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. 2008 Jul;36(7):1347-57.
doi: 10.1177/0363546508314431. Epub 2008 Feb 29.

Response of Knee Ligaments to Prolotherapy in a Rat Injury Model

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Free PMC article

Response of Knee Ligaments to Prolotherapy in a Rat Injury Model

Kristina T Jensen et al. Am J Sports Med. .
Free PMC article

Abstract

Background: Prolotherapy is an alternative therapy for chronic musculoskeletal injury including joint laxity. The commonly used injectant, D-glucose (dextrose), is hypothesized to improve ligament mechanics and decrease pain through an inflammatory mechanism. No study has investigated the mechanical effects of prolotherapy on stretch-injured ligaments.

Hypotheses: Dextrose injections will enlarge cross-sectional area, decrease laxity, strengthen, and stiffen stretch-injured medial collateral ligaments (MCLs) compared with controls. Dextrose prolotherapy will increase collagen fibril diameter and density of stretch-injured MCLs.

Study design: Controlled laboratory study.

Methods: Twenty-four rats were bilaterally MCL stretch-injured, and the induced laxity was measured. After 2 weeks, 32 MCLs were injected twice, 1 week apart, with either dextrose or saline control; 16 MCLs received no injection. Seven uninjured rats (14 MCLs) were additional controls. Two weeks after the second injection, ligament laxity, mechanical properties (n = 8), and collagen fibril diameter and density (n = 3) were assessed.

Results: The injury model created consistent ligament laxity (P < .05) that was not altered by dextrose injections. Cross-sectional area of dextrose-injected MCLs was increased 30% and 90% compared with saline and uninjured controls, respectively (P < .05). Collagen fibril diameter and density were decreased in injured ligaments compared with uninjured controls (P < .05), but collagen fibril characteristics were not different between injured groups.

Conclusion: Dextrose injections increased the cross-sectional area of MCLs compared with saline-injected and uninjured controls. Dextrose injections did not alter other measured properties in this model.

Clinical relevance: Our results suggest that clinical improvement from prolotherapy may not result from direct effects on ligament biomechanics.

Conflict of interest statement

One or more of the authors has declared a potential conflict of interest.

Figures

Figure 1
Figure 1
Study design: characterize stretch injury and investigate response from prolotherapy injection. Top 3 rows: To characterize the stretch-injury model, 12 rats underwent unilateral medial collateral ligament (MCL) stretch injury at time 0. Rats were euthanized immediately after injury and after 2 weeks and 4 weeks of healing (n = 4). The contralateral (uninjured) leg was used as control. Bottom 3 rows: To investigate the response to prolotherapy injections, 32 additional rats were used. Sixteen rats underwent bilateral MCL stretch injury at time 0. At 2 and 3 weeks after injury, rats were injected with the most commonly used solution (dextrose) or saline (control) and were euthanized 2 weeks after the second injection. Eight rats underwent bilateral MCL stretch injury with no injections and were euthanized 5 weeks after stretch injury. Seven rats did not undergo stretch injury.
Figure 2
Figure 2
Stretch-injury measurement device. The leg was placed in a reproducible position, and the device was placed under the midsubstance of the medial collateral ligament. The midsubstance was lifted medially in a “bowstring” fashion. The distance that the device moves medially until it applies 75g of force is defined as the laxity parameter.
Figure 3
Figure 3
Change in laxity parameter immediately, 2, 4, and 5 weeks after injury. Inherent laxity, as quantified by our bowstring laxity parameter, was subtracted from this same parameter at each time point to obtain the data shown in this figure. Ligaments were consistently injured. Immediately after injury, change in laxity was 0.44 ± 0.14 mm of medial displacement (*P < .0001), which was estimated to be approximately 1.6% change in total ligament length. Ligaments remained lax 2, 4, and 5 weeks after injury (*P < .05). In addition, there was more laxity 4 weeks after injection than after injury (**P < .05). Laxity was not altered by injection treatment of dextrose or saline. Mean ± standard error.
Figure 4
Figure 4
Force displacement curve comparing injured ligaments and the contralateral control immediately after injury and after 2 weeks of healing. Immediately after injury, all ligaments had a lower failure force and lower stiffness than uninjured controls. After 2 weeks of healing from injury, failure forces were similar between injured and uninjured ligaments. Injured ligaments were normalized to the force and displacement of the contralateral control because the rats were growing.
Figure 5
Figure 5
Cross-sectional area of medial collateral ligaments immediately, 2, 4, and 5 weeks after injury normalized to uninjured area. Injured (no injection) ligaments initially had a 22% smaller cross-sectional area than contralateral uninjured controls. After 2 and 4 weeks of healing, injured (no injection) ligaments had 157% and 58% larger cross-sectional areas, respectively, than contralateral uninjured controls (*P < .05 vs uninjured at the same time point). After 5 weeks of healing, dextrose-injected, saline-injected, and no injection ligaments had 90%, 46%, and 62% larger cross-sectional areas, respectively, than uninjured controls (*P < .05). In addition, injured ligaments with dextrose injection had a 30% larger area compared with injured ligaments with saline injection (**P < .05). Mean ± standard error.
Figure 6
Figure 6
Decreases in failure stress of medial collateral ligaments immediately, 2, 4, and 5 weeks after stretch injury. Immediately after stretch injury, the failure stress was similar in injured and uninjured ligaments. At 2 and 4 weeks after injury, injured ligaments failed at 63% and 42% lower stress, respectively, than uninjured controls at the same time point (*P < .05). After 5 weeks of healing, injured ligaments with dextrose injection, with saline injection, and without injection had 38%, 29%, and 34% smaller failure stresses, respectively, than uninjured controls (*P < .05). No additional differences were found between injured groups at 5 weeks. Mean ± standard error.
Figure 7
Figure 7
Transmission electron microscopy images of the cross-section of ligaments. Five weeks after injury, collagen fibril diameter and density were decreased compared with uninjured ligaments. This effect did not change with injection treatment. A, uninjured with no injection; B, injured with no injection; C, injured with saline injection; and D, injured with dextrose injection. Scale bar = 500 nm.
Figure 8
Figure 8
Collagen fibril density in ligaments 5 weeks after injury with and without injection. When grouped together, all injured ligaments (dextrose-injected, saline-injected, and no injection) had a 31% smaller collagen fibril density compared with uninjured controls (*P < .05). No differences were found between injection treatments. Mean ± standard error.
Figure 9
Figure 9
Collagen fibril diameter distributions in injured and uninjured ligaments. Five weeks after injury, there were 61% and 68% fewer larger collagen fibrils (80–120 nm and 120–160 nm) in injured ligaments (including dextrose-injected, saline-injected, and no injection) compared with uninjured ligaments (*P < .05). In addition, there was a trend toward 98% more fibrils with smaller diameters of 10 to 40 nm in injured ligaments compared with uninjured (^P = .06). Mean ± standard error.
Figure 10
Figure 10
Collagen fibril diameter distribution by treatment group 5 weeks after injury. There were no differences in collagen fibril diameter distribution between injection groups. Mean ± standard error.

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