Inflammation, cellularity, and fibrillogenesis in regenerating tendon: implications for tendon rehabilitation.

Inflammation, Cellularity, and Fibrillogenesis in Regenerating Tendon: Implications for Tendon Rehabilitation

The initial three weeks of tendon healing were followed via electron microscopy to elucidate the process of inflammation, fibrillogenesis, and the cellular and subcellular events in tenotomized Achilles tendons, a model that is commonly used to determine the biomechanical effects of electrical stimulation, physical activity, ultrasound, and other forms of physical therapy. The right Achilles tendons of 18 rabbits were tenotomized, sutured, and immobilized. On each of postoperative days 5, 7, 12, 15, 18, and 21, the right Achilles tendons of three experimental rabbits were excised and processed for electron microscopy. To compare these tendons to normal tendons, the Achilles tendons of three control rabbits were excised bilaterally without prior tenotomy and processed for electron microscopy. Electron micrographs thus obtained revealed 1) an initial period of inflammation lasting at least five days, 2) a subsequent period of fibroplasia and fibrillogenesis, and 3) a third period of progressive alignment and organization of the collagen fibrils into bundles that were oriented in the longitudinal axis of the tendon. Although healing in rabbits may not translate directly to healing in humans, the findings of this study indicate that healing begins soon after tenotomy and that the regenerating Achilles tendon undergoes different stages of healing. Because each stage entails a different set of ultrastructural events, therapeutic interventions should be modified to address the specific events of each stage. [Enwemeka CS: Inflammation, cellularity, and fibrillogenesis in regenerating tendon: Implications for tendon rehabilitation. Phys Ther 69:816-825, 1989]

Key Words: Achilles tendon; Fibroblasts; Inflammation; Microscopy, electron; Wound healing.

In clinical practice, the six to eight weeks of plaster cast immobilization required after surgical repair of Achilles tendon ruptures[1-10] frequently produce muscle atrophy,[11-15] osteo-arthritis,[16-19] atrophy and ulceration of joint cartilage,[19-21] skin necrosis,[13-22] infection,[12,15] tendocutaneous adhesion,[12,15,22] and thrombophlebitis.[12,22] These complications retard postoperative care because they must be overcome simulataneously with attempts to restore normal function. More than a decade ago, changes in surgical technique and carbon and polyester fiber implantation were simultaneously proposed as means of overcoming these complications.[22-25] Although two new surgical techniques--percutaneous approximation of tendon ends[22] and external fixation[25]--were subsequently developed and found useful in preventing skin necrosis, tendocutaneous adhesions, and rerupture, other complications of prolonged immobilization persist as eight or more weeks of immobilization remain necessary. Similarly, carbon and polyester implants reportedly enhanced healing,[23],[24] but eight or more weeks of plaster immobilization are still required.[26-29]

Because prolonged immobilization is necessitated by the slow rate of healing in tendons, the possible use of physical agents and procedures to quicken healing and hence minimize complications has been the focus of several investigations.[30-35] In these endeavors, a knowledge of the ultrastructural events associated with tendon healing is crucial to proper timing of those therapeutic interventions that may modulate healing. Although numerous studies have described these events in digital or tail tendons,[34-49] there is a dearth of similar studies on the Achilles tendon--the principal focus of these investigations. Digital and tail tendons differ from the Achilles tendon in that they transmit minimal forces, not the tremendous forces of locomotion and weight bearing.[50],[51] Such disparity in load history has been shown to produce metabolic and morphologic differences among tendons.[52-57] The rate of healing of the Achilles tendon, therefore, may differ from those of digital or tail tendons. Consequently, timing of physical therapy intervention should be based on the ultrastructural changes associated with healing of the Achilles tendon, not those of digital or tail tendons.

The ultrastructure of the healing rat calcaneal tendon has been studied in a model of complete tenotomy and repair[47]; however, previous studies on the healing calcaneal tendons of rabbits used an experimental model of partial tenotomy without cast immobilization and focused mainly on the synthesis of microfilaments, fibrils, and elastic fibers.[58-61] Little attention was paid to the processes of inflammation and cellularity. Because immobilization imposes additional constraints on the healing tendon, the ultrastructural changes reported in this model may not reflect those of complete tenotomy and immobilization, which more closely resemble the clinical situation of rupture, repair, and plaster cast immobilization in humans. The purpose of this investigation, therefore, was to study the ultrastructural changes associated with the initial three weeks of healing in tenotomized rabbit Achilles tendons. Specifically, our aims were 1) to elucidate the cellular and subcellular events that follow tenotomy and repair of the Achilles tendon, 2) to study the process of inflammation, 3) to describe fibrillogenesis, and 4) to explore the time course of these events.

Method

Animals

Twenty-one 4- to 6-month old New Zealand rabbits weighing between 1.2 and 2.5 kg were used for this study. The animals were housed one per standard 30.5- x 71- x 51-cm rabbit cage in an environment maintained at 21 [degrees] to 23 [degrees] C and fed rabbit chow and water ad libitum.

Tenotomy and Plaster Casting

Eighteen of the rabbits were weighed and then anesthetized with a mixture of 180 mg of xylazene hydrochloride, 900 mg of ketamine, and 30 mg of acepromazine maleate by intravenous injection of 1 mL per 1.5 kg of body weight. In each rabbit, the fur overlying the right Achilles tendon was shaved and the skin cleaned before a longitudinal incision was made slightly medial to the visible outline of the tendon. By blunt dissection, the tendon was separated from the adjoining tissue and severed sharply and transversely 1.5 cm above its calcaneal insertion. The severed ends of the tendon were then approximated and sutured with three loops of 3.0 surgical silk. Subsequently, the skin incision was closed by interrupted sutures and the limb immobilized in a plaster cast with the knee flexed 90 degrees and the ankle fully plantar flexed. No surgery was performed on the remaining three rabbits, which served as normal controls.

Tendon Excision

On each of postoperative days 5, 7, 12, 15, 18, and 21, the right Achilles tendons of three experimental rabbits were excised and processed for electron microscopy. Prior to tendon excision, each rabbit was again weighed and anesthetized as previously described. The skin suture of the right hind limb was carefully removed and the incision reopened. After freeing the Achilles tendon from the surrounding tissues, the tendon sutures were removed. Two sharp transverse cuts were then made to excise the neotendon formed at the site of tenotomy. Subsequently, each excised neotendon was fixed in 2% paraformaldehyde-2.5% glutaraldehyde (pH 7.4), and the animal was sacrificed with an overdose of anesthesia. To compare the tenotomized tendons of the 18 experimental rabbits with normal tendons, the Achilles tendons of the three control rabbits were excised bilaterally and immersed in a solution of the same fixative.

Tissue Processing and Transmission Electron Microscopy

The excised neotendon was then immersed in a petri dish of fixative, sliced several times to yield approximately 2- x 7-mm specimens that were fixed for two hours in 2% paraformaldehyde-2.5% glutaraldehyde (pH 7.4), buffer washed, and then postfixed for another two hours in a 1% aqueous solution of osmium tetroxide (pH 7.4). Thereafter, each specimen was washed with distilled water and dehydrated in graded alcohol before final dehydration in propylene oxide. After gradual infiltration with a mixture of propylene and EMBED 812 resin,(*) the specimen was embedded in 100% resin and kept in an oven at 60 [degrees] C for 65 to 70 hours. Each resin-embedded specimen was then trimmed and sectioned with glass knives at a thickness of 1 [Mu]m to verify the level of sectioning by light microscopy. Thereafter, a diamond knife was used to obtain representative ribbons of ultrathin (50-70 nm) silver-silver gray transverse and longitudinal sections that were stained with both uranyl acetate and lead nitrate for 10 minutes each.[62]

From each specimen, two to four light microscopy slides and five grids containing a ribbon of six or more ultrathin sections were prepared. Ultramicrotomy was performed to ensure that each set of grid-mounted ultrathin sections was separated from the next by at least 10 thin sections. Using the transmission mode of a JEOL 100CX scanning-transmission electron microscope,([dagger]) the profiles of the grid-mounted sections were visualized and photographed. A minimum of 170 electron micrographs were consistently obtained for each healing period. One third of these electron micrographs were taken at high magnification, usually in the range of 74,000 to 110,000 x. These photographs and the observations recorded when the sections were examined in the electron microscope were used to evaluate healing.

Computer Morphometry

Regardless of healing period, newly produced collagen fibrils were clearly smaller and less variable in cross-sectional area than the fibrils of the intact nontenotomized tendons. To objectively identify and distinguish these new fibrils from older ones, the high-magnification electron micrographs were placed on a Jandel Scientific digitizing tablet([double dagger]) interfaced to an IBM PC/XT computer([section]) with SIGMA-plot software([double dagger]) designed to compute morphometric measurements. To ensure accuracy of measurement, the system was first calibrated, then the electronic pen of the digitizer was used to carefully trace the outline of each fibril. Simultaneously, the cross-sectional area of each fibril was automatically computed and stored in the computer. A total of 13,379 fibrils, representing 1,400 to 3,500 fibrils per group, were measured. Because the five-day neotendons had very few and sparsely distributed new fibrils, no morphometric measurements were made on the high-magnification electron micrographs obtained from this group.

Results

The normal rabbit Achilles tendon consisted of closely packed bundles of collagen fibrils with relatively few fibroblasts and elastin bundles (Figs. 1, 2). Only in the lumen and walls of blood vessels were other types of cells seen. The fibroblasts were generally stellate in shape with a few cytoplasmic strands running between adjacent bundles of collagen. Fibroblast nuclei, remarkably varied in shape, were consistently prominent and large with several aggregates of chromatin along the inner surface of the nuclear membrane. In longitudinal section, collagen fibrils were striated with distinct light and dark bands.

Sections of regenerating tendons differed remarkably from this normal appearance. Electron micrographs prepared from transverse and longitudinal sections of specimens obtained on postoperative day 5 revealed patches of fibrin, blood clot, blood vessels, and exuded plasma cells and erythrocytes (Figs. 3-5). In addition, there was an abundance of monocytes, eosinophils, neutrophils, lymphocytes, platelets, and macrophages. Platelets and monocytes were usually found within the vicinity of several masses of fibrin, some of which they may have been phagocytizing (Fig. 5). Occasionally, ring-shaped aggregates of large lipid droplets and adipocytes were seen, as were some blood vessels and collagen fibrils. These fibrils were sparse and scattered, but had the alternating dark and light bands of mature fibrils (Fig. 3). In comparison with older fibrils, newly synthesized collagen fibrils have smaller and less variable maximum fiber cross-sectional areas (Table).[63-69]

By the seventh postoperative day, fibrils were numerous and generally lay in disarray in the extracellular compartment. Occasionally, however, pockets of well-organized, longitudinally arranged fibrils were seen (Fig. 6). Ground substance was remarkably abundant, as were cell fragments and debris. There were numerous fibroblasts, each with a well-developed nucleus and massive cytoplasm. These cells and their nuclei varied remarkably in shape, but as a rule each fibroblast had an elaborate network of rough endoplasmic reticulum (rER), several electron-lucent vesicles, and a few dark granules (Fig. 7). Only a few inflammatory cells were seen in these sections.

Electron micrographs of the 12-day neotendons contained several fragments of cells, new blood vessels, and some inflammatory cells, mostly monocytes, lymphocytes, mast cells, and macrophages. There was a preponderance, however, of multishaped fibroblasts, considered highly active on the basis of their well-developed Golgi complexes, numerous free ribosomes, and extensive networks of rER,[36],[47],[70],[71] and fibrils were numerous and scattered throughout the field. As seen in the seven-day neotendons, the fibroblasts were large with similar-sized nuclei that were characteristically rich in euchromatin (Figs. 8, 9). Their nuclear membranes had well-developed nuclear pores; occasionally, they were irregularly shaped and deeply indented at several points.

Sections of 15-day neotendons also contained monocytes and macrophages; platelets and lymphocytes were rare. Other observations made on these sections were essentially the same as those noted for the 12-day specimens, except that the fibrils were numerous and the fibroblasts were spindle-shaped with long cytoplasmic strands. In common with the 7- and 12-day neotendons, the fibroblasts in the 15-day neotendons were characterized by masses of cytoplasm that were always large and extensive with very well-developed rER (Figs. 10, 11).

Inflammatory cells were never seen in sections of 18- and 21-day neotendons. The fibroblasts in these sections were spindle-shaped, invariably had two or three cytoplasmic strands, and were mostly oriented in the longitudinal axis of the tendon (Figs. 12, 13). As in the 15-day specimens, their cytoplasm contained extensively developed rER. There was an abundance of collagen fibrils in sections of 18-day neotendons (Fig. 12). Previous morphometric measurements showed that these fibrils were also larger than those of 12-day neotendons.[69] These collagen subunits were not much different in sections of 21-day neotendons; however, grouping of fibrils into bundles was more obvious than in the 18-day neotendons.

In contrast to the normal tendon, which had few and sparsely distributed fibroblasts, 12-, 15-, 18-, and 21-day neotendons had numerous fibroblasts that appeared very metabolically active, as judged by the morphology of their organelles (Figs. 8-14).[36],[47],[70],[71]

Discussion and Conclusions

Acute inflammation entails three major events: 1) increased blood flow attributable to alterations in the caliber of the vasculature, 2) extravasation of plasma proteins and leukocytes attributable to structural changes in the microvasculature, and 3) accumulation of leukocytes at the site of injury.[72] The abundance of leukocytes and other inflammatory cells and the presence of inflamed blood vessels in five-day neotendons but not in seven-day neotendons suggest that these tendons were perhaps at the terminal stages of acute inflammation. Thus, the process of inflammation in tenotomized tendons could be prolonged, lasting as long as five days. Thereafter, however, there is rapid fibroplasia and fibrillogenesis, as shown in several low-magnification electron micrographs of the 7-day neotendons (Fig. 6).

The findings of this study suggest that as fibrils are produced, they are simultaneously arranged parallel to the longitudinal axis of the tendon. The presence of scattered fibrils, even in 21-day neotendons, suggests that this process lags behind fibril production. Herein lie some remarkable differences between healing in tendons and other soft tissues. Whereas most soft tissues heal essentially by cell proliferation (granulation), tendons require at least three separate, but related, processes: 1) cell (fibroblast) proliferation, 2) collagen fibril synthesis, and 3) alignment of fibrils with the longitudinal axis of the tendon. Because tendons are 86% collagen by dry weight,[73] the latter two processes must play a dominant role during healing. The present findings suggest that as early as seven days postinjury, the cellular and extracellular events required for tendon healing are already in progress. Thus, the notion that tendons do not actually begin to heal until at least four weeks has elapsed[74] contradicts the findings of this and other studies.[35--49]

The fibrillar changes observed were accompanied by corresponding changes in the morphology of the fibroblasts. As early as seven days postinjury, these cells had not only undergone remarkable increases in size, but had developed extensive networks of rER, large nuclei with abundant euchromatin, numerous clusters of free ribosomes, several cytoplasmic vesicles, and prominent Golgi complexes, all of which indicate active protein synthesis.[36,47,71,75--77] In general, cells produce two types of proteins--secretory proteins and those proteins required for cellular processes, mostly enzymes.[75--77] The abundance of cytoplasmic vesicles suggests that the proteins being synthesized were to be secreted, not to be used by the cells themselves. The presence of numerous cytoplasmic vesicles, well-developed rER, and prominent Golgi complexes correlates very well with the increasingly larger amounts of collagen fibrils in the extracellular compartment.

The abundance of ground substance in the seven-day neotendons, but not in those tendons examined after many more days of healing, supports previous studies that showed the production of ground substance reaches its peak quite early during healing,[36] but falls rapidly thereafter.[78--80] The presence of monocytes, lymphocytes, and macrophages in 12-day neotendons may not imply continuation of the inflammatory process because it is well known that these cells participate in the more elaborate immune response to injury.[72]

In summary, the present findings indicate that after complete tenotomy, repair, and plaster cast immobilization of the rabbit Achilles tendon, inflammation is massive and prolonged, lasting at least five days postinjury. Monocytes, eosinophils, neutrophils, lymphocytes, platelets, and macrophages abound during the initial few days of healing, but become increasingly rare as firoplasia proceeds. Although fibroblasts may be seen five days after tenotomy, they do not begin to proliferate until about the seventh postoperative day. Thus, fibroplasia commences as acute inflammation begins to subside. During the first three weeks of healing, the morphology of tendon fibroblasts differs remarkably from that of tendon fibroblasts in intact normal tendon. Unlike the fibroblasts of normal tendon, these cells were always characterized by prominent, well-developed rER, abundant free ribosomes, and euchromatin. With time, they become progressively aligned in the longitudinal axis of the tendon, assuming spindle shapes and developing a few cytoplasmic strands simultaneously as the collagen fibrils are also oriented longitudinally. These findings have also been reported by others.(~~) Newly synthesized collagen fibrils may be seen in the extracellular compartment as early as five days after surgery, but these collagen subunits are sparse and randomly oriented. By the seventh postoperative day, however, the fibrils are very numerous in this compartment. Organization of these fibrils into bundles of collagen becomes easily discernible by the 21st postoperative day. Thus, the initial three weeks of healing may be viewed as consisting of three overlapping periods: an inflammatory period lasting at least five days, a period of fibroplasia and fibrillogenesis that begins from about the seventh day, and finally a period of remodeling that overlaps fibrillogenesis but continues perhaps beyond the three-week limit of this study, as others have noted.[59-61]

Implications for Tendon Rehabilitation

Although healing in rabbits may not translate directly to healing in humans, the findings of this study indicate that the regenerating Achilles tendon undergoes different stages of healing. Because each stage involves a different set of ultrastructural events, therapeutic strategies should be modified correspondingly to address the specific events of each stage.

The importance of timing of therapeutic intervention is demonstrated by studies that have attempted to promote tendon healing via therapeutic ultrasound.[31,82-87] In one of these studies,[31] the effects of therapeutic ultrasound applied at two different time intervals were compared in 30 experimentally tenotomized and repaired rat Achilles tendons. The tendons were sonicated in the pulsed mode at 0.5 [W/cm.sup.2] for five minutes with a 1-MHz ultrasound unit, either on postoperative days 2 through 4 or on postoperative days 5 through 7. Although sonication at the earlier time period increased the tensile strength of the tendons, sonication at the latter time period reduced the strength of the tendons compared with matched controls. It may not be mere coincidence that the earlier and latter periods of sonication used in this study correspond to the periods of inflammation and of fibroplasia and fibrillogenesis, respectively, observed in the present study. The similarity of results obtained in those studies in which sonication was limited to the very early period of healing[31,84] and the contrasting but unanimous results obtained when ultrasound was applied throughout the healing period[82,83] strengthen the notion that timing of therapeutic interventions could indeed be an important determinant of treatment outcome.

There is similar evidence that mechanical stress is capable of increasing the strength of tendons during the early stage of healing and that timing of therapeutic intervention may be equally critical.[30,88-93] For example, when mechanical stress was imposed on healing rat Achilles tendons for three days, beginning five days after surgical tenotomy and repair, the tendons healed stronger than both control tendons and those tendons that were similarly stressed on postoperative days 2 through 4.[30] Subsequent studies on the rabbit Achilles tendon have shown that the increase in tensile strength is accompanied by corresponding morphological changes in the collagen matrix,[89] thus suggesting the need to apply such stress during fibrillogenesis.

Preliminary evidence also suggests that electrical stimulation augments protein synthesis and increases the strength of tendons during the early stages of healing.[32,94-96] For example, experimentally tenotomized and repaired rat Achilles tendons treated with high voltage galvanic stimulation developed higher tensile strength than controls when the positive electrode was placed at the site of tenotomy during treatment.[32] Increased protein and deoxyribonucleic acid synthesis was equally observed when cultured human fibroblasts were exposed to high voltage galvanic stimulation of various voltages and pulse rates for 20 minutes.[94] In light of the present study, it may be germane to adjust the electrical stimulation characteristics differently for the different stages of healing or to stimulate the tendon only during certain periods of healing to obtain optimal effects. In this regard, the present study provides 1) a basis for planning future studies, 2) a rationale for directing treatment toward specific ultrastructural events, and 3) a normal comparison for assessing the effects of physical modalities and procedures on the ultrastructure of the regenerating tendon. [Figures 1 to 14 Omitted] [Tabular Data Omitted]

(*)Electron Microscopy Sciences, PO Box 251, Fort Washington, PA 19034. ([single dagger])JEOL USA, Electron Optics Div, 11 Dearborn Rd, Peabody, MA 01960. ([double dagger])Jandel Scientific, 65 Koch Rd, Corte Madera, CA 94925. ([section])International Business Machine Corp, 1000 NW 51st St, Boca Raton, FL 33432. (~~)35,36,38,46,47,52-54,56,81.

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C Enwemeka, PhD, PT, is Associate Professor, Department of Orthopedics and Rehabilitation, Division of Physical Therapy, University of Miami, 5913 Ponce de Leon Blvd, Coral Gables, FL 33146 (USA). He was Assistant Professor of Physical Therapy, Assistant Professor of Cellular and Structural Biology, and Assistant Professor of Physical Medicine and Rehabilitation, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr, San Antonio, TX 78284-7762, when this article was written.