Chondroitin sulfate–coated polyhydroxyethyl methacrylate membrane prevents adhesion in full-thickness tendon tears of rabbits

Chondroitin Sulfate–Coated Polyhydroxyethyl Methacrylate Membrane Prevents Adhesion in Full-Thickness Tendon Tears of Rabbits Eftal Gu¨demez, MD, Fatih Eks¸iogˇlu, MD, Kırıkkale, Turkey, Petek Korkusuz, MD, PhD, Esin As¸an, PhD, Ankara, Turkey, I˙hsan Gu¨rsel, PhD, Bethesda, MD, Vasıf Hasırcı, PhD, Ankara, Turkey Polyhydroxyethyl methacrylate (pHEMA) membranes coated on one side with chondroitin sulfate (CS) were used to block adhesion physically and to reduce friction between healing flexor tendons and the surrounding tissue in rabbit forepaws after surgical repair. Digits with pHEMA-only, standard tendon sheath repair, and with no sheath repair were the controls. Over 12 weeks the CS-coated membranes were evaluated for joint flexion, adhesion limitation, and tendon healing progress. The membranes initially allowed for better flexion (ie, for 6 weeks), but their relative superior effectiveness faded afterward. Histology showed that adhesions were less severe and healing was better in the CS-pHEMA membranes at 3 and 6 weeks. If further studies determine precise amounts or thicknesses of CS coats that will maximize its healing properties, CS-pHEMA should prove useful in clinical settings in which restoration of tendon sheath integrity with a minimum of adhesions is not possible. (J Hand Surg 2002;27A:293–306. Copyright © 2002 by the American Society for Surgery of the Hand.) Key words: Tendon injuries, adhesion, polyhydroxyethyl methacrylate (pHEMA), chondroitin sulfate, biomaterials.

In tendon injuries and their surgical repair much attention has been focused on the sheath that protects From the Department of Orthopaedic Surgery and Traumatology, Kırıkkale University School of Medicine, Kırıkkale, Turkey; Department of Histology and Embryology, Hacettepe University, School of Medicine, Ankara, Turkey; Laboratory of Retroviral Research, Center for Biological Research, Food and Drug Administration, Bethesda, MD; and Department of Biological Sciences, Biotechnology Research Unit, Middle East Technical University, Ankara, Turkey. Received for publication December 1, 2000; accepted in revised form November 14, 2001. No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. Reprint requests: Vasıf Hasırcı, PhD, Middle East Technical University, Department of Biological Sciences, Biotechnology Research Unit, 06531 Ankara, Turkey. Copyright © 2002 by the American Society for Surgery of the Hand 0363-5023/02/27A02-0009$35.00/0 doi:10.1053/jhsu.2002.31161

the tendon and its role in healing. Peritendinous adhesions are a major problem in lacerated flexor tendons after repair, and limiting them has long been a goal. Formation of fibrous adhesions between the healing tendon and the surrounding tissues is the most common complication after a lacerated tendon repair.1 The subcutaneous tissue and the skin act as a single compartment during the healing process, and adhesion around the repair site interferes with tendon gliding, which results in contracture and decreased range of motion (ROM) of the digit.2,3 Despite technical improvements and the introduction of early controlled mobilization techniques, the final result of flexor tendon repair still is highly unpredictable.4,5 The control of excessive scar formation is essential to restore the functional integrity of a healing tendon after surgery. Continuous efforts are being made to The Journal of Hand Surgery

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reduce postoperative peritendinous adhesions in damaged flexor tendons.6 In more recent years the role and the necessity of the tendon sheath in the tendon healing process have been investigated.7,8 Consensus now leans toward the importance of avoiding inadvertent narrowing of the repaired sheath by inserting a synthetic graft and cutting down on the incidence of adhesions. The main techniques for reducing adhesions without adversely affecting the healing process itself are mechanical and biologic.2 Much of this work is focused on mechanical barriers surrounding the tendon and physically blocking adhesion between the tendon and the surrounding tissues. Alumina sheaths,3 polyethylene membranes,9 cellophane,10 Sterispon wrapping (Allen J. Hanbury, United Kingdom),11 stainless steel sheeting, silicone sheeting,12 silicone rubber envelopes,13 polytetrafluoroethylene surgical membrane,7 and numerous other materials have been used toward this end. Some materials failed because they stimulated a severe inflammatory response or allowed ingrowth of adhesions around the edges of the material; other materials prevented nutrient diffusion to the healing tendon leading to tendon necrosis. Other studies have concentrated on reducing peritendinous adhesions through biochemical means by controlling the size and quality of the newly formed collagenous scar. Cortisone,14 dextran 70,15 fibrin,16 collagen inhibitor,17 antihistamine,18 indomethacin,19 hyaluronic acid,4,20 5-fluorouracil,21 and other therapeutic agents have been tested with varying degrees of success. Combining the mechanical and biologic techniques, we designed and tested an inert, biocompatible mechanical barrier coated with chondroitin sulfate (CS) as a means of facilitating the gliding of a surgically repaired tendon lacking its natural sheath. This unique concept combines the physical separation and the chemical surface treatment approaches. Chondroitin sulfate is one of the major proteoglycan components in the body and plays a direct role in inhibiting adhesion of inflammatory cells to fibronectin matrices and type I collagen.22,23 Initial in vitro studies on the proteoglycan composition of the substratum adhesion sites of fibroblasts indicated that heparan sulfate played a direct role in mediating adhesion of cells to fibronectin matrices, whereas CS proteoglycans seemed to function in an inhibitory capacity. Studies now show that CS proteoglycans and dermatan sulfate proteoglycans inhibit the attachment of cells to type I collagen and fibronectin.23 One study has examined CS (in the form of hyal-

uronic acid/CS complex) in the prevention of adhesion.1 No study has measured the effect of CS alone or in combination with a biologic membrane in the prevention of adhesion around the repaired tendon. The development of a mechanical barrier that has chemical properties to prevent tendon adhesion is a new approach. The purpose of this study was to evaluate the effect of a CS-coated poly(2-hydroxyethyl methacrylate) membrane (pHEMA) in the prevention of adhesions in experimental, full-thickness flexor tendon tears in rabbits. The hypotheses were as follows: (1) less inflammatory response or less ingrowth of adhesions around the edges of the membrane as a result of biocompatibility of pHEMA, (2) less adhesion around the repaired tendon as a result of CS effect, and (3) less interference with the tendon healing mechanism.

Materials and Methods Polyhydroxyethyl Methacrylate Membrane Preparation The membrane preparation mixture (5 mL) contained pHEMA (2 mL), ␣,␣⬘-azoisobutyronitrile (5 mg) as polymerization initiator, and phosphate buffer (3 mL, 0.1 mol/L, pH 7). The mixture was poured into a round, glass mold (9 cm diameter) and exposed to UV radiation for 10 minutes while a nitrogen atmosphere was maintained. The membrane was washed several times with distilled water and cut into square pieces (2 ⫻ 2 cm2) with scissors.

Chondroitin Sulfate Coating of Polyhydroxyethyl Methacrylate Membranes The method of activating the alcoholic groups within the pHEMA membrane to prepare it for covalent coupling with chondroitin sulfate A (from bovine trachea; Sigma Chemical Co, St Louis, MO) involved cross-linking with epichlorohydrin. With this approach epoxy groups were incorporated covalently onto the pHEMA membrane by the nucleophilic reaction between the chloride group of epichlorohydrin and the hydroxyl group of the pHEMA molecule under alkaline conditions as follows: Each pHEMA membrane (2 ⫻ 2 cm2, thickness approximately 0.06 cm, 15 g, total surface area about 200 cm2) was immersed in a reactor containing sodium hydroxide solution (4.0% [wt/vol], 25 mL) for 1 hour; epichlorohydrin (50 mL) was added and stirred at 25°C for 6 hours; and at the end of this period the

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membranes were washed with acetone and dried at 4°C. The membranes were preserved in dry form at 4°C until use. For coating with CS, activated pHEMA membranes were placed in phosphate buffer (0.1 mol/L, pH 7.5) for 18 hours and in their swollen state transferred to CS solution (150 mg chondroitin sulfate A in 30 mL phosphate buffer). Chondroitin sulfate coating of the membranes was done at 22°C in a shaking water bath. Coating was achieved predominantly on the top surfaces of the membranes; the coating level at the bottom was hindered because of the restriction imposed on the diffusion of the macromolecular CS by the glass bottom of the container. This glass-facing (bottom) side was labeled the uncoated side of the CS-coated pHEMA membranes. Unbound CS was removed first by washing the membranes with saline solution (10 mL, 0.5 mol/L), then with phosphate buffer (20 mmol/L, pH 7.0). Close attention was paid to keeping track of the coated and uncoated sides of the membranes. The membranes were stored at 4°C in fresh buffer until use.

Implantation of Polyhydroxyethyl Methacrylate–Only and Chondroitin Sulfate–Coated Polyhydroxyethyl Methacrylate Membranes Eighteen local albino rabbits (Institute of Farming Animals, Ankara, Turkey), aged 4 months and weighing 2,000 to 2,500 g, were used. The rabbit model was chosen because it has been defined to be the best model for adhesion studies.24 The rabbits were divided into 3 equal groups and monitored for 3, 6, and 12 weeks. Anesthesia was induced with an intramuscular injection of ketamine (100 mg/kg) and xylazine (5 mg/kg). The second and fourth digits of both forepaws (in which the tendons are the same size) were used for the experiment. The surgical site was shaved and prepared with povidone-iodine solution. The flexor surfaces of the second and fourth digits of the forepaw were incised over the basal and middle phalanges. With sharp dissection, the flexor tendon sheath was identified and opened by an anterolateral incision under ⫻4 loop magnification. The flexor digitorum profundus tendon was identified between the first and second annular pulleys, lifted from between the slips of the flexor digitorum superficialis, and divided transversely with a sharp scalpel. The tendon was repaired by a modified Kessler suture with 6 – 0 Prolene (Ethicon; Johnson

& Johnson JnH, Brussels, Belgium) suture material and a continuous adaptation suture of 8 – 0 Prolene. In the right second digit, a CS-coated pHEMA membrane (CS-pHEMA), 1 cm in length, was wrapped circumferentially around the repaired tendon in place of its damaged sheath and the ends of the membrane were sutured to each other with 8 – 0 nylon. Attention was paid to closing the remaining intact portion of the tendon sheath around the edges of the membrane proximally and distally with 8 – 0 Prolene. In the right fourth digit, the same membrane without CS coating (pHEMA-only) was used, following the same procedure as in the CS-pHEMA group. In the left second digit, after tendon repair the tendon sheath was repaired (TSR) with 8 – 0 nylon. In the left fourth digit, the tendon was repaired but the sheath was not repaired (NSR). In all digits, the skin was closed with interrupted 6 – 0 PDS (Ethicon) sutures. After surgery the paws were placed in a lightweight dressing and the rabbits were allowed unrestricted motion of the digits until they were killed. Every animal had one lacerated and repaired tendon from each group, yielding a total of 4 tendons to be studied. The duration of surgery was 82 ⫾ 24 minutes (average ⫾ SD) (range, 65–165 minutes). The rabbits were caged in pairs at room temperature and were on a 12-hour day/night (light/dark) cycle. Standard laboratory food and water were provided constantly. All procedures were in full compliance with Turkish Law 6343/2, Veterinary Medicine Deontology Regulation 6.7.26, and with the Helsinki Declaration of Animal Rights.25

Joint Motion Measurement The skin was removed and all structures were divided at the proximal level of the metacarpal. The proximal portion of the flexor digitorum profundus tendon was isolated in the forepaw without disturbing the operative site and dissected free of surrounding soft tissues. Tendon rupture was not detected in any of the specimens. The lumbricals and branches to the other digits were divided. The forepaw was mounted in a testing apparatus with K-wires through the proximal and middle phalanges and the midpaw. The metacarpophalangeal and the proximal interphalangeal joints were immobilized in a straight position. A 15-g counterweight was attached to the tip of the finger to extend it fully. A clamp was applied to stabilize the proximal end of the tendon. At the beginning of the experiment, to standardize the load, a normal right second digit of a rabbit not subjected to surgery was mounted in the testing apparatus. A

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Figure 1. A schematic of the basic experimental procedure for determining joint motion measurement.

fishing line was tied to the clamp, and the load was suspended from the end of this line and wrapped around a pulley system until the distal interphalangeal (DIP) joint represented 90° flexion. This load weighed 50 g. After standardization of the load to flex the DIP joint, the 50-g weight was applied to every digit (Fig. 1). Joint motion was measured with a mechanical goniometer. Joint measurements of the angle formed between the middle and the distal phalangeal axis in the sagittal plane (flexion– extension) were done with a mechanical goniometer accurate to 1°.

Histology Histologic analysis of all repaired tendon segments and the replaced sheaths in between the first and second annular pulleys was done. Repaired tendon segments were fixed in 10% phosphate-buffered formalin (pH 7) at room temperature. After dehydration through a graded series of ethanol the specimens were embedded in paraffin. Serial sections 5 to 7 ␮m thick were stained with hematoxylin-eosin and Masson’s trichrome. Stained sections were examined for the degree of adhesion and quality of tendon healing. Histologic evaluations were blinded and made by 2 investigators. The area of tendon surface with adhesion was estimated by observing 10 slides prepared at 1.0-mm intervals for each specimen. Adhesions were quantified into 4 grades as follows: severe adhesions, ⬎66% of the tendon surface; moderate

adhesions, 33% to 66% of the tendon surface; mild adhesions, ⬍33% of the tendon surface; and no adhesions.3 Each slide was graded, and the average grade of 10 slides was calculated to assign the grade of adhesion for that specimen. The histologic quality of tendon healing was graded according to Tang et al.8 Well-re-established tendon continuity and a smooth epitenon were graded as excellent. An assessment of good was given to samples in which the intratendinous collagen bundles had healed well but the epitenon was interrupted by adhesions in some locations. Irregularly arranged and partly interrupted intratendinous collagen bundles were graded as fair. Poor samples were those evidencing repair failure, as indicated by separation of the sutured parts or by mass overgrowth of granulation on the repair site.

Statistics Three 1-way analyses of variance were done to evaluate the significant mean differences among the 4 groups for weeks 3, 6, and 12. Independent variables were CS-pHEMA, pHEMA-only, TSR, and NSR groups. The dependent variables were flexion rates at the DIP joint. Before all analyses, scores gathered at 3 different time intervals were examined through various SPSS programs for accuracy of data entry, missing values, and fit between their distributions and the assumption of univariate analysis. Normality was checked by z score, skewness, and kur-

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Table 1. Differences Between Distal Interphalangeal Joint Motion Measurements at 3, 6, and 12 Weeks CS-pHEMA (°) Mean Week 3 Week 6 Week 12

a

71.7 59.2e 56.7g

SD 6.1 20.8 12.1

pHEMA-Only (°) Mean b

57.5 31.7 49.2h

TSR (°)

SD 13.7 22.3 28.4

Mean c

44.2 47.5 71.7i

NSR (°) SD 18.8 16.0 10.3

Mean d

19.2 26.7f 35.0j

SD 23.3 17.8 10.0

a-b: p ⫽ .022. a-c: p ⫽ .003. a-d: p ⫽ .001. e-f: p ⬍ .05. g-h: p ⫽ .282. g-i: p ⫽ .022. g-j: p ⫽ .004.

tosis and found to be satisfactory. There were no univariate outliers. Follow-up tests were conducted to evaluate differences among the means of pairs. The result of Levene’s test of equality of variance, which tests the null hypothesis that the error variance of the dependent variable is equal across groups, was not significant. This result led us to assume that the variances were homogeneous, and we conducted post hoc comparisons with the Tukey HSD test, a test that assumes equal variances among the 4 groups. A paired-samples t-test was conducted to evaluate whether there was a significant difference between the means of weeks 3, 6, and 12, for each group separately. A series of Kruskal-Wallis tests was conducted to evaluate differences among the 4 groups of subjects (CS-pHEMA, pHEMA-only, TSR, and NSR) on median changes in the histologic degree of adhesions and histologic quality of tendon healing at the 3 different times. Population medians on a dependent variable were evaluated at all levels by using the same factor across results for the histologic degrees of adhesions and quality of tendon healing. Cases received scores on an independent or grouping variable and on a dependent or test variable. The frequencies of the histologic degrees of adhesions (severe, moderate, mild, and none) and quality of tendon healing (poor, fair, good, and excellent) were graded as 1, 2, 3, and 4 to create a job file for the analysis; mean ranks were calculated from these data. Follow-up tests were conducted to evaluate pairwise differences between the 4 groups, controlling for type I error across tests by Holm’s sequential Bonferroni approach.

Results Joint Motion Measurements The CS-pHEMA group had favorable flexion rates compared with the pHEMA-only, TSR, and NSR groups (p ⫽ .022, p ⫽ .003, p ⬍ .001) at week 3. At week 6, the CS-pHEMA flexion rates were still better than those of the pHEMA-only, TSR, and NSR groups. The TSR group improved to a better ROM at week 12, however. There was no significant difference between the CS-pHEMA and pHEMA-only groups at the end of 12 weeks (p ⫽ .282). The difference between the flexion rates of the CSpHEMA and the TSR and NSR groups remained significant (p ⫽.022, p ⫽ .004), with the TSR group presenting the highest ROM. The ROM of the CSpHEMA group deteriorated between weeks 3 and 12 (p ⫽ .011). The ROM of the TSR group improved significantly between weeks 3 and 12 (p ⫽ .005) and weeks 6 and 12 (p ⫽ .006). All groups except the pHEMA-only group at weeks 6 and 12 had statistically and significantly better results than the NSR group. Flexion rates at the DIP joint are shown in Table 1.

Histology At 3 weeks a thin granulation tissue layer covered the inner (tendon-facing) surface of the CS-pHEMA and pHEMA-only membranes. There was no apparent difference in the characteristics of the granulation tissue layer of both types of membranes, which were composed of fibroblastic and mononuclear phagocytic cells (Figs. 2, 3). The phagocytic cells invaded the membrane in several locations. Peripheral adhesion was mild to moderate in most specimens of the

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Figure 2. The internal surface of the CS-pHEMA membrane (M) is covered by granulation tissue composed of fibroblasts and loose collagen fibers (arrows) at week 3. (Masson’s trichrome stain; magnification ⫻10.) (A color version of this figure can be viewed at the Journal’s Web site, www.jhandsurg.org)

CS-pHEMA group but moderate to severe in the pHEMA-only group. A highly vascular granulation tissue layer containing decidual cell–like active fibroblasts with euchromatic nuclei was apparent in some specimens in the pHEMA-only group. The intratendinous collagen bundles were arranged irregularly in the CS-pHEMA and pHEMA-only groups (Figs. 2, 3). In the TSR group moderate-to-severe adhesion was apparent between the repaired sheath, the tendon, and the surrounding vascular granulation tissue. In the NSR group histologic findings also revealed severe adhesion between the tendon surface and the surrounding connective tissue. The tendon itself in the NSR group and the repaired tendon sheath of the TSR group were covered with a thick granulation tissue (Figs. 4, 5). This tissue invaded the

epitenon and the superficial layers of the repaired tendon and had poor collagen maturation. Collagen organizations of the CS-pHEMA and pHEMA-only groups were slightly better than those of the TSR and NSR groups. Statistically, at this point in the study— week 3—CS-pHEMA group proved significantly better (␹2 [3, n ⫽ 24] ⫽ 14, 98; p ⬍ .01) at limiting adhesions than either the TSR group or the NSR group. The histologic degree of adhesions was less in the CS-pHEMA group than in the TSR and the NSR groups in week 3 (Table 2). Histologic determination of the quality of tendon healing under way in week 3 also yielded statistically significant (␹2 [3, n ⫽ 24] ⫽ 9, 50; p ⬍ .05) differences for CS-pHEMA but only against the NSR group (Table 3). At 6 weeks mild-to-moderate adhesion between the CS-pHEMA and pHEMA-only membranes and the edges of the tendon sheath was observed. Granulation tissue between the membranes and repaired tendons was more apparent than that observed at week 3 in both groups (Figs. 6, 7). This granulation tissue became highly vascular, and its cellular composition changed to show more phagocytic cells. The membranes remained relatively intact with almost no giant cells in them (Figs. 6 inset, 7 inset). Collagen maturation and organization seemed better in the CS-pHEMA group than in the pHEMA-only group. Mild-to-moderate and moderate adhesions were found in the TSR and NSR groups. At this point significant differences in the degree of adhesions were not found.

Figure 3. Granulation tissue surrounds the pHEMA-only membrane (M) at week 3. Note the decidual cell–like fibroblasts (arrows) within the connective tissue layer. (Masson’s trichrome stain; magnification ⫻10.) (A color version of this figure can be viewed at the Jounral’s Web site, www.jhandsurg.org)

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299

CS-pHEMA (Fig. 10) and pHEMA-only (Fig. 11) membranes were encapsulated by a thin fibrous layer consisting mainly of flat fibroblastic cells. In the CS-pHEMA group, the granulation tissue was in continuity with this capsule and the epitenon in some locations. Collagen maturation and organization were almost similar to those of 6 weeks in the CSpHEMA and pHEMA-only groups. The degree of adhesion had decreased substantially in the TSR group, however. Proliferation of the peritenon and intratendinous collagen fiber organization were advanced compared with the degrees seen at 3 and 6 weeks (Fig. 12). Also, collagen organization in the TSR group was slightly better than that of the pHEMA-only group and comparable to that of the

Figure 4. Thick granulation tissue that invades the repaired tendon sheath (TS) and the tendon (T) is observed in the TSR group at week 3. Arrowhead shows the suture material within the tendon sheath. (Masson’s trichrome stain; magnification ⫻10.) (A color version of this figure can be viewed at the Journal’s Web site, www.jhandsurg. org)

Regarding the quality of tendon healing in week 6, poor collagen maturation and organization were seen in the TSR (Fig. 8) and NSR (Fig. 9) groups compared with those of the CS-pHEMA and pHEMAonly groups. The arrangement of intratendinous collagen bundles was irregular in the NSR group compared with all the other groups. The results of the follow-up test showed a significant (␹2 [3, n ⫽ 24] ⫽ 15, 11; p ⬍ .01) difference between the CS-pHEMA group and the TSR group and between the CSpHEMA group and the NSR group, in which the histologic quality of tendon healing was seen to be superior in the CS-pHEMA group compared with the NSR group or TSR group (Table 3). At 12 weeks inner (tendon-facing) surfaces of the

Figure 5. Thick granulation tissue covers the epitenon and the superficial layers of the tendon (T) in the NSR group at week 3. The adhesion is near the edge of the tendon (asterisk). Arrowheads show the suture material within the repaired tendon. (Hematoxylin-eosin stain; magnification ⫻10.) (A color version of this figure can be viewed at the Journal’s Web site, www.jhandsurg.org)

0 0 0

1 3 2

Mode Rate

5 2 2

Mild 0 1 2

None

Severe 2 0 0

Mean Rank 20.5a 15.8 15.2 3 5 3

Mode Rate 1 1 2

Mild

pHEMA

0 0 1

None 13.5ab 11.4 12.5

Mean Rank 3 0 0

Severe 3 4 2

Mode Rate 0 2 3

Mild

TSR

0 0 1

None 8.0b 13.3 14.2

Mean Rank 5 0 0

Severe 1 6 5

Mode Rate

0 0 1

Mild

NSR

0 0 0

None

8.0b 9.5 8.2

Mean Rank

0 1 2

0 4 4

5 1 0

1 0 0

Good Fair Poor

Excellent 0 0 1

Mean Rank 17.0a 19.3c 16.8e 0 3 4

4 3 1

2 0 0

Good Fair Poor

pHEMA

15.0ac 16.0cd 14.0e

Mean Rank

0 0 1

Excellent

0 0 5

2 3 0

4 3 0

Good Fair Poor

TSR

11.0ab 8.0d 15.4e

Mean Rank

0 0 0

Excellent

0 0 0

0 2 3

6 4 3

Good Fair Poor

NSR

7.0b 6.7d 3.8f

Mean Rank

Excellent, Continuity of the tendon is well re-established, and the epitenon is smooth; good, the intratendinous collagen bundles healed well, but the epitenon is interrupted by adhesions in some locations; fair, the intratendinous collagen bundles are arranged irregularly and interrupted partly by adhesions; poor, disconnection of the repair side or connection of the repair side to a large extent by granulation of adhesion tissues. a-b: p ⫽ .02. c-d: p ⫽ .002. e-f: p ⫽ .002.

Week 3 Week 6 Week 12

Excellent

CS-pHEMA

Table 3. Histologic Quality of Tendon Healing at 3, 6 and 12 Weeks

Severe, adhesion ⬎ 66% of the tendon surface; moderate, adhesion between 66% and 33% of the tendon surface; mild, adhesion ⬍ 33% of the tendon surface; none, no adhesion. a-b: p ⫽ .02.

Week 3 Week 6 Week 12

Severe

CS-pHEMA

Table 2. Histologic Degree of Adhesions at 3, 6, and 12 Weeks

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Figure 6. The CS-pHEMA membrane (M) provides a firm biocompatible surface for tendon (T) gliding at week 6. The collagen fibers are relatively better organized than the other groups of the same week. (Inset) Higher magnification shows the cellular composition of the highly vascular granulation tissue. Mononuclear cells and macrophages (arrowhead) are present near the membrane, within the fibrous capsule. (Masson’s trichrome stain; magnification ⫻10, inset ⫻40.) (A color version of this figure can be viewed at the Journal’s Web site, www.jhandsurg.org)

CS-pHEMA group. For the NSR group the degree of adhesions decreased slightly between weeks 6 and 12, although moderate adhesions still were present at week 12. The surrounding tissue of this group ap-

Figure 7. Granulation tissue that is continuous with the epitenon covers the internal surface of the pHEMA-only membrane (M) at week 6. The collagen fibers still are disorganized. (Inset) Higher magnification shows that the connective tissue encapsulating the membrane consists of fibroblasts and mononuclear phagocytic cells (arrowhead). (Masson’s trichrome stain; magnification ⫻4, inset ⫻40.) (A color version of this figure can be viewed at the Journal’s Web site, www.jhandsurg.org)

Figure 8. Poor collagen maturation and continuity of the granulation tissue within the epitenon (asterisk) are observed in the TSR group at week 6. Note the suture material (arrowhead) within the tendon sheath. (Masson’s trichrome stain; magnification ⫻4.) (A color version of this figure can be viewed at the Journal’s Web site, www.jhandsurg.org)

peared as a hypocellular fibrous connective tissue at the tendon repair area. The quantity of the ingrown collagen fibers into the tendon was minor, and their arrangement was irregular (Fig. 13). For the quality of tendon healing in week 12, statistical tests showed a significant difference (␹2 [3, n ⫽ 24] ⫽ 15, 32; p ⬍ .01) between the CS-pHEMA group and the NSR group. The results of the pairwise

Figure 9. Proliferation of the peritenon and poor maturation of the intratendinous collagen fibers (asterisk) in the NSR group at week 6. Arrowhead shows the suture material. (Masson’s trichrome stain; magnification ⫻10.) (A color version of this figure can be viewed at the Journal’s Web site, www.jhandsurg.org)

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tient’s chances of complete recovery, it is essential that the formation of adhesions in the healing area be avoided to the degree possible; toward this end, methods of separating the healing tendon from its surrounding tissues have been advocated.1,7,8,26 More often surgery to repair the damage is needed, however. The integrity of the protective sheath enveloping the tendon has drawn much attention because its absence or inadequacy can inhibit movement greatly. Ideally the sheath can be repaired surgically along with the tendon it contains and be restored to a “good-as-new” condition. The normal environment of the tendon is recreated, promoting healing.27,28 Depending on the type of injury that the tendon and its sheath have sustained, various approaches have been tried to make up for the loss of

Figure 10. Fibrous layer covering the CS-pHEMA membrane (M) is composed mainly of fibroblasts. The membrane still provides a firm biocompatible surface for the reorganized, relatively mature tendon (T) gliding at week 12. The paratenon regained its normal appearance (asterisk). (Masson’s trichrome stain; magnification ⫻10.) (A color version of this figure can be viewed at the Journal’s Web site, www.jhandsurg.org)

comparison analysis also indicated significant differences between the pHEMA-only group and the NSR group and between the TSR group and the NSR group. At week 12 the histologic quality of tendon healing was better in the CS-pHEMA group than in the NSR group, better in the pHEMA-only group than in the NSR group, and better in the TSR group than in the NSR group (Table 3).

Discussion Injured tendons are capable of intrinsic healing when surgery is not available or has been ruled out, provided that the damaged tissue can obtain necessary nutrients. In such cases, to maximize the pa-

Figure 11. The pHEMA-only membrane (M) represents a biocompatible material similar to that in Figure 10 at week 12. The sutures (arrowheads) are observed within the tendon sheath. (Masson’s trichrome stain; magnification ⫻10.) (A color version of this figure can be viewed at the Journal’s Web site, www.jhandsurg.org)

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tunnel had greater tendon movement than those within a narrowed sheath.7,8 At other times surgical repair of the sheath is not possible because of major defects in its substance after injury or the sheath is crushed so badly that it must be resected.3,7 In such cases attempts are made to restore the integrity of the flexor sheath or to replace it with various biologic or synthetic materials.7,16,30 Thus there are 2 different indications for barriers: a narrowed tunnel as a result of primary tendon sheath repair and a defective sheath. In contrast to sheath repair, reconstruction of the lost gliding tunnel with barriers should ensure that there is no narrowing of the space available for gliding within the fibro-osseous tunnel. Biologic barriers have included

Figure 12. A micrograph shows poor collagen organization at week 12 in the TSR group. (Masson’s trichrome stain; magnification ⫻20.) (A color version of this figure can be viewed at the Journal’s Web site, www.jhandsurg. org)

sheath tissue when a clean suturing (primary sheath repair) is not an option. These approaches have included surgical procedures that incorporate biologic or synthetic materials to reconstruct the defect in the flexor sheath.7,16,29 Such concern about the integrity of the sheath— or its substitute—is motivated by the widely held belief among clinicians that proper sheath repair and reconstruction leads to fewer adhesions and their deleterious impact on healing, as opposed to leaving a damaged sheath alone.5,8 Although it is generally agreed that primary repair of flexor tendons, with accompanying preservation or restoration of the tendon sheath, is the treatment of choice, pursuing this course can create problems. Surgical sheath closure may narrow the diameter of the gliding tunnel (eg, from scar formation). In 2 studies repaired tendons within an enlarged sheath

Figure 13. A micrograph from the NSR group shows the tendon repair area with poorly organized, relatively immature intratendinous (T) collagen fibers and the hypocellular surrounding tissue at week 12. (Masson’s trichrome stain; magnification ⫻10.) (A color version of this figure can be viewed at the Journal’s Web site, www.jhandsurg. org)

304 Gu¨ demez et al / CS-Coated pHEMA Membranes in Tendon Tears

paratenon,12 peritoneum, and tendon sheath transplantation,5 which have had variable success. These biologic tissues have the disadvantages of an extra incision and removal of sound, functional tissues. Synthetic materials have included metal tubes,3 cellophane,10 polyethylene,9 polytetrafluoroethylene,7 and silicone sheeting.13 Some of these materials failed because they stimulated a severe inflammatory response or allowed ingrowth of adhesions around the edges of the material. Other materials prevented nutrient diffusion to the healing tendon leading to tendon necrosis. We treated irreparable tendon sheaths with a mechanical barrier consisting of a biocompatible, nonresorbable pHEMA membrane coated with CS. The expectation was that these CS-pHEMA membranes are better than a pHEMA-only membrane at preventing adhesion in full-thickness flexor tendon tears in which the original sheath could not be saved. We also assessed the CS-pHEMA membrane in comparison with cases in which the original sheath had been susceptible to surgical repair and cases in which no repair of the sheath had been tried (ie, the controls). The results at 3 weeks indicated that CS-pHEMA membranes had acted as superior mechanical barriers as shown by flexion rates, with their CS components apparently enabling the repaired tendon to function as close to normal (Table 1) as shown by ROM figures of 72% compared with 58% for pHEMA-only membranes, 44% for standard tendon sheath repair (TSR), and 19% for no sheath repair (NSR). At 3 weeks a thin layer of granulation tissue covered the inner surface of the CS-pHEMA membranes, indicative of natural healing. This granulation tissue was highly vascular, facilitating the healing of the repaired tendon abutting it. Tenocytes were more active in the CS-pHEMA group at 3 weeks than in the other 3 groups, signaling that intrinsic cellular response had not been affected. For the degree of adhesions, histologic study showed that CS-pHEMA at 3 weeks far exceeded the 3 other treatments in inhibiting the development of these healing blockers: only mild-to-moderate peripheral adhesions were present in these rabbits. At weeks 6 and 12, the CS-pHEMA membranes’ beneficial effects on flexion (ROM) seemed to slip, decreasing from 72% at 3 weeks to 60% at 6 weeks and 57% at 12 weeks. The effectiveness of pHEMAonly membranes declined from 58% to 49% by week 12. Conversely, the TSR group started out at 44% at week 3 but moved steadily upward, reaching 48% at week 6 and 72% at week 12, the same percentage

that CS-pHEMA had at the first measurement marking at 3 weeks. The control group (NSR) started out poorly at 19% at week 3 and managed a climb only to 35% by the time the study ended in week 12. Macroscopic histologic evaluation showed that in adhesion minimization the CS-pHEMA membrane performed well, but its effectiveness seemed to wane after being in first place among the 4 groups at week 3. At 12 weeks CS-pHEMA was equal to pHEMAonly in this criterion, still ahead of the NSR group, but had fallen behind the TSR group. More specifically at week 12, connective tissue proliferation was marked at the edges of the sheath in the CS-pHEMA specimens with mild adhesions. Collagen fiber arrangement on the tendon and the peritendinous elements were recovering well. For the CS-pHEMA and pHEMA-only membranes, few macrophages and no giant cells were present on their outer surfaces. No significant foreign body reaction was detectable at any time in either membrane; this was attributed to their biocompatibility. Overall this status was considered an indication of the quality of tendon healing, and CS-pHEMA showed promising results, especially at week 6, compared with the others but fell behind by the end of the study. At 12 weeks the internal surface of the CSpHEMA membrane was encapsulated with a thin fibrous layer consisting of mainly flat fibroblastic cells. The granulation tissue was in continuity with this capsule and the epitenon in several locations. Both the membranes had protected their tendons, as evidenced by the absence of phagocytic cells. In comparisons between the CS-containing and CSlacking membrane study groups, the intratendinous collagen bundle organization was more regular and the tenocytes were more active in the former group than in the latter. The presence of CS on the membrane is suggested to provide a macromolecular network that functions as a diffusion barrier for the fibrinogen-rich and fibronectin-rich wound exudate. This barrier would limit the development of a fibrin/fibronectin network connecting the tendon. The scaffold for the formation of granulation tissue would be prevented from forming by the physicochemical properties of CS without interference with the healing of the tendon repair. Chondroitin sulfate and hyaluronic acid have been used as a compound in the prevention of adhesion in tendon healing. Local administration of CS/hyaluronic acid reduced the incidence of adhesion formation after tendon tears of the rabbit plantaris ten-

The Journal of Hand Surgery / Vol. 27A No. 2 March 2002

don.1 Initially in our study CS-pHEMA–implanted digits had better flexion rates than those of the pHEMA-only, TSR, and NSR groups. Although the ROM of the CS-pHEMA group decreased at week 6, results still were better than those of the pHEMAonly, TSR, and NSR groups. At the end of the experiment the beneficial effect of the CS-pHEMA membrane was lost, as measured by mechanical evaluation. Its positive effect on the prevention of adhesion and on the healing of the tendon continued, as measured by histologic evaluation. One of the important aspects of this study is that the rabbits were allowed free motion immediately after surgery. Mechanical and histologic evaluations were done on the same digits, and tendon rupture was not detected in any of the specimens. In the preliminary study, hindpaws were used and all tendons were ruptured. Forepaws were used in a second preliminary study with no tendon rupture recorded. Consequently, the current study used forepaws. Perhaps no tendon rupture was detected on the forepaws because rabbit forepaws bear less weight than hindpaws. Mechanical and histologic evaluation on the same specimens may be a limitation. Because immediate motion is the ultimate goal of tendon repairs, however, rabbits were allowed free motion immediately after surgery; this would be another way to test the membrane because allowing unrestricted motion immediately after surgery would cause a mechanical stress (tension) at the repaired tendon site. A shear force would occur between the repaired tendon and the sheath. If the application of this unrestricted repetitive mechanical force throughout the follow-up period on the repaired tendon and at the sheathtendon junction had affected the repair site, we believe the subsequent mechanical testing would not have produced any additional impact. Mechanical and histologic evaluation of the same specimens probably would not be different if histologic and mechanical evaluations had involved different specimens. Many studies have emphasized the importance of early controlled mobilization rather than considered the importance of repair of the synovial tendon sheath. Gerard et al31 reported that there were no tendon ruptures during early active mobilization after repair of complete sections of digital flexor tendons. Halikis et al32 studied the energy required to flex the digit against tendon adhesion after surgery; the immobilization protocol required more energy than immediate mobilization to regain acceptable flexion rates. Suturing the tendon sheath and narrowing the

305

diameter of the canal may cause progressive inflammation and impair tendon gliding, however.33 The explanation for the discrepancy between the results of histologic and mechanical testing of joint movement may be found in the length of time that the healing tendon is exposed to CS and CS coating’s half-life— both unknown parameters. Molecular weight, concentration, and viscosity all are variables that may need to be improved for CS to have a sustained beneficial effect. Although the clinical end point of tendon repair is to regain ROM, the use of ROM for determining the degree of adhesion in experimental studies is controversial. Range of motion may not be an adequate tool to assess tendon adhesion levels.34,35 Such controversy may lend further credibility, however, to the histologic determinations in experimental studies, marking them out as a better criterion in such studies. The CS-coated pHEMA membrane was successful in decreasing postoperative tendon adhesion, especially for the first 6 weeks. Although restrictive adhesions may develop after 6 weeks, we consider this unlikely. Because of the presence of the ionic, hydrophilic polysaccharide, the CS-coated membrane seems to serve its function well. The soft jelly-like layer of the polysaccharide probably absorbs significant amounts of water and creates a water-rich surface. Such highly swollen structures are mechanically weak, however, and may wear off in time as a result of mechanical abrasion. Future studies should concentrate on optimizing the CS concentration on the coated sides for best antiadhesion results. Further investigations involving CS should include improvement of its duration of action, molecular weight, concentration, and stability of the coat to better show its beneficial effects. The membrane showed good biocompatibility, and the CS-pHEMA membrane seems to be a promising material for use in flexor tendon surgery in cases in which the original tendon sheath cannot be restored. The authors thank Assistant Professor Yakup Arıca, Kırıkkale University, for his contribution to the synthesis of the pHEMA membranes; and Professor Feza Korkusuz, Dr Sedat Is¸ıklı, and Steven Riva (Middle East Technical University) for their contributions to the experimental studies, statistics, and editing.

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