Healing Response of Knee Ligaments to Prolotherapy in a Rat Model

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Abstract

Purpose: Prolotherapy is an alternative medicine technique used to treat joint pain. Prolotherapy involves a series of injections of irritant solutions that are thought to cause inflammation, reenergize the healing process, tighten lax ligaments, and, thereby, reduce pain. However, to our knowledge, no study has been performed to show that inflammation occurs, and no study has investigated the mechanical effects of prolotherapy injections on lax ligaments. This thesis addresses some of these issues in two studies in a rat model.

Study 1 - Methods: The medial collateral ligaments of 56 rats were injected at the tibial and femoral insertions. Immunohistochemistry (IHC) was utilized to determine the inflammatory response 6, 24, and 72 hours after injection of dextrose compared to saline and no injection controls. At 24 hours, sodium morrhuate, P2G, and needle stick control were also analyzed. Results: At 6 hours, macrophages are increased in dextrose and saline injection groups at the tibial and femoral insertions compared to uninjected controls. At 24 hours, neutrophils are increased in sodium morrhuate and P2G injected ligaments at the tibial insertion and midsubstance, in saline injected ligaments at the tibial insertion, and in needle stick injected ligaments at the midsubstance compared to no injection. Also at 24 hours, tissue macrophages are increased in dextrose, sodium morrhuate, and needle stick injected groups at the femoral insertion compared to uninjected controls. At 72 hours, the inflammatory response is resolved. This study supports the concept that prolotherapy injections produce an inflammatory response. Interestingly, the inflammatory response of prolotherapy solutions is not different than saline and needle stick controls.

Study 2 - Methods: Rat medial collateral ligaments (MCL) were stretch-injured and the induced laxity measured. After two weeks of healing, two sets of injections were performed one week apart of dextrose, saline control, or no injection control. Two weeks after the last injection (5 weeks after injury), laxity was measured, and rats were euthanized. Five weeks after injury, pull to failure tests to determine ligament strength (n=8 for all groups, except n=7 for uninjureduninjected), transmission electron microscopy to determine fibril diameter and density (n=3), and IHC to determine prolonged inflammatory response (n=5, except n=4 for uninjured-uniniected) were performed. Results: Laxity is consistently created but is not altered by prolotherapy (dextrose) or control (saline) injections. The failure force is similar between dextrose injected and the three controls (saline injection, no injection, and uninjured-uninjected ligaments). Cross-sectional area is increased with dextrose compared to saline and uninjured controls (30 and 90% increase, respectively, p < 0.05). Collagen fibril diameter distribution is decreased in injured ligaments compared to uninjured controls with fewer fibrils in the 80-120 nm and 120-160 nm fibril diameter classes (61 and 68% decrease, respectively, p < 0.05). Collagen fibril density is decreased by 31% in injured ligaments compared to uninjured control (p < 0.05). Collagen fibril diameter and density are not altered with injection. The inflammatory response is alleviated. This study finds that prolotherapy injections do not alter laxity or mechanical properties but do increase the cross-sectional area of the ligament in this model.

Conclusion: Prolotherapy solutions and saline and needle stick control all create an inflammatory response that is resolved by 3 days after injection. Since dextrose injections do not improve the clinically related outcomes (laxity and strength), the biomechanical clinical benefit of prolotherapy is not supported in this study. Further experiments are needed to determine if the improved strength found with previous animal studies using sodium morrhuate injections on healthy ligaments create a similar result in an injured model. Further human studies should also be performed to determine the effects of prolotherapy on joint pain, which is an important clinically relevant outcome that cannot be directly assessed in an animal model.

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