This Article Abstract Full Text (PDF) Free Letters to the Editor: Submit a response Alert me when this article is cited Alert me when Letters to the Editor are posted Alert me if a correction is posted Services E-mail this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My File Cabinet Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by JÜRGENSEN, K. Articles by HUNZIKER, E. B. Search for Related Content PubMed PubMed Citation Articles by JÜRGENSEN, K. Articles by HUNZIKER, E. B. Social Bookmarking                   What's this?

A New Biological Glue for Cartilage-Cartilage Interfaces: Tissue Transglutaminase*

K. JÜRGENSEN, M.D., D. AESCHLIMANN, PH.D., V. CAVIN, , M. GENGE, and E. B. HUNZIKER, M.D., BERN, SWITZERLAND

Investigation performed at the M. E. Müller Institute for Biomechanics, University of Bern, Bern

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we used an in vitro model to test the capacity of tissue transglutaminase to increase the adhesive strength at a cartilage-cartilage interface. Full-thickness cartilage-bone cylinders were prepared from fresh adult bovine shoulder joints, and the superficial half of the hyaline cartilage was then removed to provide a plane surface. Tissue transglutaminase was applied to the freshly cut surface of one cylinder, and a calcium-chloride solution (to act as an activating agent) was applied to that of the other. The cartilage surfaces were immediately apposed, one on top of the other, and an eighty-gram weight was applied to the upper cylinder for ten minutes at 37 degrees Celsius under defined humidity conditions. A measured force was then applied transversely to the upper cylinder until it was displaced from the lower one (which was clamped in a holding device), and the force recorded at this point was taken as a measure of the adhesive strength achieved at the cartilage-cartilage interface. The adhesive strength increased linearly with an increasing concentration of tissue transglutaminase (0.25 to 2.75 milligrams per milliliter) and was enhanced by increasing the duration of incubation, but it was not influenced by the level of humidity. The adhesive strength was improved by as much as 40 per cent when the cartilage surfaces had been pretreated with chondroitinase AC or hyaluronidase to remove glycosaminoglycan chains of proteoglycans, which are largely responsible for the intrinsic anti-adhesive properties of cartilage. CLINICAL RELEVANCE: The mechanical fixation of cartilage fragments in diarthrodial joints and the fixation and immobilization of cartilage transplant materials or biodegradable matrices containing chondrogenic cells pose serious problems for the orthopaedic surgeon. In the present study, the adhesive strength achieved with use of tissue transglutaminase at the cartilage-cartilage interface was greater than that obtained with use of Tissucol, a commercially available fibrin sealant. Tissue transglutaminase thus has great promise for practical application in clinical orthopaedics. Because this material has a relatively simple single-polypeptide-chain structure, production of the substance in large quantities with use of recombinant DNA technology is feasible. As a biological adhesive, it thus offers new possibilities for improving the repertoire for the treatment of chondral lesions.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
All biological adhesives used in orthopaedic operations are fibrin-based, but none has given truly satisfactory results^{15,16,24,38,40}. Fixation involving lesions of articular cartilage is particularly problematic. Such defects may be of traumatic origin (chondral fractures), but they are more frequently encountered during the early stages of osteoarthrosis. With a defect of traumatic origin, the fracture fragment may be glued back in its original location; the success of this depends, of course, on the adhesive strength afforded by the glue preparation and on its compatibility with the healing process. With the clefts and fissures that are seen in the early stages of osteoarthrosis, the tissue repair process may be induced by the application of biodegradable matrices (such as fibrin, gelatin, or collagen) containing chondrocytes or repair cells with chondrogenic potential (mesenchymal stem cells or perichondral fibroblasts³⁹). Since one of the properties of articular cartilage is its low-friction, anti-adhesive surface, the adhesion between native and implanted repair materials is usually inadequate.

The anti-adhesiveness of cartilage matrix is largely attributable to its high proteoglycan content, and these molecules have been removed from tissue surfaces to promote the attachment of repair materials^18,20. However, even if the surface of the lesion is depleted of proteoglycans, the degree of adhesiveness has still been insufficient for clinical purposes; the establishment of a cross-linked interface between native and implanted material therefore appears to be necessary.

Transglutaminases are a large family of enzymes that catalyze the calcium-dependent formation of covalent -glutamyl--lysine isopeptide bonds between proteins, thereby leading to the generation of stable polymer networks². Enzymes of this class are widely distributed among tissues and body fluids. Proteins modified by transglutaminases are found throughout the body in locations where high tensile strength and resilience are needed, such as in fibrin clots and cornified epidermis² ; in their absence, wound-healing is severely compromised^6,17. Tissue transglutaminase is expressed in various differentiated cartilages, where it is involved in the cross-linking of extracellular matrix molecules after its release from an intracellular pool during the maturation of chondrocytes^3,4. Tissue transglutaminase is a monomeric globular protein with a molecular mass of about seventy-seven kilodaltons, and it exhibits a high degree of sequence similarity to other transglutaminases—for example², the -subunit of plasma factor XIII. It requires no proteolysis for activation.

The objective of this study was to examine the effectiveness of tissue transglutaminase as a biological glue in the treatment of cartilaginous lesions. A new in vitro system was developed to evaluate quantitatively the adhesive strength achieved at a cartilage-cartilage interface as a function of enzyme concentration, duration of incubation, and relative humidity during incubation. Not only was the adhesive strength of tissue transglutaminase superior to that of a commercially available fibrin-glue preparation, but also the gluing reaction itself was found to be less susceptible to changes in physical parameters such as humidity.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 

Preparation of Cartilage-Bone Cylinders
Two cartilage-bone cylinders, each with a diameter of 3.9 millimeters and a cartilage thickness of 1.5 to 2.5 millimeters, were obtained from bovine shoulder joints with use of a hollow myelotomy drill (Straumann Institute, Waldenburg, Switzerland). The superficial half of the articular cartilage was removed with use of a guided razor blade (Fig. 1 and Fig. 3, A, and B, broken line). One cylinder was immobilized in a holding device, and its freshly cut surface was treated with tissue transglutaminase; calcium-chloride solution was applied to the cartilage surface of the other cylinder. The two cartilage surfaces were then apposed, one on top of the other, and an eighty-gram weight was applied to the upper cylinder for ten minutes at 37 degrees Celsius in a humidity-controlled (50 per cent humidity) chamber.

View larger version (46K): [in this window] [in a new window]   Fig. 1 Scheme summarizing the preparation of cartilage-bone cylinders for treatment with tissue transglutaminase and the testing of the adhesive strength at the glued cartilage-cartilage interfaces. Full-thickness cartilage-bone cylinders were obtained from bovine shoulder joints with use of a hollow myelotomy drill (1). The height of the layer of articular cartilage (solid area) was measured in each specimen, and the superficial half was removed to provide a plane surface for gluing (2). Two cartilage-bone cylinders were coupled, and one of them was immobilized by clamping the subchondral bone (speckled area) in a holding device. Four microliters of tissue transglutaminase solution (tTG) was applied to the freshly cut cartilage surface of the clamped cylinder, and the same volume of 0.1-molar calcium-chloride solution was applied to the surface of its counterpart (3). The two cylinders were immediately joined, and an eighty-gram weight was applied. Polymerization was effected in a humidity chamber (50 per cent humidity) at 37 degrees Celsius for ten minutes (4). The adhesive strength at the cartilage-cartilage interface was determined by measuring the force required to displace the upper cylinder from the lower one by application of lateral pressure (5).

View larger version (97K): [in this window] [in a new window]   Fig. 3 Sections of a cartilage-bone cylinder (A and B) and of the cartilage-cartilage interface between two cylinders after application of tissue transglutaminase (C and D). A: A 200-micrometer-thick section of a methacrylate-embedded cartilage-bone cylinder prepared with a milling cutter and stained with fuchsin red and McNeil tetrachrome. The bar represents one millimeter. B: Higher magnification of the specimen depicted in A. The superficial half of the cartilage layer was removed by cutting along the plane indicated (arrow and broken line), thus providing a plane surface for gluing. The bar represents 200 micrometers. C: A five-micrometer-thick cryosection stained with Mayer hematoxylin, depicting the interface (arrow) between two cartilage-bone cylinders glued with tissue transglutaminase. Tissue transglutaminase was detected with use of affinity-purified antibodies and visualized by an immune peroxidase reaction (brown stain). D: A control section that had been incubated with non-specific rabbit immunoglobulin G. The arrow indicates the tissue interface. The bar represents thirty micrometers.

Measurement of Adhesive Strength
After the eighty-gram weight was removed, a linearly increasing force (0.29 newton per twelve square millimeters per second) was applied to the upper cylinder through an electromagnetic coil and a ferromagnetic bar (Fig. 1). The force was regulated by a signal generator (Kontron Electronics, Zurich, Switzerland) and an amplifier. Calibration experiments demonstrated that when the ferromagnetic bar was placed against an undeformable object, the force generated was proportional to the magnitude of the applied current. The application of a linearly increasing current results in a proportionally increasing shear force at the cartilage-cartilage interface until the point of failure is reached and the upper cylinder is displaced from the lower one (which is clamped in the holding device). The shear force at the cartilage-cartilage interface was measured with a specially designed load-cell (with a precision of 1.0 X 10^-3 newton), which was connected to a dynamic strain-gauge amplifier (DMD 20 A; Hottinger Baldwin, Messtechnik, Darmstadt, Germany). Over a three-second period, 240 values per second were recorded on a data-logger (Mikromec, Suprag, Switzerland) and were transmitted to a computer for statistical evaluation. The applied current and resulting force were visualized on an oscilloscope for online monitoring. The shear force at failure, which is equivalent to the maximum force of resistance of the glued interface, was considered as a measure of the adhesive strength.

At least ten specimens were evaluated for each parameter that was tested, and the results are given as the mean and the standard deviation. Cartilage-bone cylinders for each experimental group were prepared from at least three different bovine shoulder joints. Significance was determined according to the two-group unpaired nonparametric Mann-Whitney U test (StatView II; Abacus Concepts, Berkeley, California).

Treatment of Cartilage Surfaces with Tissue Transglutaminase
Tissue transglutaminase was prepared from a guinea pig liver homogenate, as previously described^1,9. It was concentrated to one milligram per milliliter in ten-millimolar Tris-acetate-buffer solution containing one-millimolar EDTA and 0.15-molar sodium chloride (pH 6.0). The enzyme was either diluted (in the same buffer solution) or further concentrated with use of microconcentrators (Centricon 10; Amicon, Beverly, Massachusetts). Tissue transglutaminase was activated by mixing equal volumes of the enzyme preparation with 0.1-molar calcium-chloride solution (in ten-millimolar Tris-hydrogen chloride, containing 0.3-molar sodium chloride [pH 7.4]). To avoid premature activation of the enzyme, the tissue transglutaminase was applied to one cylinder and the calcium-chloride solution, to its counterpart; the two surfaces were then immediately apposed. The batch-to-batch variation in enzyme activity was less than 12 per cent; nonetheless, in order to exclude the possibility that differences in the absolute shear force values were attributable to the use of different enzyme preparations, the measurements under standard conditions (enzyme concentration, one milligram per milliliter) always were made in parallel and the results were normalized. The depicted relative results thus reflect real differences between a given condition and the standard one.

The adhesive strength of tissue transglutaminase was compared with that of Tissucol (Immuno AG, Vienna, Austria), a commercially available fibrin-glue preparation. This product consists of two solutions that are mixed before application. One solution contains fibrinogen (seventy to 110 milligrams per milliliter), plasma fibronectin (two to nine milligrams per milliliter), plasma factor XIII (ten to fifty units per milliliter), and plasminogen (forty to 120 micrograms per milliliter). The other solution consists of four international units of thrombin in forty-millimolar calcium-chloride solution (manufacturer's specification). The individual solutions were warmed to 37 degrees Celsius and were then mixed and applied immediately to the cartilage surfaces.

Tris-calcium-buffer solution (ten-millimolar Tris-hydrogen chloride, 0.3-molar sodium chloride, and 0.1-molar calcium chloride [pH 7.4]), enzyme solvent (ten-millimolar Tris-acetate-buffer solution, one-millimolar EDTA, and 0.15-molar sodium chloride [pH 6.0]), and 0.15-molar sodium-chloride solution served as controls. The adhesive strength at untreated cartilage-cartilage interfaces also was determined, to test for stickiness caused by dehydration.

Pretreatment of Cartilage Surfaces with Glycolytic Enzymes
To depolymerize glycosaminoglycans, cartilage surfaces were treated for five minutes with chondroitinase AC (Sigma Chemical, St. Louis, Missouri), diluted to a concentration of one, ten, or 100 units per milliliter in phosphate-buffered saline solution (Gibco Life Technologies, Gaithersburg, Maryland). The influence of a prolonged incubation (duration, fifteen minutes) with chondroitinase AC (one unit per milliliter) or hyaluronidase (one unit per milliliter; Fluka Chemicals, Buchs, Switzerland) also was investigated.

Analytical and Biochemical Methods
Transglutaminase activity was determined as the incorporation of [1,4-³H]putrescine (Amersham International, Buckinghamshire, United Kingdom) into N,N-dimethylcasein (Serva, Heidelberg, Germany), as previously described¹. The protein concentrations were determined with use of the reagent for bicinchoninic acid (Pierce Chemical, Rockford, Illinois). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis²⁷ was performed with use of 4 to 20 per cent gradient gels; proteins were reduced with 1 per cent (volume per volume) 2-mercaptoethanol and were visualized with use of Coomassie brilliant blue R-250. For immunohistochemical evaluation, the cartilage cylinders were glued with tissue transglutaminase under standard conditions, embedded in TissueTek (Miles Scientific, Naperville, Illinois), and frozen on dry ice. Tissue transglutaminase was detected subsequently in cryosections with use of affinity-purified antibodies against guinea pig tissue transglutaminase¹ and the peroxidase protocol, as previously described⁴. Control sections were incubated with non-specific rabbit immunoglobulin G (Fig. 3, D). Tissue preparation for histological examination has been described in detail elsewhere²¹.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Force-versus-time plots were constructed (Fig. 2). The shape of the curve after treatment with tissue transglutaminase (Fig. 2, line a) represented a typical "all-or-nothing" type of interaction, which is characteristic of covalent bonding. Three phases could be distinguished: (1) a linear increase in force (approximately 0.29 newton per twelve square millimeters per second) to the point of failure, (2) a steep decrease at the point of failure (approximately -7.89 newtons per twelve square millimeters per second in the experiment represented by Figure 2), and (3) a gradual decline to the baseline level.

View larger version (22K): [in this window] [in a new window]   Fig. 2 Graph of the adhesive strength of tissue transglutaminase (one milligram per milliliter) (a), Tissucol (b), and Tris-calcium-buffer solution (c). Each specimen was incubated for thirty minutes at a humidity of 50 per cent and a temperature of 37 degrees Celsius, and the resistance to a linearly increasing shear force applied to the upper of the two glued cartilage-bone cylinders was measured. The point of failure (indicated by arrows) was taken as a measure of the adhesive strength at the cartilage-cartilage interface. The shape of the curve is a reflection of the individual molecular interactions established in each specimen (see text).

The treatment of the cartilage surfaces with the Tris-calcium-buffer solution alone did not lead to substantial adhesion (Fig. 2, line c).

Analysis of the tissue transglutaminase and Tissucol preparations with use of sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed a single protein band for tissue transglutaminase. The major constituents of Tissucol were fibrinogen and fibronectin; the relative amount of the active protein cross-linking component, plasma transglutaminase factor XIIIa, was less than 0.5 per cent. The differences in the profiles of force-versus-time plots for tissue transglutaminase and for Tissucol reflect the individual molecular mechanisms characterizing the interactions established in each case. Tissue transglutaminase itself acts as a glutaminyl substrate, thereby catalyzing rapid autocatalytic cross-linking¹. The primary reaction occurring on activation of Tissucol by thrombin is the non-covalent polymerization of fibrin to form a clot³⁴, which, with time, becomes covalently cross-linked by factor XIIIa.

Evaluation of cryosections prepared from cylinders glued with tissue transglutaminase revealed a close connection between cartilage surfaces in congruent regions (Fig. 3, C). Small gaps did, however, occur along the interface, and these may account for the variability in the adhesive strength among the specimens. Immunostaining for tissue transglutaminase was restricted to the interface between glued cartilage cylinders, thereby demonstrating autocatalytic cross-linking of the enzyme onto the surfaces; tissue transglutaminase did not penetrate into deeper tissue zones (Fig. 3, C).

The adhesive strength of tissue transglutaminase had an approximately linear dependence on the enzyme concentration (range, 0.25 to 2.75 milligrams per milliliter), and the maximum value after a ten-minute incubation was 0.43 ± 0.13 newton per twelve square millimeters (Fig. 4). Preliminary experiments with use of a crude commercial tissue transglutaminase preparation (Sigma; approximately eleven milligrams per milliliter, containing approximately 50 per cent tissue transglutaminase as assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis) with an activity/protein weight value of 46 per cent (compared with our pure enzyme preparation) demonstrated that the adhesive strength was greater (0.52 ± 0.15 newton per twelve square millimeters) with higher enzyme concentrations. The adhesive strengths achieved after use of the control solutions (Tris-calcium-buffer solution, enzyme solvent, and physiological saline [0.15-molar sodium chloride]) solution were significantly lower than the adhesive strengths achieved with use of the glue preparations (p < 0.01 for tissue transglutaminase at concentrations of 0.5 milligram per millimeter and higher), as can be seen by comparing Figures 4 and 5, even though there were differences among the three (Fig. 5). The joining of untreated cut surfaces resulted in dehydration and deformation of the tissue, as well as in increased stickiness (Fig. 5).

View larger version (20K): [in this window] [in a new window]   Fig. 4 Graph depicting the adhesive strength (maximum resistance to shear force) achieved at the cartilage-cartilage interface with use of tissue transglutaminase (tTG) at various concentrations and under standard incubation conditions (ten minutes at a humidity of 50 per cent and a temperature of 37 degrees Celsius).

View larger version (15K): [in this window] [in a new window]   Fig. 5 Graph depicting the adhesive strength (maximum resistance to shear force) achieved at the cartilage-cartilage interface in the control groups under standard incubation conditions (ten minutes at a humidity of 50 per cent and a temperature of 37 degrees Celsius). The control solutions included 0.15-molar sodium-chloride solution, Tris-calcium-buffer solution (ten-millimolar Tris-hydrogen chloride, 0.3-molar sodium chloride, and 0.1-molar calcium chloride [pH 7.4]), and enzyme solvent (ten-millimolar Tris-acetate-buffer solution, one-millimolar EDTA, and 0.15-molar sodium chloride [pH 6.0]). One control group was left untreated.

View larger version (33K): [in this window] [in a new window]   Fig. 6 Graph depicting the adhesive strength (maximum resistance to shear force) achieved at the cartilage-cartilage interface with use of tissue transglutaminase (tTG) (one milligram per milliliter), Tissucol, or Tris-calcium-buffer solution, measured as a function of the duration of incubation (at a humidity of 50 per cent and a temperature of 37 degrees Celsius).

View larger version (31K): [in this window] [in a new window]   Fig. 7 Graph depicting the influence of carbohydrate digestion before the application of tissue transglutaminase. The adhesive strength (maximum resistance to shear force) of tissue transglutaminase (one milligram per milliliter) was determined after the cartilage surfaces had been pretreated with chondroitinase AC (CAC) (at a concentration of one or ten units per milliliter for five or fifteen minutes) or with hyaluronidase (H) (at a concentration of one unit per milliliter for fifteen minutes). The column designated as tissue transglutaminase (tTG) represents the results for specimens that were not pretreated.

View larger version (25K): [in this window] [in a new window]   Fig. 8 Graph depicting the influence of the humidity during incubation on the adhesive strength (maximum resistance to shear force) achieved at the cartilage-cartilage interface with use of tissue transglutaminase (tTG), Tissucol, or Tris-calcium-buffer solution (incubated at 37 degrees Celsius for ten minutes).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The inherent capacity of tissue transglutaminase to act as a biological glue at a cartilage-cartilage interface was assessed in this study with use of an in vitro system for quantifying adhesive strength. At a concentration of 2.75 milligrams per milliliter, tissue transglutaminase had an adhesive strength (maximum, 0.43 ± 0.13 newton per twelve square millimeters) that was approximately 62 per cent greater than that of Tissucol, a commercially available fibrin-glue preparation. The adhesive strength of each glue was enhanced by about 50 per cent when the duration of incubation was increased from ten to thirty minutes. A similar time-dependence has been reported for fibrin adhesives used in skin-grafting³⁵.

In a study by Claes et al.⁸, bovine patellar cartilage surfaces (diameter, fifteen millimeters) were glued with Tissucol and then subjected to a pressure of 1.5 newtons per square centimeter for thirty minutes at 21 degrees Celsius (the relative humidity during incubation was not defined). Under such conditions, the strength-to-shear stress was 4.6 ± 1.6 newtons per square centimeter, as calculated by assuming proportionality between the determined force and the area of the glued surface (176.7 square millimeters). An analogous calculation based on our own measurements (surface area of twelve square millimeters) demonstrated an adhesive strength of 4.3 ± 0.9 newtons per square centimeter with use of Tissucol. The good agreement between these values indicates that the surface area and resistance to shear stress are linearly related and that this method for testing adhesive strength yields accurate and reliable results.

The differences in the shapes of the curves for tissue transglutaminase and Tissucol in the force-versus-time plots suggest that the mechanisms of interaction at the cartilage-cartilage interface differ. Both curves exhibited a coincident initial linear increase in force with time until the point of failure, indicating that there was no interfacial motion between the glued surfaces at this stage. Thereafter, the relatively slow and variable decrease in force that was observed for Tissucol is consistent with the gradual disruption of both covalent (factor XIIIa-catalyzed cross-linking) and non-covalent (polymerization of fibrin) interactions. In contrast, the sharp decrease in force for tissue transglutaminase suggests breakage of only covalent bonds.

Controlled digestion of the cartilage surfaces with chondroitinase AC or hyaluronidase before gluing with tissue transglutaminase was effected to decrease the intrinsic anti-adhesive property of the extracellular matrix, which is partially attributable to its richness in various types of proteoglycan molecules³¹. Such an enzymatic treatment has been shown to have a beneficial effect on the adhesion of cells^18,20 and biomatrices during the repair of cartilage²⁰. The treatment of cartilage surfaces in this manner enhanced the adhesive strength of tissue transglutaminase in a time-dependent way; this indicates that additional substrate sites for the enzyme, presumably present on collagenous constituents (type-II and XI collagen^3,25) and glycoprotein constituents (such as fibronectin, osteonectin, and osteopontin^3,11,14,36) of the extracellular matrix^4,11, are made available after the removal of glycosaminoglycan side chains from proteoglycans.

There is a pressing need for suitable adhesives for the treatment of lesions of articular cartilage. There have been various clinical and experimental attempts to improve the repair of cartilage, but they have had limited success thus far^{10,12,22,28-30,32,33,37}. In many of the procedures adopted, transplanted materials, such as pure cartilage, bone-cartilage composites, and perichondral or periosteal tissue, are used either to fill the defects or to trigger a healing process⁷. More recently, it has been proposed that non-biodegradable or biodegradable matrices be seeded with one or several types of cells⁷, such as chondrocytes, chondroblasts, or mesenchymal stem cells²⁴, to elicit a repair response. However, both the initial attachment of matrices and the bonding between the implanted material and the healthy surrounding tissue pose serious problems. Biomatrix adhesion assumes an even greater importance when the defect is superficial (that is, when it does not penetrate to the subchondral bone). Recent experiments on animals in our laboratory have indicated not only that bonding between the matrix and native tissue was improved in the presence of tissue transglutaminase (unpublished data) but also that this glue does not interfere with repair processes; indeed, it may even be involved in the activation of transforming growth factor-ß²⁶ (playing a role in chondrogenesis and cartilage differentiation^4,19,23).

The data related to the adhesive strength achieved with use of tissue transglutaminase are very encouraging, but long-term in vivo experiments are needed to establish whether it will be of real value as a biological glue in orthopaedic operations. Unfortunately, no calculations pertaining to the adhesive strength necessary to resist forces occurring in a diarthrodial joint during movement are available, to our knowledge, although Claes et al.⁸ estimated the forces to be in the range of 4.3 ± 0.9 newtons per square centimeter. The adhesive strength that was achieved in our experiments could be enhanced with use of higher enzyme concentrations or with a combination of tissue transglutaminase and specifically designed substrate molecules (such as polyamines), thereby bridging the microgaps between surfaces. One of the prerequisites for carrying out the necessary experiments is the availability of this enzyme in large quantities and at very high concentrations. As this protein has a relatively simple single-polypeptide-chain structure (no complex post-translational modifications) and has been cloned¹³, essential preconditions have already been fulfilled for in vitro production of this material with use of recombinant DNA technology. Moreover, with this means of producing the glue, human sources can be avoided, as can the problems of transmitting the human immunodeficiency virus or hepatitis and of inducing clotting inhibitors⁵. Tissue transglutaminase recently has been shown to be expressed in a variety of differentiated cartilages⁴ and to catalyze physiologically the formation of cross-links in the extracellular matrices of cartilage³. This glue thus mimics a naturally occurring process and, as both the enzyme and the products of its reaction are present in normal cartilage, a therapeutic contraindication appears unlikely. Tissue transglutaminase thus holds great promise for future application in the treatment of chondral lesions.

NOTE: The authors are grateful to M. Paulsson, M.D., for critical discussions both during the experimental stage of this study and during the preparation of the manuscript, and to E. Berger, O. Kaupp, and U. Rohrer for their technical assistance. The authors also are indebted to Ceri England for her constructive contribution to the organization of the manuscript and the editing of the text.

	Footnotes

 
*One or more of the authors has received or will receive benefits for personal or professional use from a commercial party related directly or indirectly to the subject of this article. In addition, benefits have been or will be directed to a research fund, foundation, educational institution, or other non-profit organization with which one or more of the authors is associated. Funds were received in total or partial support of the research or clinical study presented in this article. The funding source was Orthogene, Sausalito, California.

M. E. Müller Institute for Biomechanics, University of Bern, P.O. Box 30, CH-3010 Bern, Switzerland.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Aeschlimann, D, and |and |Paulsson, M.: Cross-linking of laminin-nidogen complexes by tissue transglutaminase. A novel mechanism for basement membrane stabilization. J. Biol. Chem., 266: 15308-15317, 1991.[Abstract/Free Full Text]
2. Aeschlimann, D., and |and |Paulsson, M.: Transglutaminases: protein cross-linking enzymes in tissues and body fluids. Thromb. and Haemost., 71: 402-415, 1994.[Medline]
3. Aeschlimann, D.; Kaupp, O.; and |and |Paulsson, M.: Transglutaminase-catalyzed matrix cross-linking in differentiating cartilage: identification of osteonectin as a major glutaminyl substrate. J. Cell Biol., 129: 881-892, 1995.[Abstract/Free Full Text]
4. Aeschlimann, D.; Wetterwald, A.; Fleisch, H.; and |and |Paulsson, M.: Expression of tissue transglutaminase in skeletal tissues correlates with events of terminal differentiation of chondrocytes. J. Cell Biol., 120: 1461-1470, 1993.[Abstract/Free Full Text]
5. Bänninger, H.; Hardegger, T.; Tobler, A.; Barth, A.; Schüpbach, P.; Reinhart, W.; Lämmle, B.; and |and |Furlan, M.: Fibrin glue in surgery: frequent development of inhibitors of bovine thrombin and human factor V. British J. Haematol., 85: 528-532, 1993.[Medline]
6. Board, P. G.; Losowsky, M. S; and |and |Miloszewski, K. J.: Factor XIII: inherited and acquired deficiency. Blood Rev., 7: 229-242, 1993.[Medline]
7. Buckwalter, J. A.; Rosenberg, L. C.; and Hunziker, E. B.: Articular cartilage: composition, structure, response to injury, and methods of facilitating repair. In Articular Cartilage and Knee Joint Function: Basic Science and Arthroscopy. 19-56. Edited by J. W. Ewing. New York, Raven Press, 1990.
8. Claes, L.; Burri, C.; Helbing, G.; and |and |Lehner, E.: Biomechanische Untersuchungen zur Festigkeit verschiedener Knorpelklebungen. Helvetica Chir. Acta, 48: 11-13, 1981.
9. Connellan, J. M.; Chung, S. I.; Whetzei, N. K.; Bradley, L. M.; and |and |Folk, J. E.: Structural properties of guinea pig liver transglutaminase. J. Biol. Chem., 246: 1093-1098, 1971.[Abstract/Free Full Text]
10. Coutts, R. D.; Woo, S. L.-Y.; Amiel, D.; von Schroeder, H. P.; and |and |Kwan, M. K.: Rib perichondrial autografts in full-thickness articular cartilage defects in rabbits. Clin. Orthop., 275: 263-273, 1992.
11. Fésus, L.; Metsis, M. L.; Muszbek, L.; and |and |Koteliansky, V. E.: Transglutaminase-sensitive glutamine residues of human plasma fibronectin revealed by studying its proteolytic fragments. European J. Biochem., 154: 371-374, 1986.[Medline]
12. Furukawa, T.; Fyre, D. R.; Koide, S.; and |and |Glimcher, M. J.: Biochemical studies on repair cartilage resurfacing experimental defects in the rabbit knee. J. Bone and Joint Surg., 62-A: 79-89, Jan. 1980.[Abstract/Free Full Text]
13. Gentile, V.; Saydak, M.; Chiocca, E. A.; Akande, O.; Birckbichler, P. J.; Lee, K. N.; Stein, J. P.; and |and |Davies, P. J.: Isolation and characterization of cDNA clones to mouse macrophage and human endothelial cell tissue transglutaminases. J. Biol. Chem., 266: 478-483, 1991.[Abstract/Free Full Text]
14. Hohenadl, C.; Mann, K.; Mayer, U.; Timpl, R.; Paulsson, M.; and |and |Aeschlimann, D.: Two adjacent N-terminal glutamines of BM-40 (osteonectin, SPARC) act as amine acceptor sites in transglutaminase-catalyzed modification. J. Biol. Chem., 270: 23415-23420, 1995.[Abstract/Free Full Text]
15. Homminga, G. N.; Bulstra, S. K.; Bouwmeester, P. S. M.; and |and |van der Linden, A. J.: Perichondral grafting for cartilage lesions of the knee. J. Bone and Joint Surg., 72-B(6): 1003-1007, 1990.[Abstract/Free Full Text]
16. Homminga, G. N.; van der Linden, T. J.; Terwindt-Rouwenhorst, E. A.; and |and |Drukker, J.: Repair of articular defects by perichondrial grafts. Experiments in the rabbit. Acta Orthop. Scandinavica, 60: 326-329, 1989.[Medline]
17. Huber, M.; Rettler, I.; Bernasconi, K.; Frenk, E.; Lavrijsen, S. P.; Ponec, M.; Bon, A.; Lautenschlager, S.; Schorderet, D. F.; and |and |Hohl, D.: Mutations of keratinocyte transglutaminase in lamellar ichthyosis. Science, 267: 525-528, 1995.[Abstract/Free Full Text]
18. Hunziker, E. B., and |and |Rosenberg, L. C.: Biological basis for repair of superficial articular cartilage lesions. Trans. Orthop. Res. Soc., 17: 231, 1992.
19. Hunziker, E. B., and |and |Rosenberg, L.: Induction of repair in partial thickness articular cartilage lesions by timed release of TGFß. Trans. Orthop. Res. Soc., 19: 236, 1994.
20. Hunziker, E. B., and |and |Rosenberg, L. C.: Repair of partial-thickness defects in articular cartilage: cell recruitment from the synovial membrane. J. Bone and Joint Surg., 78-A: 721-733, May 1996.[Abstract/Free Full Text]
21. Hunziker, E. B.; Wagner, J.; and |and |Zapf, J.: Differential effects of insulin-like growth factor I and growth hormone on developmental stages of rat growth plate chondrocytes in vivo. J. Clin. Invest., 93: 1078-1086, 1994.
22. Itay, S.; Abramovici, A.; and |and |Nevo, Z.: Use of cultured embryonal chick epiphyseal chondrocytes as grafts for defects in chick articular cartilage. Clin. Orthop., 220: 284-303, 1987.
23. Joyce, M. E.; Roberts, A. B.; Sporn, M. B.; and |and |Bolander, M. E.: Transforming growth factor-beta and the initiation of chondrogenesis and osteogenesis in the rat femur. J. Cell Biol., 110: 2195-2207, 1990.[Abstract/Free Full Text]
24. Kaplonyi, G.; Zimmerman, I.; Frenyo, A. D.; Farkas, T.; and |and |Nemes, G.: The use of fibrin adhesive in the repair of chondral and osteochondral injuries. Injury, 19: 267-272, 1988.[Medline]
25. Kleman, J. P.; Aeschlimann, D.; Paulsson, M.; and |and |van der Rest, M.: Transglutaminase-catalyzed cross-linking of fibrils of collagen V/XI in A204 rhabdomyosarcoma cells. Biochemistry, 34: 13768-13775, 1995.[Medline]
26. Kojima, S.; Nara, K.; and |and |Rifkin, D. B: Requirement for transglutaminase in the activation of latent transforming growth factor-beta in bovine endothelial cells. J. Cell Biol., 121: 439-448, 1993.[Abstract/Free Full Text]
27. Laemmli, U. K: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685, 1970.[Medline]
28. Mitchell, N., and |and |Shepard, N.: The resurfacing of adult rabbit articular cartilage by multiple perforations through the subchondral bone. J. Bone and Joint Surg., 58-A: 230-233, March 1976.[Abstract/Free Full Text]
29. Mitchell, N., and |and |Shepard, N.: Healing of articular cartilage in intra-articular fractures in rabbits. J. Bone and Joint Surg., 62-A: 628-634, June 1980.
30. Mitchell, N., and |and |Shepard, N.: Effect of patellar shaving in the rabbit. J. Orthop. Res., 5: 388-392, 1987.[Medline]
31. Morgelin, M.; Heinegard, D.; Engel, J.; and |and |Paulsson, M.: The cartilage proteoglycan aggregate: assembly through combined protein-carbohydrate and protein-protein interactions. Biophys. Chem., 50: 113-128, 1994.[Medline]
32. O'Driscoll, S. W.; Keeley, F. W.; and |and |Salter, R. B.: Durability of regenerated articular cartilage produced by free autogenous periosteal grafts in major full-thickness defects in joint surfaces under the influence of continuous passive motion. A follow-up report at one year. J. Bone and Joint Surg., 70-A: 595-606, April 1988.[Abstract/Free Full Text]
33. Schmid, A., and |and |Schmid, F.: Results after cartilage shaving studied by electron microscopy. Am. J. Sports Med., 15: 386-387, 1987.
34. Shainoff, J. R.; Urbanic, D. A.; and |and |DiBello, P. M.: Immunoelectrophoretic characterizations of the cross-linking of fibrinogen and fibrin by factor XIIIa and tissue transglutaminase. Identification of a rapid mode of hybrid alpha-/gamma-chain cross-linking that is promoted by the gamma-chain cross-linking. J. Biol. Chem., 266: 6429-6437, 1991.[Abstract/Free Full Text]
35. Sierra, D. H.; Feldman, D. S.; Saltz, R.; and |and |Huang, S.: A method to determine shear adhesive strength of fibrin sealants. J. Appl. Biomater., 3: 147-151, 1992.[Medline]
36. Sorensen, E. S.; Rasmussen, L. K.; Moller, L.; Jensen, P. H.; Hojrup, P.; and |and |Petersen, T. E.: Localization of transglutaminase-reactive glutamine residues in bovine osteopontin. Biochem. J., 304: 13-16, 1994.
37. Speer, D. P.; Chvapil, M.; Volz, R. G.; and |and |Holmes, M. D.: Enhancement of healing in osteochondral defects by collagen sponge implants. Clin. Orthop., 144: 326-335, 1979.
38. Tilling, T.: Follow-up after reattachment of chondral and osteochondral fragments of the knee joint. In Traumatology: Orthopaedics, pp. 74-78. Edited by G. Schlag and H. Redl. New York, Springer, 1986.
39. Wakitani, S.; Goto, T.; Pineda, S. J.; Young, R. G.; Mansour, J. M.; Caplan, A. I.; and |and |Goldberg, V. M.: Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage. J. Bone and Joint Surg., 76-A: 579-592, April 1994.[Abstract/Free Full Text]
40. Widenfalk, B.; Engkvist, O.; Ohlsén, L.; and |and |Segerström, K.: Perichondrial arthroplasty using fibrin glue and early mobilization. An experimental study. Scandinavian J. Plast. and Reconstr. Surg., 20: 251-258, 1986.

CiteULike

Connotea

Del.icio.us

Facebook

Technorati

Twitter

This article has been cited by other articles:

				S. K. Lee, J. G. Chi, S. C. Park, and S. I. Chung Transient Expression of Transglutaminase C During Prenatal Development of Human Muscles J. Histochem. Cytochem., November 1, 2000; 48(11): 1565 - 1574. [Abstract] [Full Text]

				M. T. Kaartinen, A. Pirhonen, A. Linnala-Kankkunen, and P. H. Maenpaa Cross-linking of Osteopontin by Tissue Transglutaminase Increases Its Collagen Binding Properties J. Biol. Chem., January 15, 1999; 274(3): 1729 - 1735. [Abstract] [Full Text] [PDF]

				A. P. Newman Articular Cartilage Repair Am. J. Sports Med., March 1, 1998; 26(2): 309 - 324. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Free Letters to the Editor: Submit a response Alert me when this article is cited Alert me when Letters to the Editor are posted Alert me if a correction is posted Services E-mail this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My File Cabinet Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by JÜRGENSEN, K. Articles by HUNZIKER, E. B. Search for Related Content PubMed PubMed Citation Articles by JÜRGENSEN, K. Articles by HUNZIKER, E. B. Social Bookmarking                   What's this?

Stanford University Libraries' HighWire Press (TM) assists in the publication of JBJS Online.
Copyright © 1997 by The Journal of Bone and Joint Surgery, Inc. Online ISSN: 1535-1386.
The Journal of Bone and Joint Surgery®, JBJS®, JB&JS®, and eJBJS® are registered in the U.S. Patent and Trademark Office.