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Effect of Hyperbaric Oxygen on the Ligament Healing Process in Rats

Mashitori, Hirotaka*; Sakai, Hiroya*; Koibuchi, Noriyuki; Ohtake, Hideki; Tashiro, Toshiyuki; Tamai, Kazuya*; Saotome, Koichi*

Clinical Orthopaedics and Related Research: June 2004 - Volume 423 - Issue - p 268-274
doi: 10.1097/01.blo.0000128970.27390.f5
SECTION II: ORIGINAL ARTICLES
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Animal experiments were done to investigate whether administration of hyperbaric oxygen promotes scar tissue formation, increases expression of the Type I procollagen gene, and improves the tensile properties of healing ligament. In 76 Sprague-Dawley rats, a 2-mm segment of the medial collateral ligament was removed. Thirty-eight rats were exposed to hyperbaric oxygen at 2.5 atmospheres absolute for 2 hours 5 days per week (Group H), whereas the remaining rats were exposed to room air (Group C). The animals were sacrificed at 3, 7, 14, and 28 days postoperatively. In situ hybridization histochemistry was done to examine the Type I procollagen gene expression in healing ligaments in 40 rats, whereas a tensile failure test was done in the remaining rats. The amount of scar tissue was greater in Group H than in Group C. Type I procollagen gene expression at 7 or 14 days was significantly greater in Group H than in Group C. The ultimate load and stiffness in Group H were significantly greater than in Group C at 14 days. Administration of hyperbaric oxygen promotes scar tissue formation and increases Type I procollagen gene expression in healing ligaments. These effects are associated with the improvement of their tensile properties.

From the *Department of Orthopaedic Surgery; †Department of Physiology, Dokkyo University School of Medicine, Tochigi, Japan; and the ‡Department of Orthopaedic Surgery, The University of Tokyo, Tokyo, Japan.

Received: October 7, 2002

Revised: March 19, 2003; June 30, 2003

Accepted: July 2, 2003

Correspondence to: Hiroya Sakai, MD, PhD, Department of Orthopaedic Surgery, Saitama Medical Center, Saitama Medical School, 1981 Kamodatsujido, Kawagoe, Saitama 350-8550, Japan. Phone: 81-49-228-3627; Fax: 81-49-223-8426; E-mail: hsakai@saitama.med.ac.jp.

Hyperbaric oxygen (HBO) therapy has been used as a primary treatment of decompression sickness, air embolism, carbon monoxide poisoning,15,25 and as an adjunct for several other conditions, including burns, crush injuries, and compartment syndrome.8,31,33,34 Hyperbaric oxygen therapy also has been used by some professional teams to speed recovery of injured athletes, hoping that it could help soft tissue injuries heal more rapidly.20,27 However, the literature contains conflicting results regarding the efficacy of HBO for sports-related soft tissue injuries. Some studies have supported its benefit,2,17,19 whereas others have shown negative results.3,14,24,26

Ligament injury is one of the most common soft tissue injuries among athletes.11 It is important for injured athletes to recover from such injuries and to resume sporting activities as soon as possible. Collagen is the most abundant substance in ligaments,12 and the mechanical properties of ligaments depend primarily on collagen fibers.37 More than 90% of collagen in normal ligaments is Type I collagen.12 Type I collagen synthesis plays an important role in the healing of ruptured ligaments, which is initiated with the expression of the Type I procollagen gene. Ligament injuries heal by scar formation rather than true ligament regeneration.12 Therefore, in addition to examination of the amount of the newly created scar tissue, examination of the expression of the Type I procollagen gene in the scar tissue is an appropriate method for assessment of the early phase of the ligament healing.29,38 However, the functional recovery of healing ligaments is seen most directly by examination of their tensile properties. Ultimate load and stiffness are the most common values used for assessment of the tensile properties of healing ligament.4,5,12,16,17,35 Ishii et al18,19 showed that administration of HBO promoted scar tissue formation and increased the expression of the Type I procollagen gene in lacerated patellar tendons. Horn et al17 showed that administration of HBO increased the ultimate load in lacerated medial collateral ligaments (MCL), despite no significant difference in the stiffness. We investigated whether administration of HBO could promote ligament healing. Our hypothesis was that administration of HBO increases the amount of scar tissue and expression of the Type I procollagen gene in the scar tissue, and that these effects are associated with improvement of the ultimate load and stiffness of healing ligaments.

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MATERIALS AND METHODS

Animals and Treatments

Institutional review board approval was obtained before beginning all animal studies. Seventy-six male Sprague-Dawley rats (age, 11 weeks; weight, 369 ± 9 g) were used. Each animal was anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg). The MCL of the left knee was exposed through a longitudinal medial skin incision, and a 2-mm segment of the midportion of the MCL (centered over the knee line) was resected horizontally according to the method of Chimich et al5 with some modification. The ligament was separated completely and a gap was left between the retracted ends of the ligament. The skin then was closed with continuous nylon sutures. No immobilization was done after surgery and the animals were returned to their cages.

Thirty-eight rats were housed in individual cages in normal room air (Group C), whereas the remaining 38 rats were exposed to HBO from the day of surgery (Group H). In Group H, the animals were placed in a cylindrical pressure chamber (P-5100S; Hanyuda Iron Works Ltd, Tokyo, Japan) with a volume of 15.2 L and exposed to HBO at 2.5 atmospheres absolute (ATA) for 2 hours 5 days per week.

By administration of a lethal intraperitoneal overdose of pentobarbital, the animals were sacrificed at 3, 7, 14, and 28 days postoperatively. In the animals euthanized at 28 days, HBO only was done during the first 2 weeks.

In 40 of the rats (five each from each group euthanized at 3, 7, 14, and 28 days postoperatively), the MCLs were identified carefully and the amount of the scar tissue created between both ends of the cut ligament was inspected macroscopically. Then the midportion of the healing ligament, centered on the gap or on the scar, and a comparable portion of the contralateral unoperated MCL, were excised and immediately fixed in 4% paraformaldehyde/0.1 mol/L phosphate buffer for 12–24 hours at 4°C. The fixed tissues were dehydrated, embedded in paraffin, and stored at 4°C.

In the other 36 rats (six each from each group euthanized at 7, 14, and 28 days postoperatively), the hind limbs were disarticulated at the hip, sealed in plastic bags, and stored in a freezer at –20°C until the tensile failure test.

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Preparation of Probes

The protocol for probe preparation was reported previously.29 In brief, a larger EcoR I fragment of the cDNA for human Type I (α 1(I)) procollagen mRNA (Hf677)6 was subcloned into the pBluescript SK II (+) transcription vector (Stratagene, LaJolla, CA). The vector was linearized with either Not I or Sal I restriction endonuclease for the antisense or sense probe, respectively, and each riboprobe was transcribed using T7 or T3 RNA polymerase, respectively. [35S]UTP was used to label the probes. Probes then were hydrolyzed to a length of approximately 300 nucleotides according to the procedure of Cox et al.10

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Tissue Preparation and In Situ Hybridization

The protocol for tissue preparation and for in situ hybridization essentially was the same as reported previously.22,29 In brief, paraformaldehyde-fixed and paraffin-embedded ligament tissues were cut into coronal sections on a cryostat at a thickness of 10 μm. The sections were mounted onto organosilane-coated glass slides and prehybridized for 1.5–2 hours at room temperature with 40 μL of prehybridization buffer.22 The prehybridization solution then was removed and 40 μL of hybridization buffer22 containing the RI-labeled antisense or sense riboprobe (3 × 105 cpm/section) was pipetted onto each section. Hybridization was done for 24 hours at 50°C.

After hybridization, the sections were treated with ribonuclease A (RNase A), rinsed with RNase-free buffer, and dehydrated through a graded series of ammonium acetate with ethanol, followed by rinsing in absolute ethanol. Sections then were dipped in Kodak NTB3 autoradiography emulsion (Eastman Kodak Co, Rochester, NY) (45°C), and stored at 4°C for 2 weeks. Next, the slides were developed, stained with hematoxylin and eosin, and cover-slipped.

The localization of silver grains was determined by microscopy. To analyze the results of in situ hybridization semiquantitatively, six areas of 30,338 μm2 (197 μm × 154 μm) were selected randomly in the scar tissue between the two cut ends of the ligament on each section. Grains and cell nuclei were counted in each area using a personal computer with image analysis software (Mac SCOPE, Version 2.56; Mitani Corp, Fukui, Japan). The value for each section was determined as an average of the numbers of grains or cell nuclei in six areas.

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Tensile Failure Test

Each hind limb was defrosted overnight at 4°C and thawed at room temperature inside the plastic bag on the day of testing.40

An Instron-type testing machine (Tensilon RTM-500, Toyo Baldwin, Tokyo, Japan) was used for the tensile failure test.28 Under a dissecting microscope, all soft tissue was removed from the femur and tibia, leaving only the cruciate ligaments, collateral ligaments, and menisci. The specimens then were mounted in a specially designed device using acrylic resin.28 The femur and tibia were oriented at an angle of 60° and 0° from the loading axis, respectively, so that the load was directed along the longitudinal axis of the MCL.17 The remaining soft tissues around the knee, except for the MCL, were divided sharply.

The test was done at a displacement rate of 10 mm/minute. A load-deformation curve was recorded, from which the ultimate load and the stiffness were measured, and the mode of failure was observed macroscopically.

Analysis of variance with independent measures was used to compare results between the groups and with time with p < 0.05 considered significant.

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RESULTS

Weight gain in Group H was 4.4 ± 4.2 g at 3 days, 12.0 ± 4.0 g at 7 days, 22.0 ± 11.3 g at 14 days, and 46.3 ± 14.8 g at 28 days, whereas weight gain in Group C was 10.0 ± 8.0 g at 3 days, 24.1 ± 12.6 g at 7 days, 36.9 ± 8.0 g at 14 days, and 60.1 ± 7.8 g at 28 days. There was a significant difference in weight gain between the groups at 7, 14, and 28 days postoperatively (p = 0.010, 0.002, and 0.015, respectively). There were no skin or soft tissue infections after surgery.

The amount of the scar tissue was greater in Group H than in Group C (Fig 1). On macroscopic inspection, the gap in the MCL was filled with scar tissue in each animal, which reached a maximum amount 14 days postoperatively in both groups. The healing MCL had more scar tissue in Group H than in Group C at each time, but the difference between the groups was greatest at 7 days (Fig 1).

Fig 1.

Fig 1.

The histologic findings were similar in both groups. Three days postoperatively, inflammatory cells dominated the scar tissue, but decreased later, so that active fibroblasts dominated most fields by 2 weeks. Cells were arranged randomly at first, but there was some evidence of longitudinal alignment of the nuclei by 28 days.

Hybridization signals representing mRNA expression were observed as dark silver grains over specific cells in the sections incubated with the antisense probe. Such specific signals were not seen with the sense probe (data not shown).

Type I procollagen gene expression in the scar tissue increased more in Group H than in Group C at 7 and 14 days (Fig 2). Three days postoperatively, increased expression of the Type I procollagen gene was observed in the scar tissue filling the gap in each ligament, and there was no difference in the level of gene expression between the two groups. At 7 days, the healing ligaments showed increased expression in both groups compared with that at 3 days, but a more prominent increase was observed in Group H. The strongest expression was detected in the scar tissue developing between both cut ends of each ligament. A similar increase also was observed in the region of the ligament adjacent to the scar tissue. At 14 days, the level of gene expression was higher than at 7 days in both groups. Type I procollagen gene expression still was higher than in the MCL that was not treated surgically at 28 days, but lower than at 7 or 14 days (Fig 2). A low level of expression of the Type I procollagen gene was observed in the MCL of each group that was not treated surgically.

Fig 2.

Fig 2.

Semiquantitative analysis showed that the grain counts at 7 and 14 days were significantly greater in Group H than in Group C (p < 0.001 at 7 days, p = 0.005 at 14 days), and the difference between the groups was the most prominent at 7 days (Fig 3). The grain count at 7 days was significantly greater than at 3 days in both groups (p = 0.011 in Group C, p < 0.001 in Group H). At 14 days, the grain count was significantly greater than at 7 days in Group C (p < 0.001), but there was no significant difference between 7 and 14 days in Group H (p = 0.412). At 28 days, the grain count was significantly lower than at 7 or 14 days in both groups (p < 0.001 in both groups). There was no difference of the nuclear count between the groups or with time (Fig 4).

Fig 3.

Fig 3.

Fig 4.

Fig 4.

Tensile failure test showed that the ultimate load and stiffness of the healing MCL specimens in Group H was significantly greater than that in Group C at 14 days (p = 0.009, and 0.002, respectively) (Figs 5, 6). The ultimate load and stiffness for the healing MCL specimens improved with time in both groups, although none of the specimens reached a level equivalent with those of the side that was not treated surgically.

Fig 5.

Fig 5.

Fig 6.

Fig 6.

On the side that was not treated surgically, there was no significant difference between the groups or with time, except that the specimens from Group H at 7 days had a significantly lower ultimate load compared with those from Group C (p = 0.032) (Figs 7, 8).

Fig 7.

Fig 7.

Fig 8.

Fig 8.

All the healing MCL specimens failed in the midsubstance of the healing ligament, whereas all the MCL specimens that were not surgically treated failed by the ligament detaching from the tibia.

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DISCUSSION

The purpose of this study was to investigate whether administration of HBO has a beneficial effect on the ligament healing process. In addition to the amount of the scar tissue, we examined the expression of the Type I procollagen gene, because this is the initial event of Type I collagen synthesis and its increase as affected by the change in a milieu, such as administration of HBO, is likely to be detected even in the early phase of the ligament healing. We also examined the ultimate load and stiffness of the healing ligament to confirm that the increased expression of the Type I procollagen gene, together with the increased amount of the scar tissue, led to its early functional recovery.

One of the limitations of the current study was a relatively short followup for the final experiments. However, considering the great healing capacity of the MCL in rats,4,35 the results up to 28 days are significant. Other limitations include macroscopic investigation of the amount of the scar tissue. Cross-sectional area is the most common objective measurement for assessment of the amount of the ligament tissue in larger animals such as rabbits.5,12,16,17 However, the size of the chamber for HBO therapy led us to choose rats for experiment animals, in which the size of the MCL was too small to measure the cross-sectional area of the scar tissue.4,17,35 Therefore, the amount of the scar tissue was examined only macroscopically, and the detailed description of the macroscopic findings was avoided in the current study.

There have been only a few experimental studies about the effect of HBO on healing of ligaments or tendons. Ishii et al18,19 studied scar tissue formation and expression of the Type I procollagen gene by Northern blot hybridization in rats with lacerated patellar tendons, and found promotion of scar tissue formation and increased gene expression 7–14 days after laceration. Horn et al17 did tensile failure tests at 2, 4, 6, and 8 weeks after surgical laceration of the MCL in rats, and showed that the ultimate load of specimens from animals exposed to HBO was greater at 4 weeks than that of specimens from animals without HBO. They also showed that 33 of 48 (69%) failures were by the ligament detaching from the tibia, with only 15 specimens (31%) failing in the midsubstance of the ligament.17 The high incidence of the ligament detaching from its insertion probably was attributable to the greater healing capacity of the MCL in rats,4,35 and also indicated that the tensile properties of the healing ligament were not evaluated properly.

Horn et al17 used standardized surgical laceration of the MCL as their model of ligament injury, whereas we used a gap model in which the midsubstance of the MCL was removed. The reason for this was twofold. First, functional recovery of the injured ligament is slower when a segment is removed. The healing process of the gap model is known to be essentially the same as that of the laceration model, but healing takes much longer.5 All of our surgically treated MCL specimens failed in the midsubstance of the ligament, indicating that we succeeded in obtaining a midsubstance failure model at each assessment and that the tensile properties of the healing ligament were evaluated properly. Second, we assessed procollagen gene expression in scar tissue by in situ hybridization. Using the gap model, it was easier to distinguish the newly created scar tissue from normal ligament tissue on histologic sections.

Our study showed that the amount of scar tissue in the gap was greater in Group H than in Group C, indicating that HBO promoted scar tissue formation. Semiquantitative analysis by in situ hybridization showed the number of cell nuclei per unit area was equivalent in both groups, suggesting the scar tissue in both groups contained approximately the same density of cells. Therefore, a larger number of cells were considered to have formed in the scar tissue of Group H. This probably was attributable to HBO promoting fibroblast proliferation and angiogenesis.1,21,23,36

Our results on the serial changes of Type I procollagen gene expression in the healing MCL were essentially the same as those of Wiig et al38 and our previous study using a rabbit model.29 Administration of HBO increased expression of the Type I procollagen gene at 7 and 14 days, although there were no differences between the two groups 3 and 28 days postoperatively. These results also were consistent with those of Ishii et al.18 Our semiquantitative analysis showed more grains in Group H at 7 and 14 days compared with Group C, but there was no difference in the cell nucleus count. These results indicated that the fibroblasts in the scar tissue were stimulated to produce more Type I procollagen mRNA in Group H during this period.

Increased procollagen gene expression attributable to HBO strongly suggested that Type I collagen synthesis also was promoted,23 which would contribute to the recovery of the mechanical properties of the MCL in Group H. Together with the greater amount of scar tissue, this probably was the cause of the superior structural properties of the femur-MCL-tibia complex in Group H at 14 days.

Although the difference between the groups in the amount of the scar tissue and Type I procollagen gene expression was most prominent at 7 days, the difference in tensile properties was not significant at 7 days, but was significant at 14 days. This delay probably was attributable to the time difference between gene expression and protein synthesis. An enhancing effect of oxygen on collagen maturation also could have contributed.7

The optimal protocol for administration of HBO to treat ligament injury, including the pressure and duration, has not been determined. In this study, we selected 100% oxygen at 2.5 ATA for 2 hours per day, using previous studies as a reference.17,18,36 Ishii et al19 compared the effect on ligament healing of four treatments: normobaric room air, HBO at 1.5 ATA for 30 minutes daily, HBO at 2 ATA for 30 minutes daily, and HBO at 2 ATA for 60 minutes daily, and concluded that all HBO regimens were effective in promoting ligament healing, with HBO at 2 ATA for 60 minutes (the highest pressure and longest duration) being the most effective. The pressure and duration of HBO used in the current study were greater than in their study,19 therefore a more positive effect potentially could be produced, but there also is a possibility of negative effects because of longer exposure to a higher pressure. In our study, the ultimate load of the unoperated right femur-MCL-tibia complex was lower in Group H than in Group C at 7 days, which returned to the normal level at 14 and 28 days. The reason for this difference cannot be determined from the current study. However, it was not attributable to the direct effect of oxygen toxicity, because the toxic effects of oxygen are known to occur after exposure to high pressure for 3 hours or longer.9,32 Instead, the relative inactivity, poor food intake, or both, attributable to the stress of HBO could be the reason for the inferior tensile properties of the unoperated MCL at 7 days.39 The lower weight gain in rats in Group H might support this explanation.

However, additional studies are necessary to determine the optimal protocol for administration of HBO to treat ligament injuries before it is used clinically, because safety must be considered. Considering the possible adverse effect on the contralateral uninjured side shown in this study, the protocol for HBO may need to be revised (shorter duration or lower pressure or both, such as 2 ATA for 1 hour), although Staple and Clement32 recommended 1.5–2 hours of HBO daily for at least the first week after injury.

Another candidate therapy for enhancement of ligament healing is application of growth factors.13,16,30 There have been several reports describing favorable effects,13,16,30 but many problems remain to be solved before clinical use, including the cost and the method of administration. Hyperbaric oxygen therapy has been widely and reliably used for other conditions. Therefore, after the optimal protocol for application of HBO is determined, it can be used for treatment of ligament injuries.

Results of animal experiments like those of the current study cannot be applied directly to the clinical situation, but we hope that our findings provide encouragement to injured athletes who wish to return to sport as soon as possible. In the current study, a difference in tensile properties was seen only at 14 days and no longer was seen at 28 days. However, these results still are encouraging because better structural properties of the injured ligaments in the early phase allow acceleration of the rehabilitation program, even if the properties are lower than the normal values.

Administration of HBO promotes scar tissue formation and increases expression of the Type I procollagen gene in the scar tissue, and these effects are associated with improvement of the tensile properties of the healing ligament.

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Acknowledgment

The authors thank Y. Imaizumi from the Department of Physiology, Dokkyo University School of Medicine, for assistance in the experiments.

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