The use of autogenous tendon grafts to replace injured digital flexor tendons is a common clinical procedure. Although extrasynovial tendons commonly are used (such as palmaris longus tendons), numerous complications have been reported including the formation of adhesions, joint stiffness, and poor digital function.1,8,27,29 Tendon grafts of intrasynovial origin, however, show cell survival, intrinsic revascularization, minimal adhesions, and adequate function in this anatomic site.13,14,26 However, the availability of autogenous intrasynovial tendons for use as grafts in intrasynovial sites such as the digital sheath is limited, and alternate graft materials are needed.
A potential strategy for tendon grafting is the use of natural extracellular matrices (ECMs) as scaffold materials.3 Conceptually, these materials provide temporary functional support, possess inherent biologic properties that favorably influence cell attachment and tissue development, involve no donor site morbidity, and are biocompatible, bioresorbable, and immunologically safe. Small intestinal submucosa (SIS) is one ECM that has been shown to promote healing and site-specific regeneration of neotissue in various applications including vessels,5,19,24 bladder,17,18 dura,10,11 abdominal wall,9,22 ligament,2 and tendon.6,12
Small intestine submucosa is a collagenous biomaterial consisting of the tunica submucosa layer of the porcine small intestine. Small intestine submucosa has a thickness of approximately 80–100 μm and contains predominately Type I collagen and fibronectin, chondroitin sulfate, heparin, heparin sulfate, hyaluronan, and numerous growth factors (such as FGF-2, TGF-β, and VEGF).15,16,21,28 To date SIS has shown promise as a grafting material for various orthopaedic soft tissue applications. Specifically, in rabbit Achilles6 and canine infraspinatus12 tendon models, SIS grafting was associated with rapid angiogenesis, host tissue ingrowth followed by constructive neotendinous remodeling, and little to no evidence of chronic inflammation or peripheral adhesions. Although the ultimate failure loads of SIS-regenerated and control-repaired tendons were significantly less than native tendons, long-term assessments (greater than 1 year) were not done in these animal models.
To our knowledge, the use of SIS as a graft material for flexor tendon repair and regeneration has not been evaluated. It was our objective to investigate the use of SIS for flexor tendon grafting in a canine model. We hypothesized that at 6 weeks after implantation, SIS grafts undergo host cell infiltration, neovascularization, and show evidence of replacement by host neotendon. We also hypothesized that the SIS grafts would be incorporated by the host without extensive adhesions to surrounding tissues and that the SIS grafts would maintain normal digit function (the ability to translate independently with respect to the flexor digitorum superficialis [FDS] tendon as determined by the ability to rotate the distal interphalangeal [DIP] joint). An intrasynovial tendon autograft was used as the control or gold standard because of its documented ability to remain viable, heal without the ingrowth of fibrovascular adhesions, and maintain independent function in this animal model.26
MATERIALS AND METHODS
The overall study design, animal model, and procedures used were based on a well-established canine flexor tendon model.7,13,14,25,26 All procedures were done in accordance with the American Association for Accreditation of Laboratory Animal Care standards. Six adult female mixed breed dogs (age range, 2–4 years; weight range, 30–35 kg) were used. Small intestine submucosa grafts (7 cm × 2.5 mm × 1 mm) were used to replace the flexor digitorum profundus (FDP) tendon in the intrasynovial region of the fifth digit of the left forepaw (Fig 1A). The intrasynovial tendon from the fifth digit was moved to replace the second digital tendon of the left forepaw and served as an autograft control. After surgery, the forelimb was placed in a cast and the animals were allowed weightbearing and unrestricted cage activity. Postoperative rehabilitation consisted of passive flexion of the casted carpus and digits twice daily, 6 days per week. The animals were sacrificed 6 weeks postoperatively and the grafts were assessed by gross observation for adhesions and functionality. The grafted digits then were excised with their surrounding tissues for histologic evaluation.
Small intestine submucosa grafts were fabricated by DePuy Orthopaedics (Warsaw, IN). Small intestine submucosa was obtained from the small intestine of specific pathogen-free pigs. The tunica submucosa layer was isolated from the mucosal, muscularis, and serosal layers by gentle abrasion. The SIS was disinfected and made acellular by washing in peracetic acid and ethanol.5,24 The grafts consisted of 25 layers of SIS, stacked such that the luminal side of one layer was apposed to the abluminal side of the adjacent layer. This laminate was vacuum dried, cut to 2.5 mm in width, wrapped with a piece of hydrated SIS (luminal side inward), and lyophilized. Grafts were E-beam irradiated (18.3–22.3 kGy, TitanScan Technologies, Lima, OH) and shipped to our institution. Before implantation, grafts were rehydrated for approximately 20 minutes in sterile saline. Graft dimensions were chosen to be similar in size to the native canine tendon (approximately 2.5 mm wide × 1 mm thick). A 7- to 8-cm length was used.
The following surgical preparation and anesthesia procedures were used. Thirty minutes before anesthetic induction, dogs were given an intramuscular injection of acepromazine (0.15 mg/kg). Dogs initially were anesthetized with an intravenous dose of thiopental (20 mg/kg) to effect. Penicillin benzathine and procaine (20,000 IU/kg) were administered intramuscularly at induction. Dogs were intubated orotracheally and maintained on isoflurane in oxygen, titrated to effect (0.5–5%). Dog forelimbs were shaved and prepared with iodophor povidone iodine (Operand, Aplicare, Inc, Branford, CT). The surgery was done using tourniquet control.
The surgical approach to the proximal and distal FDP tendon was similar to that described previously.26 Proximally, a 1.5-cm volar incision was made over the midmetacarpal level, well proximal to the digital flexor tendon sheath. The FDP tendons to the second and fifth digits were isolated beneath the flexor digitorum superficialis (FDS) tendons. Distally, the insertions of the FDP tendons of the second and fifth digits in the left forepaw were approached by a midlateral incision at the border of the distal fat pad and extended proximally in a Brunner zigzag fashion for approximately 1.5 cm. The fat pad and pulp were sharply elevated off the distal flexor tendon sheath taking care not to damage the branched neurovascular bundle. The sheath was sharply entered at the level of the DIP joint, and the FDP tendon was detached from the base of the distal phalanx. The surface of the phalanx at the insertion site was abraded with a scalpel blade to remove host tendon and prepare the bone surface.
For SIS graft placement, a 4–0 Ethibond suture (Ethicon, Inc, Piscataway, NJ) was placed in the distal tendon stump of the fifth digit with a modified-Kessler stitch, and tied leaving a long loop. A looped 4–0 Supramid suture (S. Jackson Inc, Alexandria, VA) was placed in one end of the SIS graft with a Krakow stitch and placed through the suture loop in the distal tendon stump. The SIS graft was advanced through the synovial sheath, as the native FDP tendon was pulled out of its sheath in a distal-to-proximal direction. The SIS graft was oriented so that the luminal side of the 25 layers was deep against the bone. The FDP tendon was transected 1 cm distal to the common tendon quadrification and preserved in saline for grafting into the second digit. A looped 4–0 Supramid suture was placed in the remaining FDP tendon stump with a modified-Kessler stitch. Proximal suture of the SIS graft (4–0 Supramid suture and Krakow stitch) to the native FDP tendon (4–0 Supramid suture and modified-Kessler stitch) was done in an end-to-end fashion creating a four-strand repair.30 A 6–0 Prolene (Ethicon, Inc) continuous, locking epitendinous stitch was placed to reinforce the repair. Distally, the SIS graft was cut to a length to achieve appropriate tension using the flexion cascade of the native toes as a guide. A 4–0 Ethibond suture was placed in the end of the SIS graft with a Krakow stitch. The suture was passed through drill holes in the distal phalanx and tied over a button placed on the toenail.
For placement of the native tendon autograft, after incising the distal phalangeal insertion of the second digit FDP tendon, a tag suture was placed leaving a long loop. A looped 4–0 Supramid suture was placed in the proximal free end of the fifth digit autograft with a modified-Kessler stitch and threaded through the suture in the distal tendon stump. The autograft was advanced through the synovial sheath as the native FDP tendon was pulled out of its sheath in a distal-to-proximal direction. First, the distal repair was done as described above. Then the second digit FDP tendon was cut proximally, removed, and discarded. The location of the cut was approximately 1 cm distal to the common tendon quadrification. Tension was gauged using the other digits as a guide. A looped 4–0 Supramid suture was placed in the remaining FDP tendon stump with a modified-Kessler stitch. Proximal suture of the autograft (4–0 Supramid suture and modified-Kessler stitch) to the native FDP tendon (4–0 Supramid suture and modified-Kessler stitch) was done in an end-to-end fashion with a four-strand repair. The same epitendinous stitch previously described was used for reinforcement.
All wounds were irrigated and closed with 3–0 Vicryl sutures (Ethicon, Inc). Antibiotic cream, gauze, cast padding, and a compressive bandage were applied. Postoperative analgesia consisted of buprenorphine (0.3 mg) administered subcutaneously on the day of surgery and the following day if indicated.
Postoperatively, the surgically treated limbs were subjected to casting and a defined rehabilitation protocol. A fiberglass shoulder-spica cast was applied to the left forelimb, with the shoulder in approximately 30° abduction, the elbow in approximately 70° flexion, and the carpus in approximately 30° flexion.26 Dogs were allowed immediate weightbearing, as permitted by the cast, and unrestricted cage activity. The volar ½ of the forelimb portion of the cast was removable to allow for controlled passive mobilization exercises, starting on the first postoperative day. Rehabilitation consisted of manually applied passive flexion and extension of the carpus and digits at 0.5 Hz, 5 minutes, 2 times daily, 6 days per week for 6 weeks. With this protocol the digits were brought to full extension, however, the carpus never reached the full extension because of the limits of the cast.
The dogs were sacrificed 6 weeks postoperatively by an intravenous lethal injection (approximately 0.25 mL/kg) of pentobarbital sodium with phenytoin (Beuthansia-D, Schering-Plough Animal Health Corp, Union, NJ). At necropsy the graft proximal repair sites were grossly assessed for connectivity and adhesions.
To assess whether grafts maintained normal digit function (the ability to translate independently with respect to the FDS tendon), a rudimentary test was done. The middle phalynx was secured with hemostats while the native FDP tendon was secured proximal to the graft repair site and pulled along its axis of loading. If DIP joint rotation was observed, the graft was noted to function independently from the FDS tendon (normally). Alternately, if pulling the graft produced only the same motion as when pulling the FDS tendon (rotation of the PIP joint but not the DIP joint), the graft was noted as not having independent function.
The grafted digits then were excised en bloc with their surrounding synovial sheath, superficial FDS tendon, and distal phalynx for histologic evaluation. The harvested digits were cut into three segments along their length (Fig 1B). Zone I contained the distal phalynx and distal repair site, whereas Zone III contained the extrasynovial region and proximal repair site.26 All segments immediately were fixed for 48 hours in 10% zinc formalin. Graft segments from Zones I and II contained bone and subsequently were decalcified in Accumate RDO Rapid Decalcifying Solution (Sigma-Aldrich, Inc, St Louis, MO) for 20–24 hours. Tissues were then processed routinely, embedded in paraffin, sectioned (4 μm), and stained with hematoxylin and eosin. Histologic sections were evaluated by three observers. Cellularity, vascularity, inflammatory cells, and adhesions of the graft to surrounding tissues were noted. In addition, a representative canine digit was harvested and processed for observation of the histologic features of normal flexor tendon.
All dogs had normal recovery from surgery, adjusted quickly to their cast, and were standing within 24 hours. Two dogs had urinary tract infections develop during the experiment and were treated with a 14-day course of oral antibiotics (enrofloxacin, 272 mg/day). One dog pulled out of the cast 16 days postoperatively for as much as 5 hours. This dog was recasted but not excluded from the study.
At necropsy, all grafts appeared grossly intact at their proximal repair sites. In four of the autografts and two of the SIS grafts, the repair sites were easily delineated with minimal to no adhesions to surrounding tissues (Fig 2A). In two autografts and three SIS grafts, the repair sites were identifiable but not readily separable from surrounding tissues (Fig 2B). In both cases the autografts were scarred to the overlying FDS tendon, whereas the SIS grafts were scarred to the overlying FDS tendon, deep muscle, and/or adjacent FDP or FDS tendons. In one SIS graft, the proximal repair site was indistinguishable from the FDS tendon and surrounding tissues (Fig 2C).
Four of the six autografts had independent function as determined by DIP joint rotation. The two autografts that did not had experienced surgical trauma to the distal pulley or sheath during surgery. Five of six of the SIS grafts did not rotate the DIP joint, and the function of the sixth was so minimal that we considered the assessment inconclusive.
Histologic sections of an autograft (normal FDP tendon) and a 25-layer SIS graft, as used for implantation, are shown in Figure 3. The inner layers and outer wrap of the SIS graft can be seen (Fig 3B).
At 6 weeks the autograft tendons were cellular and seemed to have maintained viability along their length. At the proximal repair site (Zone III), all autografts appeared integrated with the native tendon. The proximal repairs were hypercellular and regions of lymphocytes, plasma cells, macrophages, and spindle-shaped cells (presumed fibroblasts or mesenchymal cells) were observed (Fig 4A). At the proximal repair site the grafts were infiltrated by neovasculature and regions of neotissue. Away from the repair site and in all zones throughout their length, autografts were avascular, and contained similar numbers of cells and had similar morphologic features as normal FDP tendon (Fig 4B). No inflammatory cells were observed.
At 6 weeks the SIS grafts were infiltrated by neovasculature and had greater than normal numbers of spindle-shaped nuclei (probable fibroblasts or mesenchymal cells) in all zones along their length (Fig 4C–D). At the proximal repair site (Zone III), the SIS grafts were integrated with the native FDP tendon but often appeared adhered to adjacent tissues such as the FDS tendon. Focal regions of lymphocytes, plasma cells, and macrophages were observed in all zones (Fig 4C). In all zones the SIS grafts contained regions of organized, wavy tissue, oriented in the direction of graft loading (Fig 4C–D).
Evidence of fibrovascular adhesions could be identified most readily from the Zone II histologic sections. In Zone II, all autografts appeared to remain distinct from the synovial sheath and FDS tendon (Fig 5A). Minimal to no adhesions were observed to the surrounding tissues. However, in Zone II the SIS grafts from all six dogs appeared to be adhered to the surrounding FDS tendon and sheath (Fig 5B–C).
At the distal repair site (Zone I), all autografts were integrated with bone. Increased vascularity, tissue remodeling, and occasional inflammatory cells were observed. In Zone I the SIS grafts were integrated with bone, but the remodeling SIS neotissue was scant and difficult to identify in the fat pad region.
One of the six dogs had a marked inflammatory response to the SIS graft at 6 weeks. This response was evident along the length of the graft, and was composed of numerous lymphocytes, plasma cells, and necrobiotic granulomas.
The SIS grafts used for this study consisted of 25 layers of SIS, which were vacuum-pressed, cut to 2.5 mm in width, wrapped with hydrated SIS (luminal side inward), and lyophilized. This graft design involved several considerations. First, graft dimensions were chosen to be similar in size to the native canine tendon (approximately 2.5 mm wide by 1 mm thick). The grafts were vacuum-pressed similar to the fabrication methods of the Restore™ Orthobiologic Implant (DePuy Orthopaedics). Vacuum-pressing secures the laminate structure, stiffens the material, improves the suture pull-out strength over fresh SIS, but also compacts the tissue structure. Therefore, the surfaces of our grafts were wrapped with a layer of fresh SIS and subsequently lyophilized to impart a modest surface porosity that might be more favorable for cell attachment than the vacuum-pressed surface. It has been reported, however, that lyophilized and rehydrated SIS is still less conducive to cell attachment than fresh SIS.20
The mechanical stiffness and overall mass of a tissue-engineered graft are also important design parameters and must be considered in light of the particular biomaterial in consideration. Specifically, the logic of using SIS as a tissue-engineering scaffold is that it has been reported to elicit a robust healing response and undergo rapid incorporation by the host in wide range applications.2,5,6,9,10,11,12,17,18,19,22,24 By 14 days after implantation, SIS grafts become intensely vascularized and undergo progressive replacement by immature, host neotissue.3 In a canine urinary bladder model, only scattered remnants of SIS grafts were present 4 weeks after implantation.4 By 3 months, less than 10% of the SIS graft material remained.23 The implications of these findings are twofold. First, the mass of SIS may need to be optimized for a desired outcome. For example, adding more layers of SIS to a graft to increase its stiffness may increase the incorporation time and adversely affect the result. Second, because of the rapid host response, the initial stiffness or strength of an SIS graft may not be as important as its biologic properties. Specifically, it is unknown how the initial stiffness of the graft relates to its rapidly decreasing postimplantation stiffness and concomitant neotissue loading.
In pilot studies, we determined that SIS grafts fabricated for this study were an order of magnitude less stiff and less strong than native FDP tendons (autograft controls). Size constraints of the synovial sheath restricted us from adding more mass to the grafts to increase their stiffness. Other fabrication methods (such as braiding) to increase graft stiffness were considered, however, suture pullout strength was severely compromised. Chemical cross-linking to increase stiffness was not considered because we were interested in studying native SIS in this site. We chose to use the SIS grafts, despite their relatively compliant nature, based on the documented ability of similarly fabricated SIS grafts to promote neotendinous tissue formation in other applications6,12 and the absence of data to support the logic that the grafts had to be as stiff or as strong as native tendon to produce a desirable outcome.
An intrasynovial tendon autograft was used as the control for this study because of its documented ability to remain viable, heal without the ingrowth of fibrovascular adhesions, and maintain independent function in this animal model.26 The autograft results from this study support previous work with this animal model, showing that intrasynovial tendons are especially well suited for survival and functionality after grafting into the digital sheath.26 At 6 weeks the intrasynovial tendon autografts remained viable, contained normal numbers of cells along their length, and had minimal peritendinous adhesions. Chronic inflammation was isolated to the repair sites and probably was related to surgical insult or suture material. Four of six autografts had independent (normal) function.
The two autografts that did not maintain independent function were adhered to surrounding tissues at the proximal repair site and had experienced surgical trauma to the distal pulley or sheath during surgery. We speculate that the surgical complications involving the distal sheath may have induced adhesions between the FDP graft and FDS tendon just proximal to the DIP joint. These adhesions could have limited FDP graft excursion with respect to the FDS tendon and contributed to greater scarring. Further, the wound healing response in these two animals may have been exceptionally robust, as these dogs had more of an inflammatory response to the SIS graft than three of the other four animals.
The healing of the SIS grafts differed from that of the intrasynovial tendon grafts. The SIS grafts were hypercellular and vascularized in all zones along their length. Such a hypertrophic response is similar to what has been reported for early times in previous implantation studies using this material.6 The SIS grafts also contained wavy fibers, predominantly oriented along the axis of load. It was reported that by 6 weeks in a canine urinary bladder model, SIS grafts were largely replaced by host tissue.4,23 Although it is possible that SIS resorption kinetics in the intrasynovial sheath differ from other sites, presumably at 6 weeks in the current study, at least some of the observed tissue was host-derived.
Small intestine submucosa graft incorporation occurred without evidence of a hyperimmune response such as ischemic necrosis, vasculitis, or vascular intimal hyperplasia that can be seen when preexisting antibodies are present in a sensitized host. Nevertheless, chronic inflammation persisted throughout the SIS grafts at 6 weeks. Although this study was not designed to determine the natural history of the inflammatory reaction, one might expect the inflammation to gradually disappear as the xenograft tissue is completely replaced by the host.12
One of the six dogs had a marked inflammatory response to the SIS graft. This response was composed of numerous lymphocytes, plasma cells, and necrobiotic granulomas. Necrobiotic granulomas of this type in humans are associated with rheumatoid arthritis (rheumatoid nodules), granuloma annulare, and immune reactions to foreign material or to organisms. The significance of the excessive inflammatory response in the canine is unclear. Special stains for bacteria, fungal organisms, and acid-fast bacilli were negative. An inflammatory reaction of this magnitude would be expected to eventually compromise the mechanical integrity of this graft.
At 6 weeks, normal function (rotation of the DIP joint) was not achieved with the SIS grafts, and adhesions to surrounding tissues were observed histologically along their length. Although the functional test we did was relatively crude, because of these ubiquitous adhesions, we speculate that normal function would not be attained with time. However, the presence of significant adhesions is contrary to previous reports of SIS in other tendon applications6,12 and similar to the outcome when using an extrasynovial tendon autograft in this model.14,26 As in the case of extrasynovial tendon grafts, the peripheral adhesions observed with SIS in this model may be attributed to the obligatory cell and vascular invasion of the grafts from host tissues, in such particularly close proximity to the surrounding tendon sheath from which it originates.
It is possible that inadequate excursion of the SIS grafts compounded the extensive adhesions observed. As stated previously, SIS grafts in their hydrated form were an order-of-magnitude less stiff and less strong than native FDP tendon. This inherent compliance of the SIS graft compared with native tendon made it challenging to judge whether appropriate tension was achieved during graft placement. Because a less than optimally tensioned SIS graft would undergo less excursion than a well-tensioned autograft during the same rehabilitation regimen, it is possible that inadequate excursion also played a role in SIS graft adhesions. However, consider the outcome of extrasynovial tendon grafting in the synovial sheath. Extrasynovial tendon grafts have similar stiffness as the native tendon, can be appropriately tensioned, and presumably undergo similar excursion as intrasynovial tendon grafts with the rehabilitation protocol used.26 However, extrasynovial grafts become scarred to the synovial sheath whereas intrasynovial grafts do not. Combined with the results of our study, these results support the hypothesis that irrespective of optimal graft tension or rehabilitation, adhesions will be difficult to avoid in this tendon site when using any graft that requires ingrowth of peripheral vessels and cells for incorporation.
The current study is limited by the lack of differential staining to assess the degree of SIS graft replacement by the host, and the relatively short time evaluated (6 weeks). Because we did not do differential staining in our study, we were unable to conclude that the wavy, oriented tissue observed with the SIS grafts at 6 weeks was host-derived neotendon. Based on previous studies in other sites,4,23 however, we presume that at least some of it was host-derived. Had we evaluated the grafts at a longer time, for example, at 6 months, the histomorphometric features and biomechanics of the then relatively mature, host-derived, neotissue could have been more quantitatively evaluated. Despite these limitations, the histologic observations in this study suggest a likely conclusion regarding the use of SIS as a full-length intrasynovial graft.
Porcine-derived SIS was evaluated as a scaffold for use in flexor tendon grafting. The choice of SIS was based on its purported ability to promote site-specific tissue regeneration and function.5,6,12 With the exception of a striking inflammatory reaction in one sample, the SIS grafts in this study were associated with a generally favorable biologic response including host cell infiltration, neovascularization, and the observation of wavy, oriented tissue. However, ubiquitous adhesions, together with impaired function in all cases, suggest that SIS grafts in the configuration used were not suitable as full-length intrasynovial grafts in this tendon and animal model. A more effective indication for SIS in flexor tendon repair might be to embed a small segment of SIS at the interface of FDP tendons undergoing primary repair within the synovial sheath to enhance their intrinsic healing kinetics.
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