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Herbal remedies improve the strength of repairing ligament in a rat model.

Abstract

Herbal remedies have been reported to be effective in controlling inflammation for acute soft tissue injuries. There exist, however, no reports of their effects on collagen production and remodeling; thus mechanical strength studies of the tissues have not been reported. This study tested the effects of a herbal remedy on the strength of healing medial collateral ligaments (MCL) in rats. Sixteen rats receiving surgical transection to their right MCLs and eight receiving sham operation were tested. Eight of the MCL-injured animals were treated with an adhesive herbal plaster application to their right knees, while the other eight in the MCL injured group and the sham group were treated with plain adhesive plaster to their right knees. The MCLs were harvested and tested at either 3 or 6 weeks post-operation. The ultimate tensile strength (UTS) and stiffness normalized to the uninjured side of each animal of the herb and sham groups were significantly larger than those of the control at both 3 and 6 weeks (p = 0.001). No significant difference was found in stiffness between the herb and sham groups (p > 0.05). We concluded that the herbal remedy improves the UTS and stiffness of repairing MCLs at 3 and 6 weeks after injury.

[c] 2004 Elsevier GmbH. All rights reserved.

Keywords: Healing; Injury; Tissue biomechanics; Herbal medicine; Rat

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Introduction

The use of herbal remedies in Chinese medicine has a long history of over three thousand years (Wong and Dahlen, 1999). In modern Chinese medical practice, the traditional remedies have been merged into Western-based medical practice for clinical evaluation and management of different conditions, so as to provide an evidentiary basis for Chinese medicine (Cai, 1988; Chi, 1994; Chan, 1995; Hesketh and Zhu, 1997).

Various herbal prescriptions have been found effective in reducing swelling, pain and improving joint mobility in acute joint sprains (Lam, 2001; Ko and Wong, 2002). However, the effects of herbal treatments on the quality of the healing tissues have not been reported. Previous studies have found that tendon and ligament scars have inferior mechanical properties (Frank et al., 1992; Reddy et al., 1999). With the positive reports of the clinical efficacy of herbal remedies, there is a need to evaluate whether herbal treatment improves the biomechanical strength of the healing tissues.

Previous studies have explored the effects of individual herbs and found that Dryobalanops aromatica, Capsicum frutescens, Cinnamomum cassia and Panax notoginseng have anti-inflammatory effects on various soft tissue conditions (Akira, 1987; Kubo et al., 1996; Li and Chu, 1999; Han et al., 2001). Some recent studies have suggested that the anti-inflammatory effects of herbs such as Eugenia caryophyllata and Cinnamomum cassia were attributed to the scavenging of nitric oxide free radicals by these herbs in the tissues (Yokozawa et al., 2000; Toda, 2001). Takei et al. (1996) found a basic fibroblast growth factor (bFGF)-like molecule in P. notoginseng which has immunoreactivity characteristics and molecular weight similar to human bFGF, and bFGF has been reported to enhance the healing of ligaments in vitro and in vivo (Fukui et al., 1998; Tang et al., 2001), which explains the pharmaceutical actions of P. notoginseng when used for treating ligament injuries.

In light of the potential therapeutic effects of individual herbs, we were interested in investigating a commercial herbal remedy (Chongqing Peidu Pharmaceutical Factory China, 1999) of the composition shown in Table 1. According to a high performance liquid chromatography (HPLC) analysis of this herbal remedy (Tong, 2000), Ginsenoside Rb1 was identified as one major effective constituent. Due to the complexity of this remedy, which consists of 12 different herbs, further chemical analysis may be required to acquire a full "fingerprint". In light of clinical interest, however, we decided to test the mixed remedy as a whole, instead of its individual constituents.

We have reported recently that low energy laser therapy (LLLT) is effective in improving the biomechanical properties of the healing medial collateral ligament (MCL) of rats (Fung et al., 2002). Adopting the same model and methodology, we aimed to examine if the herbal remedy shows the same effects on tissue strength.

Soft tissue healing comprises three phases, namely, inflammation, repair and remodeling (Enwemeka, 1989; Oakes, 1994). Each phase has its unique timing and function in preparation for the next phase. We are interested in the repair (3 weeks) and early remodeling (6 weeks) phases, in which fibroblast proliferation, fibrillogenesis and fibril remodeling are most active (Enwemeka, 1989; Oakes, 1994).

The present study was aimed at testing the effect of an externally applied herbal remedy (Chongqing Peidu Pharmaceutical Factory China, 1999) on the biomechanical performance of healing ligaments at 3 weeks and 6-weeks post-injury in a rat model.

Materials and methods

Twenty-four mature, male Sprague-Dawley rats with mean body weight of 302 g each (range 275-326 g) and aged 12 weeks at surgery were used for the study. The Animal Subjects Ethics Review Committee of the administering institution approved this study. The animals were allocated randomly into three groups with two testing times (Table 2).

All surgical procedures were carried out under general anesthesia with intra-peritoneal injection of a mixture of ketamine (100 mg/ml) and xylazine (20 mg/ml). The dosages of the drugs applied were calculated with the ratios of 8 mg ketamine and 0.8 mg xylazine per 100 g animal weight. For the herb and control groups, MCL of the right knee was exposed and transected completely at the middle portion with a scalpel. For the sham group, only a skin wound was induced but the MCL was not injured. The skin wounds were sutured immediately after surgery. A remedy plaster with 12 herbs (Table 1) was applied over the skin of the medial knee and secured further with adhesive bandaging in the herb treatment group. This herbal plaster was changed on alternate days to prevent drying of the medication. The size of each plaster was 3.5 cm X 5 cm, the total weight 0.75 g. Neglecting the weight of the adhesive plaster base (0.28 g), the weight of the herbs applied was 0.47 g. Therefore, the weight of each herb ingredient applied can be calculated by multiply its weight % (Table 1) by 0.47 g. The HPLC-fingerprint analysis of the testing herbal remedy was performed according to Tong (2000). For the HPLC-data and the fingerprint-chart contact the authors.

According to the manufacturer, the herbal remedy was made from the dried plant parts of the different herbs in Table 1. With the proportion according to the dry weight, the herbs were mixed and grounded into a powder form. This herbal powder was than mixed with a rubber base, from which an herbal remedy plaster was formed (Chongqing Peidu Pharmaceutical Factory China, 1999; Ko and Wong, 2002).

For the control and sham groups, the surgical procedures were the same as for the herb treatment group, except the adhesive plaster bandaging without any herbal medication was applied to the operated knees. The bandaging was changed on alternate days for both groups.

The rats were maintained in cages without restraint, inside a room with a 12-hour daylight cycle. Food and water were given ad libitum. The rats were euthanized at either 3 or 6 weeks post-surgery, by double-dose injection of anesthetics. Both lower limbs were harvested by dis-articulation at the hip joint, sealed in a plastic bag and stored in a freezer at -40[degrees]C.

Biomechanical testing procedures

At 6 h before testing, the legs were retrieved from the freezer and thawed at room temperature inside the plastic bag. Each leg specimen was carefully dissected to remove all soft tissues in the knee joint, leaving only the femur-MCL-tibia complex intact. The length of the MCL was measured with a pair of vernier calipers. The room temperature was controlled at 25[degrees]C and the specimen was kept moist with normal saline throughout the tests.

The biomechanical tests were done following our previous study of the same MCL model (Fung et al., 2002). The free ends of the femur and tibia were potted in small metal cylinders with a non-exothermic, easy-setting polymer. The cylinders were then mounted on two specially designed clamps, fixed on a MTS Synergie 200 machine (MTS Systems Corporation, Minnesota, USA) at 5[degrees] of knee flexion. A sliding table of the x-y plane was built for the lower cross-head of the MTS machine, so that the tibial clamp could be adjusted and aligned with the femur. A MTS 634.12F-24 extensometer (MTS Systems Corporation, Minnesota, USA) was attached to the lower end of the femur and the upper end of the tibia for measuring the local displacement.

Testing for viscoelasticity

After the specimen was mounted on the material testing machine, it was pre-conditioned with 10 oscillation cycles of 2.5% strain at a rate of 10 mm/min to minimize the effect of deep freezing on the tissue (Woo et al., 1986). After pre-conditioning, the specimen was elongated to 2.5% strain and maintained for 10 min to test for its load-relaxation behavior (Ng et al., 1996). The loads were recorded throughout the test at a sampling rate of 5 Hz. The difference between the initial and final load was expressed as a percentage of the initial load to represent load-relaxation.

Testing for stiffness and UTS

After the above testing, the specimen was unloaded, moistened with normal saline, and left for 5 min on the machine to allow the ligament to resume its original length. Afterwards, it was subjected to ultimate tensile testing at an elongation rate of 500 mm/min until failure. Load and displacement data were recorded at a sampling rate of 50 Hz. The maximum load recorded represents the UTS, and the gradient in the linear portion immediately after the toe region of the load-displacement curve represents the stiffness. The UTS and stiffness values of the right legs were normalized against the left leg of each animal before using them for data analysis.

Statistical analyses

Two-way analysis of variance (two-way ANOVA) was used to analyze the load-relaxation, UTS and stiffness data, with treatment group and sacrifice time as the independent factors. Post-hoc linear contrast was conducted for the significant ANOVA results and [alpha] was set at 0.05 for all the tests.

Results

On visual examination, all the repairing MCLs in the herb and control groups showed signs of repair with thick fibrous scar formation at the injury sites. The right MCLs in the herb and control groups appeared to be larger than that of the sham groups. The anatomical orientations of all the MCLs were comparable at the macroscopic level.

Fig. 1 reveals that both the herb and sham groups had lower load-relaxation values than the control group (p = 0.072). For UTS, the herb and sham groups values were significantly higher than the control group value (p = 0.001) (Fig. 2), and the sham group value was higher than that of the herb group (p = 0.019). Although no statistically significant difference was found between the UTS results of the two tested time frames (p = 0.334), a sign of increase UTS with time was observed in both the herb and control groups, and the herb group appeared to have improved UTS more rapidly than the control group in the studied period (Fig. 2).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

The herb and sham groups had significantly higher normalized stiffness than the control group (p = 0.001) (Fig. 3). No significant difference was found in stiffness between the herb and sham groups (p > 0.05). Across the two testing periods, no significant differences were found in stiffness within each group.

Discussion

This study aimed to examine the effects of herbal remedies on the biomechanical properties of repairing ligament. The results have demonstrated that external herbal application can promote recovery of surgically transected MCL in terms of viscoelastic and biomechanical behaviors.

[FIGURE 3 OMITTED]

Load-relaxation is a parameter used to evaluate the viscoelastic behavior of soft tissue under sustained loading (Ozkaya and Nordin, 1991). Previous studies have shown that scar tissues load-relaxed more rapidly than normal tissues (Frank et al., 1985, Ng et al., 1996), and this is detrimental to the joint because the load not taken up by the scar would overload other joint structures. In the present study, the herbal treatment group demonstrated less load-relaxation than the control specimens under a sustained loading condition (Fig. 1) (p = 0.072). This suggests that the herbal medication has improved the viscoelastic properties of the repairing ligaments.

Both the normalized UTS and structural stiffness of the herb group are better than the control (Figs. 2 and 3), and the herb group has comparable structural stiffness to the sham group (Fig. 3). These findings demonstrate that herbal remedies not only have anti-inflammatory effects in the initial phase after injury (Akira, 1987; Kubo et al., 1996; Li and Chu, 1999; Han et al., 2001), but also affect the later fibrillogenesis and remodeling phases by improving tissue biomechanical strength.

Our findings have important clinical implications, because tendons and ligaments are known to be weakened after injury (Frank et al., 1992, Reddy et al., 1999). If herbal medications can improve the strength of the scars, they can salvage the other joint structures and increase the safe functional limit for the joint. Although findings in rats may not be directly applicable to humans, their soft tissue healing processes are similar and the present findings suggest that the therapeutic effects of these herbal remedies could also be demonstrated in man, which warrants further investigations.

Enwemeka (1989) demonstrated that an injured rabbit Achilles tendon took 3 weeks to achieve a regular morphological pattern for the newly formed collagen fibrils. Parry and Craig (1977, 1978) reported positive relationships between collagen fibril size and strength in various tissues of different animals. Ng (1995) studied the ultrastructural collagen diameter in injured ACLs in goats and found that the collagen fibrils had gradually increased in size over time and there was a concomitant increase in breaking strength of the ligament. Based on these reports (Parry and Craig, 1977, 1978; Ng, 1995), it may be postulated that improvements in biomechanical strength of the MCLs in the present study are due to a facilitation in production and remodeling of the collagen. We are currently conducting a study examining the ultrastructural collagen fibril morphology of herbal treated MCLs so as to study the mechanisms underlying the improved strength.

We have previously reported that low energy laser therapy improved the mechanical properties of surgically transected MCLs in the same animal model and time intervals (Fung et al., 2002). When comparing the data of that study with the present study, the effect of herbal remedies is shown to be better than that of laser therapy in structural stiffness (Fig. 4). This is an exciting finding and promising for the future development of herbal remedies and their applications.

[FIGURE 4 OMITTED]

Most studies on the effects of herbal remedies have been conducted in China and reported in the Chinese literature (So, 2000; Yeung, 2000; Lam, 2001; Ko and Wong, 2002). The majority of these studies compared the clinical signs and symptoms in patients with a mix of peripheral joint symptoms before and after treatment. These studies might be criticized for not having a homogeneous group and some even lacked control groups. In the present study, we applied the concept of a randomized, controlled trial with a homogeneous injury model and the experimental procedures are well-established. Our study addresses the basic scientific effects of herbal medications and will pave the way for further clinical studies and evaluations to justify the use of herbal remedies.

Conclusion

We have shown that external herbal application can improve viscoelastic load-relaxation behavior and restore the ultimate tensile strength and stiffness of repairing MCL after complete transection in rats at 3 and 6 weeks post-injury.
Table 1. Ingredients of the tested herbal remedy (Chongqing Peidu
Pharmaceutical Factory China, 1999)

Herb ingredient Plant part % by dry weight

Panax notoginseng (Burk.) F.H. Root 8
Chen
Dryobalanops aromatica Gaertn. F. Resin 8
Eugenia caryophyllata Thunb. Flower bud 4
Cinnamomum cassia Presl. Bark 6
Mentha haplocalyx Briq. Leaf 13
Capsicum frutescens L. Fruit 8
Ilex pubescens Hook. et Arn. Root 13
Sparganium stoloniferum Buch.- Rhizome 8
Ham.
Aconitum kusnezoffli Reichb. Rhizome 8
Zanthoxylum nitidum (Roxb.) D.C. Root 8
Curcuma phaeocaulis Valeton Rhizome 8
Aquilaria sinensis (Lour.) Gilg. Resin 8

Table 2. The herb, control and sham groups of the study

Group Surgery (right MCL) Treatment Time of
 sacrifice
 (weeks after
 surgery)

Herb (n = 4) Transected Herb remedy with 3
 plaster bandaging
Control (n = 4) Transected Plaster bandaging 3
 only
Sham (n = 4) Exposed only Plaster bandaging 3
 only
Herb (n = 4) Transected Herb remedy with 6
 plaster bandaging
Control (n = 4) Transected Plaster bandaging 6
 only
Sham (n = 4) Exposed only Plaster bandaging 6
 only


Acknowledgements

We acknowledge The Hong Kong Polytechnic University Area of the Strategic Development Fund and the Research Grant Council of Hong Kong for financial support of this study. The technical support of Dr. Mason Leung of the Department of Rehabilitation Sciences, The Hong Kong Polytechnic University, and Dr. David Tay of the Department of Anatomy, The University of Hong Kong, is gratefully acknowledged.

Received 10 September 2003; accepted 23 October 2003

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D.T.C. Fung, G.Y.F. Ng*

Department of Rehabilitation Sciences, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong

*Corresponding author. Fax: +852-23308656.

E-mail address: rsgng@polyu.edu.hk (G.Y.F. Ng).
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Author:Fung, D.T.C.; Ng, G.Y.F.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Date:Jan 1, 2005
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