Ann Thorac Surg 2002;73:1747-1751
© 2002 The Society of Thoracic Surgeons
Original article: general thoracic
Survival analysis of rats implanted with porous titanium tracheal prosthesis
Philippe Schultz, MDa,
Dominique Vautier, PhDb,
Johanna Chluba, PhDb,
Luc Marcellin, MDc,
Christian Debry, MD, PhD*a
a Department of Otolaryngology-Head and Neck Surgery, Hautepierre Hospital, Strasbourg, France
b Department of Anatomo-Pathology, Hautepierre Hospital, Strasbourg, France
c INSERM 424, Centre de Recherche Odontologique, Strasbourg, France
Accepted for publication February 26, 2002.
* Address reprint requests to Prof Debry, Hôpital de Hautepierre, Avenue Molière, 67098 Strasbourg Cedex, France
e-mail: christian.debry{at}chru-strasbourg.fr
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Abstract
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Background. Surgical treatment of a malignancy in the trachea may lead to a long resection that has to be reconstructed with an artificial prosthesis. However, most of the available prostheses encounter inflammatory rejection and mechanical constraint problems. To improve tracheal rehabilitation a porous titanium prosthesis was developed. The aim of this study was to test the biocompatibility of this novel material.
Results. Fibroblast colonization of titanium pores and a ciliary cylindrical epithelial layer developed on the endoluminal side of the prosthesis and the inflammatory reaction was minimal.
Conclusions. The results of this short-term study validate, from surgical and histologic standpoints, the usefulness of a porous titanium tracheal prosthesis.
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Introduction
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Invasion of the trachea by tumors, causing asphyxia or hemorrhage, is responsible for up to 47% of disease-related death in patients presenting with head and neck tumors [1]. Radical long-segment resection of trachea is still the predominant method in the treatment of tumors for operable patients [2].
At present, most attempts to reconstruct an artificial trachea have rarely achieved long-term success. Inflammatory tissue invasion as a result of the biomaterial itself [3] and mechanical constraints [4] were the main problems encountered for rejection of the prosthesis.
In our study we selected titanium, a metal known to be well tolerated [5] and chemically resistant [6] to the corrosive and oxidative activity of various agents. These features make titanium a commonly used metal in implantology, and most particularly in odontology [7].
This study reports on the design of a new type of prosthesis made of titanium manufactured in collaboration with ONERA (Office National d’Etudes et de Recherches Aérospatial) and its evaluation as a tracheal implant. To test the biocompatibility of this material, first we evaluated in vitro the fibroblast morphology at the surface of titanium. Then, we investigated in vivo the tissue colonization after the implantation of the titanium tracheal prosthesis in rats.
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Material and methods
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Production of the prosthesis
Assembled titanium prostheses were made of Ti-40 by ONERA (Palaiseau, France), containing amounts of carbon, iron, and nickel in conformity with the AFNOR (Association Française de NORmalisation) standards. The prostheses are made with spherical titanium beads of 400 to 500 µm in diameter. The beads were placed into a mold and fused by condensed electrical discharges. The porous space between each contiguous fused bead was about 150 µm. Prosthesis sizes were adjusted on the mean values of trachea diameter and length previously determined from identical rats in age and weight to those used for in vivo experimentation. The prostheses were cylindrical tubes of 1 cm in length corresponding to six tracheal rings. They were 1.3 to 1.4 mm thick with three layers of spheres of 5.7 mm outer and 3 mm inner diameters. On the prosthesis, a 0.8-µm longitudinal open slot and a 1-mm diameter hole was added at 1 mm from the end of each tube (Fig 1).
Mechanical resistance was assessed by crush tests conducted at the ONERA laboratory. Before implantation, the titanium prostheses were sterilized under ultraviolet light (255 nm) for 1 hour.
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Fig 1. Final appearance of the titanium prosthesis with (a) longitudinal slot and (b) holes at the extremities of each tube.
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Scanning electron microscopy
Qualitative information on the morphology of the cells at the surface of prosthesis was obtained using scanning electron microscopy. Rat fibroblast (rat 1 cell lineage), were cultured in GIBCO BRL RPMI 1640 medium containing 10% fetal calf serum (Life Technologies, Cergy Pontoise, France), 50 U/mL penicillin, and 50 µg/mL streptomycin (Biowhittaker) in an atmosphere of 5% CO2 and 95% air at 37°C. After reaching confluence, the cells were dissociated with 1x trypsin/EDTA solution (Life Technologies) and plated at a density of 1 x 105 cells/mL on porous titanium samples placed in 24-well plastic plates (NUNC, Roskilde, Denmark). After 4 hours of culture, cells attached to the substrate were washed in phosphate-buffered saline (GIBCO BRL) at 37°C, two washes of 2 minutes each. Fibroblasts were fixed for 20 minutes in 2.5% glutaraldehyde in phosphate-buffered saline at room temperature. The samples were dehydrated in an ascending ethanol series (50%, 70%, 90%, and twice at 100% for 10 minutes each). Specimens were mounted on sample holders with silver print, sputter-coated with a gold-palladium alloy in an Hummer JR unit (Siemens, Karlsruh, Germany), and visualized by scanning electron microscopy with a JEOL JSM 35 C (JEOL USA, Peabody), operating at 25 kV. At least two substrates were observed.
In vivo experiments
All animals received care in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication 85-23, revised 1985). Wistar rats (4 to 5 months old, about 500 g in weight) were used. Male rats received an intraperitoneal injection of an anesthetic solution, 1/5 of 2% (vol/vol) Rompun (xylasin chlorhydrate 2% and methyl parabenzoate 0.1%) and 4/5 of 5% (vol/vol) Imalgen 1000 (pure ketamine). A vertical median cervicotomy was performed from the sternum up to the jaw. The subhyoid muscles and nerves were separated from the trachea. The recurrent nerves were identified and carefully kept unharmed. The trachea was incised from the second to the eighth tracheal ring. To prevent retraction of the tracheal extremities a thin band of tracheal membrane was conserved. The longitudinal slot of the prosthesis was slide into the posterior trachea. The tube was rotated 45° to place the slot in a lateral position maintaining the extremities of the prosthesis in the aerial axis (Fig 2).
Thus, the posterior trachea membranous band situated into the prosthesis lumen contributes to its positional stability. The extremities of the prosthesis were then joined with tracheal tips, using the holes in the prosthesis and Prolene 6-0 (Johnson & Johnson, Springfield) sutures. Finally, the subhyoid muscles were replaced as cover and sutured together on the median lane. The animals were caged in a controlled environment with 12-hour on/off cycles. Food and water were provided ad libitum.
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Fig 2. Titanium prosthesis positioned in the rat larynx (a). Ends of the prosthesis were joined at the tracheal ends, using the holes in the prosthesis (b).
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Histologic analysis
After an observation period ranging from 2 to 6 weeks, the rats were sacrificed by intraperitoneal injection of phenobarbital. A large exeresis was performed including the cervical region into a single block to avoid mechanical disjunction of the prosthesis--biological tissue interface. All histologic analyses were performed in the Orthopedic Research Laboratory of Paris VII University using the following technique: specimens were fixed, dehydrated, and immersed into three successive methylmetacrylate baths containing increasing concentrations of catalyzing agent. The last bath was placed at 37°C until full polymerization was completed; 200-µm sections were prepared using a LEICA 1600 microtome (LEICA, Wetzlar, Germany), and stained with Stevenel’s blue-de Van Gieson’s picrofuchsin for microscopic analysis.
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Results
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Microscopic aspect of the titanium beads and early cellular attachment
Microscopic features of the welded titanium beads were observed in vitro using scanning electron microscopy (Fig 3).
Beads presented a quasi-uniform size with some surface irregularities (Fig 3A). Fibroblasts were attached after 4 hours of culture on the titanium surface. Cells appeared to have a normal cellular shape with lamellipodia and pseudopodia (Fig 3B). Titanium, therefore, seemed sufficient to trigger cell adhesion and spreading of fibroblasts.
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Fig 3. Scanning electron micrograph showing morphologies of rat 1 cells cultured 4 hours on titanium prosthesis surface. (A) Titanium beads on the prosthesis surface. (Bar, 1000 µm). (B) Rat 1 cells exhibit normal cellular shape with lamellipodia/pseudopodia. (Bar, 10 µm).
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Animal survival
Subsequent to the prosthesis implantation, 11 of the 17 rats used in this study survived for a period ranging from 22 to 41 days (Table 1).
To verify that the prosthesis implantation did not alter the animal’s normal food intake, the rats’ weights were measured. A variation in weight could be the result of a compression of the prosthesis on the esophagus. We observed a reduction in the animal weight up to 25 days after implantation. After 25 days rats regained between 40 and 140 g. There were two exceptions: one animal gained 50 g at day 22, and another gained only 5 g after 36 days. Except for these outliers, the animals regained a normal weight after few weeks. Thus, it seems that the variation of weights was independent of the prosthesis compressing the esophagus.
We performed autopsies on the six rats that died before the end of the study and the causes of death are as follows: overdose of anesthetic agent (one rat), incorrect positioning and displacement of the prosthesis (two rats), wound of the recurrent nerve during operation with immediate acute dyspnea (two rats), sealing of the prosthesis resulting in respiratory insufficiency and loss of weight (one rat).
Histologic analysis
The histologic results of the prosthesis section of the 11 rats implanted after a period of 15 to 41 days were similar. On a global section of the cervical region (Fig 4)
the prosthesis made of titanium beads could be seen ahead of a cervical vertebra and the esophagus. The posterior endoluminal side of the implant was filled with the membranous tracheal part. The external side of the prosthesis was surrounded by a thin cuff of richly vascularized fibrous tissue presenting numerous neocapillary vessels, fibroblasts, and few lymphocytes (not shown).
This connective and vascular tissue was infiltrating the porous spaces between the titanium beads, the entire prosthesis wall thickness up to the internal side, which was fully coated (Fig 5).
Titanium beads were entirely coated with a layer of giant multinuclear cells largely visible when beads were eliminated upon polishing. Macrophage and giant cell granuloma were also present in the connective and vascular sheet covering the internal side of the prosthesis, under the ciliary cylindrical epithelial layer lining the open tracheal lumen (Fig 6).
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Fig 4. Transverse section of the cervical area 2 weeks after implantation showing (p) prosthesis made of titanium beads, (ve) cervical vertebra, and (e) esophagus. The posterior tracheal membrane (pmt) was inserted into the longitudinal slot of the prosthesis. The submaxillary glands (smg) are located in the most anterior position of the section.
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Fig 6. The titanium beads 1 month after implantation. (a) The endoluminal side of the prosthesis is coated with fibrous and vascular tissue spotted with clusters of macrophages and resorption of multinuclear giant cells; (b) a few lymphocytes are present. (c) A new respiratory high ciliary cylindrical epithelial layer surrounds the tracheal lumen.
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Fig 5. Fibrosis around the implant 3 weeks after implantation. (a and b) Fibrosis within spaces directly in contact with the titanium surface. (b) Fibroblasts in the deeper layers are still present, but in low density.
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Comment
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Prosthesis tolerance under septic environmental conditions remains a major challenge in thoracic or ear, nose, and throat operations, especially when dealing with replacement of the trachea, cricoid cartilage, or mandibular bone. Prostheses made of polyethylene terephthalate, Dacron, or silicone for tracheal and larynx implants produce mitigating results such as prosthesis extrusion, infection, and tracheal stenosis caused by major inflammatory reaction; these types of implants seem to be restricted to short-term or palliative implantation [8]. Septic rejection is caused both by immunologic incompetence of the prosthetic environment [9] and the pathogenic potential of microorganisms [10]. Autologous transplantation seems to give better results but can only be used as tracheal support, not for partial replacement [11].
Tracheal prostheses designed to be successfully integrated into biological tissues should meet the following specific criteria: (1) mechanical stability, (2) no inflammatory rejection, and (3) tissue colonization and vascularization. In the search for this ideal material, thoracic surgeons experimented with various metals [12] and polymers [13].
During the past few years, we developed a porous Ti-40 prosthesis presenting with the mechanical features requested by our ear, nose, and throat specialty field. This prosthesis is characterized by its rigidity, providing support for the tracheal and laryngeal cartilage and bone structures (mandible or dome of the skull).
Nonreactivity of implanted titanium
The crucial issue was to obtain a nonresorbing exogenous material resistant to the aggressive environment of the larynx and aerial tract, which includes a variable pH, air, and humidity that induce corrosion and oxidation, and the presence of bacteria and fungi. Tissue colonization of the prosthesis may serve to restore immunocompetence of the prosthetic environment. However, this requires a better knowledge of the microbial adhesion mechanisms leading to an alteration of the prosthetic structure, and reasons for the periprosthetic immunodeficiency [14]. To circumvent these problems different approaches have been used. To inhibit microbial adhesion at the biomaterial-tissue interface we used a smooth and nonreactive surface. The degree of porosity of the material might also play a major role. Recent studies have assessed on cell behavior on polished or granulous porous titanium. The results indicated that polishing led to a change in the local production of growth factors. The roughness of the material increases enzymatic synthesis, cellular proliferation, and collagen production [15–18]. Today, induced biomimetic properties of the material surface [19], such as adhesion [20], cellular proliferation [21], and differentiation [22] has become a major challenge. Interestingly, our implant did not induce an inflammatory reaction, as shown by the few lymphocytes present in the tissue surrounding the prosthesis. These data were consistent with previous studies [23, 24]. The tissular release of titanium particles was also described but was not associated with important local reactions [25].
Cellular adhesion and proliferation on titanium
Our histologic studies show colonization on the external side of the prosthesis by fibrous tissue and fibroblasts all around the prosthesis and within the empty spaces between the titanium beads. These tissues were well vascularized, as seen by a large number of blood vessels present in the histologic section. Around the prosthesis, in the tracheal lumen, we observed a colonization of the mucosa with fibroblasts. Interestingly, we also observed a layer of ciliary cylindrical epithelial cells. The presence of this layer can be explained by the following hypotheses: colonization by epithelial cells from the prosthesis extremities or from the band of tracheal membrane voluntarily preserved, or colonization by fibroblasts differentiated from the epithelial lining. Such epithelium prevents the build up of obstructive granuloma, which could be responsible for airway obstruction, and explain the good respiratory tolerance of the surviving animals. Consistent with our data, Laing and colleagues [25] observed in rabbits two distinct tissular areas segregated within prosthesis: one was made of a cell cluster adjacent to the titanium implant forming a pseudomembrane, and the other was fibrous and fatty tissue substituting for muscle.
In conclusion, we studied both the mechanical and biological characteristics of porous titanium in a rat model. We also performed in vitro and in vivo experimental tests of the biomaterial to assess its capacity to integrate into biological tissues.
The long-term survival of rats after replacement of their cervical trachea by this porous prosthesis represents an encouraging result for clinical repair of tracheal defects, and could, in the future, be used as part of the larynx replacement. However, additional study with a longer period of observation is necessary with prostheses of longer size. Studies addressing the formation of the ciliary cylindrical epithelial cells in the tracheal lumen are ongoing in our laboratory. In addition, to confirm the minimal inflammatory reaction observed in our histologic analysis, cytokine measurement is being investigated. It would be interesting, in the future, to add an antiinflammatory factor to our prosthesis.
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Acknowledgments
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We wish to acknowledge Prof Joëlle Ogier and Dr Christophe Egles for critical comments. We are also grateful to André Walder for providing the titanium prosthesis (ONERA, Palaiseau, France). This work was supported by grants from "Ligue Contre le Cancer-Alsace-France."
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Abstract
Introduction
