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Fibroblast responses to variation in soft tissue mobilization pressure


Medicine & Science in Sports & Exercise: April 1999 - Volume 31 - Issue 4 - p 531-535
Basic Sciences: Original Investigations

Fibroblast responses to variation in soft tissue mobilization pressure. Med. Sci. Sports Exerc., Vol. 31, No. 4, pp. 531-535, 1999. Augmented soft tissue mobilization therapy (ASTM), a newly developed massage therapy, has been successfully used in the treatment of chronic tendinitis patients. We theorized that the ASTM technique promotes healing through a controlled application of microtrauma.

Purpose: The purpose of this study was to determine morphologic changes in the rat Achilles tendon after enzyme-induced injury with collagenase and subsequent pressure variations in ASTM therapy.

Methods: Thirty male white rats were randomly assigned to one of five groups with six animals per group: tendinitis (A), tendinitis plus light ASTM (B), tendinitis plus medium ASTM (C), tendinitis plus extreme ASTM (D), and control with surgery only (E). ASTM was performed for 3 min, for a total of six treatment sessions. The Achilles tendons of each group were harvested 1 wk after the last ASTM treatment. Fibroblast numbers were assessed by light microscopy. An electron microscope was used to observe enlargement of fibroblasts.

Results: Statistical analysis of the number of fibroblasts present indicated a significant difference (P < 0.00) between group D and all other groups.

Conclusion: The morphological evidence indicated that the application of heavy pressure promoted the healing process to a greater degree than light or moderate pressure.

Biomechanics Laboratory, Department of Health and Physiology, Ball State University, Muncie, IN 47306

Submitted for publication March 1998.

Accepted for publication June 1998.

The authors would like to thank Thomas Sevier, M.D., and Performance Dynamics (Muncie, IN) for their financial support and the utilization of the ASTM instruments.

Address for correspondence: Gale Gehlsen, Director, Biomechanics Laboratory, School of Physical Education, Ball State University, Muncie, IN 47306. E-mail: GGEHLSEN@WP.BSU.EDU.

Several forms of massage or soft tissue mobilization techniques have been developed and utilized for the treatment of acute and chronic tendinitis (1). Cyriax and Russell (2) have used a deep friction massage to affect musculoskeletal ligament, tendon, and muscle structures. How soft tissue mobilization specifically functions to foster improved healing is not clearly understood. According to Norris (15), the purpose of frictional massage is to promote a local hyperemia, massage analgesia, and reduction of adherent scar tissue. Further, Prentice (17) has hypothesized that frictional massage may facilitate tendon healing by augmenting the inflammatory process to completion so the later states of healing can occur. Davidson et al. (3) support Prentice's theory and found that augmented soft tissue mobilization (ASTM) promotes healing via increased fibroblast recruitment.

In a clinical setting ASTM has been successfully used in the treatment of tendinitis. According to Sevier et al. (19), ASTM is a modification of traditional soft tissue mobilization and utilizes specifically designed solid instruments. These instruments are used, rather than hands and fingers of the therapist, to provide the contact mobilization force in the treatment of tendinitis. ASTM treatments like friction massage treatments require the therapist to apply a considerable amount of pressure. We hypothesize that the magnitude of pressure is related to the number of fibroblasts. Although much has been done in the identification of the physiological mechanisms involved in tissue repair, the magnitude of microtrauma necessary to induce change is not known. The purpose of this study was to determine morphologic response variations in ASTM pressure applied to the rat Achilles tendon after enzyme-induced injury with collagenase. Three different pressures levels (0.5 N·mm−2, 1.0 N·mm−2, and 1.5 N·mm−2) were applied and monitored during ASTM therapy.

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Animals. Male white Sprague-Dawley rats (30 wk of age) weighing between 225 and 263 g were used in this study. The animals (N = 30) were housed six per standard rat cage and fed rat chow and water ad libitum. The animals were randomly assigned to one of five groups with six animals per group: tendinitis (A), tendinitis plus light ASTM (B), tendinitis plus medium ASTM (C), tendinitis plus extreme ASTM (D), and control with surgery only (E). This study design and procedures were approved by the Ball State University Institutional Review Board in conformance with the guidelines for animal experimentation published by Medicine and Science in Sport and Exercise.

Achilles tendon injury. The left Achilles tendon was injected with collagenase to induce tendinitis. The direct injection of collagenase into the Achilles tendon provides useful model of tendinitis (7,23) Before the injection, the animals were anesthetized intramuscularly with a cocktail solution of Ketamine and Xylazine at respective dosages of 60 mg·kg−1 and 5 mg·kg−1. Next, the hair overlying the left Achilles tendon was removed with a topical depilatory agent. After cleaning the skin, a longitudinal incision was made slightly medial to the visible outline of the tendon. Using blunt dissection, the tendon was exposed distally near the calcaneal insertion. Under direct visualization, 30 μL of collagenase (10 mg·mL−1) was injected throughout the body of the Achilles tendon from the tendon muscle junction to the Achilles insertion. The incision was closed with several simple sutures of 5-0 Ethilon and a single 0.2-cc dose of Bicillin (300,000 U·mL−1) was administered intra-muscularly for prophylaxis against infection. The surgical site was allowed to heal for 3 wk before applying ASTM.

Augmented soft tissue mobilization (ASTM). A solid ASTM instrument was modified specifically for applying ASTM to the rat's Achilles tendon. A pressure transducer was embedded into the ASTM instrument and interfaced to a computer. With the aid of a computer program, pressure curves were displayed on the monitor and used to regulate treatment pressure. Animals in group B underwent light pressure (0.5 N·mm−2) ASTM; group C underwent medium pressure (1.0 N·mm−2) ASTM; group D underwent extreme pressure (1.5 N·mm−2) ASTM. To administer ASTM, animals were anesthetized via inhalation with metofane and placed in a prone position with the left rear foot elevated for access to the Achilles tendon. Cocoa butter was used as a lubricant between the skin and the instruments. The tendon was then massaged with the ASTM instrument in a longitudinal plane, moving distal to proximal and proximal to distal along the length of the tendon. ASTM was performed on animal groups B, C, and D using three distal to proximal and three proximal to distal massage movements every 4 d for a total of six treatments.

Light microscopy. One week after the last ASTM treatment, the tendons were prepared for light microscopy to detect structural changes. The animals were sacrificed by a lethal intraperitoneal injection of Ketamine combined with Xylazine and followed by thoracotomy. To facilitate identification and the structural integrity, the Achilles tendons were harvested by excision of the lower limb superior to the knee joint. The foot and fur was removed from the excised limb. The limb was then fixed overnight in 10% buffered formalin and subsequently decalcified in two changes of 1 N HCL for 6 d. The specimen was then bisected along the midsagittal plane. The resultant specimens were then dehydrated through a graded alcohol series and xylene, and embedded in paraffin. Tissue sections were cut at 4 μ, stained with hematoxylin, counterstained with eosin, mounted, and observed with a bright field microscope. Fibroblasts were identified by their spindle shape and characteristic pale staining, large round nucleus.

Fibroblast numbers were assessed by light microscopy. The specimens were randomly assigned numbers and blindly counted. Fibroblast counts were performed on 10 tendon specimens from each group. Counts from 10 random, 450× microscopic fields were recorded. The specimens were coded with an assigned number and randomly counted.

Electron microscopy. To study fibroblast structure with the transmission electron microscope, specimen reprocessing was required. The Achilles tendon was cut free from the paraffin blocks with a razor blade. The tendon was then deparaffinized in xylene, rehydrated, cut into 1 mm3 blocks, postfixed in 1% osmium tetroxide in 0.1 M cacodylate buffer, dehydrated in a graded series of ethanols, and embedded in LX-112 (Ladd Research Industries, Burlington, VT). After polymerization at 60°C for 24 h, the cured blocks were trimmed and 1-μm-thick sections were cut with glass knives on a LKB ultratome. The thick sections were stained with toluidine blue and surveyed with the bright field microscope. Favorable blocks were retrimmed and 800-900 nm ultrathin sections obtained with a diamond knife. The thin sections were picked up on 200 mesh copper grids, stained with 2% uranyl acetate, counterstained with lead citrate, and examined with a Hitachi H-600 transmission electron microscope.

Statistical analysis. The mean fibroblast count for each group was calculated and the significant difference (P < 0.05) between groups was determined by Kruskal-Wallis one-way ANOVA and Student, Newman, Keuls methods. Alpha level was set at 0.05.

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Light microscopy. With the light microscope, structural differences were observed in the tendons of the five groups. As illustrated in Figure 1, the control tendons (group E) exhibited parallel collagen fibers and a few elongated fibroblasts with oval nuclei and little cytoplasm. In comparison, the tendons of group A with collagenase-induced tendinitis demonstrated fiber misalignment and an increased number of fibroblasts (Fig. 2A). These fibroblasts had prominent, large round nuclei and more cytoplasm. Groups B, C, and D with tendinitis and treated with ASTM at different pressures (Fig. 2, B-D) also exhibited disrupted, randomly arranged collagen fibers with an apparent increase in the number of fibroblasts. The tendon fibroblasts of groups B, C, and D were often enlarged and displayed round nuclei and an abundance of cytoplasm. The tendons of group C treated with moderate ASTM pressure when compared with the tendons of group B treated with light ASTM pressure appeared to exhibit a greater number of fibroblasts. As compared with the other groups, the tendons of group D (Fig. 2D) treated with heavy ASTM pressure displayed the greatest number of fibroblasts.

Figure 1

Figure 1

Figure 2

Figure 2

Fibroblast counts. The observable differences in fibroblast numbers as seen by the light microscopy among the groups were more accurately assessed by fibroblast counts. The mean fibroblast cell counts of 10 microscopic fields at 450× for the five specimens from each group is depicted in Figure 3. Kruskal-Wallis one-way ANOVA on ranks indicated a significant difference (P < 0.00) between groups. Statistical power was 0.74. Post-hoc analysis (Newman-Keul) indicated a significant difference (P < 0.05) between group D mean fibroblast counts and all other group means. Significant differences (P < 0.05) were also demonstrated between groups A and E, groups B and E, as well as groups C and E. No significant difference were identified between groups B and C.

Figure 3-Fib

Figure 3-Fib

Electron microscopy. The electron microscope was utilized to determine whether the observed enlargement of fibroblasts in the tendinitis and variable pressure groups was due to fibroblast activation. The abundant presence of rough endoplasmic reticulum within the cytoplasm of fibroblasts is a structural feature associated with fibroblast activation and active collagen synthesis (3,11,24). Although the reprocessing of preserved tissues for electron microscopy is less than ideal, some fibroblasts examined in this study were sufficiently preserved to permit the identification of rough endoplasmic reticulum. Only tendons of groups A, D, and E were examined; group D was selected to represent the variable pressure group. The fibroblasts of the control tendons (group E) contained few cytoplasmic components and no observable organized rough endoplasmic reticulum (Fig. 4A). On the other hand, fibroblasts from group D (Fig. 4B) exhibited an abundance of rough endoplasmic reticulum within the cytoplasm signifying activation and protein synthesis. To a lesser degree, activated fibroblast were also observed in the tendons of group A.

Figure 4-E

Figure 4-E

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The results of this study indicated that ASTM not only stimulates fibroblast proliferation but that this proliferative response is apparently dependent upon the magnitude of the applied ASTM pressure. Fibroblast proliferation and activation play a key role in the tendon healing process. Fibroblasts are responsible for the further production of cellular mediators of healing and proteinaceous synthesis of collagen fibers (6,11,24). Activated fibroblasts are commonly characterized by an increase in rough endoplasmic reticulum, a prominent round nucleus, and an increase in ribosomes. Such morphological features of fibroblasts are associated with collagen synthesis (3,4). The observance of activated fibroblasts with well develop rough endoplasmic reticulum observed in the present study tends to indicate that ASTM facilitates tendon healing by the recruitment and activation of fibroblasts. The results of this study support the work of Davidson et al. (3), who also reported significant increase of fibroblasts in rat Achilles tendon with the application of ASTM.

The corresponding changes in applied therapeutic pressure and fibroblast proliferation found in this study suggest that pressure may provide the initial stimulus for the healing cascade. An alternate form of pressure, ultrasound, has also been shown to facilitate fibroblast proliferation and protein synthesis (22,26). Madden and Smith (13) states that cyclic effects of ultrasound are related to oscillatory movements accompanied by waves of pressure that are repeated at each wave cycle. According to Madden and Smith (13), ultrasound induces a kind of "micro-massage," which could facilitate tissue repair by reducing edema. It has also been reported that during the remodeling process, the application of physical force through both stress and motion help to modulate the synthesis of proteoglycans and collagen by the fibroblasts (16,20,25). Stearns (20) observed the fibroblastic activity in the healing of connective tissue as well as possible scar formation, were related to effects of tissue movement. Cyriax and Russell (2) contended that movement encouraged realignment and lengthening of the fibers.

In recent studies, the issue of mechanical force eliciting cellular responses has gained considerable support. For example, mechanical stretching of fibroblasts has been shown to stimulate their proliferation (9) and affect their synthesis of extracellular matrix proteins (21). As reviewed by Glanz (5), mammalian cells may have force-carrying connections that extend from the cell membrane to the nucleus. Ingber (8) has postulated that the cytoskeleton may function like a tensegrity structure to transmit forces. Because a tensegrity structure act like force-carrying network, forces applied to the cell surface would quickly be propagated by the cytoskeleton into the interior of the cell. Maniotis et al. (14) have shown that mechanical forces acting though integrin receptors on the surface of bovine capillary endothelial cells were transferred by cytoskeletal components and caused realignment of nuclear structures. Mechanical stimuli have been shown to alter many cellular functions including ion transport (18), release of second messengers (12), protein synthesis (21), secretion (23), and even gene expression (10). From a clinical standpoint, the mechanical stimulation applied to cells by soft tissue mobilization may play a significant role in modifying cellular functions. In support of this suggestion, the present study has demonstrated that fibroblast proliferation and activation increases with the magnitude of the applied ASTM force.

In conclusion, the results of this study support the notion that ASTM has a positive effect on healing of collagenase induced rat Achilles tendon tendinitis. The results show that the proliferation and activation of tendon fibroblast is dependent upon the mechanical force applied during the ASTM treatment. When compared with light and moderate pressure, the application of heavy pressure appears to best promote fibroblast proliferation, which may serve to augment the healing process. Much research needs to be done, but the clinical implications of this study to the health practitioner are exciting. Insurance organizations are beginning to dictate the number of visits and length of stay of rehabilitation patients. It is therefore incumbent upon health care practitioners to find more effective and efficient means of nurturing the healing process. Improved fibroblast response may lead to improved tensile strength of the healing tissue and identifying the optimal pressure to facilitate this response may lead to fewer treatment sessions. This response may be of particular significance for patients suffering from cumulative trauma disorders such as Achilles tendinitis, rotator cuff tendinitis, patellar tendinitis, golfer's elbow, and tennis elbow. By applying ASTM in a manner that best stimulates the healing response, the goal of decreasing the cost of health care while optimizing patient outcome can be achieved.

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