concentrations ≥30 ng/mL had more menisci repair procedures during anterior cruciate ligament reconstruction. Based on more menisci repair procedures and the subsequent standard of care of limiting range of motion and weight-bearing activity after meniscus repair, one would assume a slower recovery in muscular strength after anterior cruciate ligament surgery in participants with plasma 25-hydroxyvitamin D concentrations ≥30 ng/mL, especially as early as 3 months postsurgery. However, our results suggest otherwise.

In summary, we provide unique data showing that during inflammatory challenge, strength recovery following anterior cruciate ligament reconstruction was lower in participants with lower vitamin D levels. Based on these results, we conclude that low vitamin D could be detrimental to muscle strength recovery shortly (ie, months) after anterior cruciate ligament surgery. Future intervention studies determining if improvements in vitamin D status can remedy muscular weakness following an anterior cruciate ligament injury and surgeries are warranted. Future studies should use a safe and cautious approach when investigating the therapeutic influence of different vitamin D interventions (ie, dietary intake, supplemental intake, or sun exposure) on strength recovery following anterior cruciate ligament surgery.

Footnotes

Acknowledgments

We would like to thank Penny Snow and Kristi Thunell (The Orthopedic Specialty Hospital, Intermountain Healthcare, Murray, UT) for phlebotomy; Ronda Ingram and Graham Burdett for participant recruitment; and all those who participated in this study.

TB, TBM, HRH, CRK, RHT, LKW, and MGT contributed to the writing of this manuscript and data interpretation. TB, RHT, LKW, and MGT designed the study. TB performed all data and statistical analyses. TBM performed the cytokine measurements.

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

This work was funded in part by USANA Health Sciences, Inc. (Salt Lake City, UT, USA) and the Deseret Foundation (Intermountain Healthcare, Salt Lake City, UT, USA).

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Low Vitamin D Impairs Strength Recovery After Anterior Cruciate Ligament Surgery

Abstract

Keywords

Introduction

Methods

Study Protocol

Blood draws and sample handling

Single-leg strength testing

Nonsteroidal anti-inflammatory drugs

Anterior cruciate ligament reconstructive surgery

Analytical Procedures

Plasma 25-hydroxyvitamin D total concentrations

Plasma cytokine concentrations

Single-leg peak isometric forces

Statistical Analyses

Results

Participant Characteristics

Plasma 25-Hydroxyvitamin D Concentrations

Cytokine Concentrations

Single-Leg Forces

Discussion

Footnotes

Acknowledgments

References