Effects of methionine restriction and endurance exercise on bones of ovariectomized rats: a study of histomorphometry, densitometry, and biomechanical properties.

To investigate the effects of dietary methionine restriction (MetR) and endurance exercise on bone quality under a condition of estrogen deficiency, female Sprague-Dawley rats (36-wk-old) were assigned to a sham surgery group or one of five ovariectomized groups subjected to interventions of no treatment (Ovx), endurance exercise (Exe), methionine restriction (MetR), methionine restriction plus endurance exercise (MetR + Exe), and estrogen treatment (Est). Rats in the exercise groups were subjected to a treadmill running regimen. MetR and control diets contained 0.172 and 0.86% methionine, respectively. After the 12-wk intervention, all animals were killed, and serum and bone tissues were collected for analyses. Compared with estrogen treatment, MetR diet and endurance exercise showed better or equivalent efficiency in reducing body weight gain caused by ovariectomy (P < 0.05). Whereas only the Est group showed evidence for reduced bone turnover compared with the Ovx group, MetR diet and/or endurance exercise demonstrated efficiencies in downregulating serum insulin, leptin, triglyceride, and thiobarbituric acid reactive substances (P < 0.05). Both the Exe and MetR groups showed higher femoral cortical and total volumetric bone mineral density (vBMD), but only the Exe and Est groups preserved cancellous bone volume and/or vBMD of distal femora (P < 0.05) compared with the Ovx group. After being normalized to body mass, femora of the MetR and MetR + Exe groups had relatively higher bending strength and dimension values followed by the Sham, Exe, and Est groups (P < 0.05). In conclusion, both MetR diet and endurance exercise improved cortical bone properties, but only endurance exercise preserved cancellous bone under estrogen deficiency.

A methionine-restricted diet and endurance exercise decrease bone mass and extrinsic strength but increase intrinsic strength in growing male rats.

Dietary methionine restriction (MR) has been suggested to be comparable to endurance exercise with respect to its beneficial effects on health. To further investigate the effects of MR and endurance exercise on growing bone, 7-wk-old male Sprague-Dawley rats were fed different l-methionine (Met)-containing diets with or without endurance exercise intervention (Ex; 0.86% Met, 0.52% Met, 0.17% Met, 0.86% Met-Ex, 0.52% Met-Ex, and 0.17% Met-Ex groups). After an 8-wk intervention period, exercise-trained rats had a 9.2% lower body weight (BW) than did sedentary rats (P < 0.05). Additionally, 0.17% Met-fed rats had 32% lower BW when compared with rats fed the other 2 diets (P < 0.05). Serum osteocalcin was lower in the 0.17% Met-Ex group compared with the other 2 exercise groups and the 0.17% Met group (P < 0.05). Serum concentrations of C-terminal telopeptide of type 1 collagen were lower in exercise-trained and 0.17% Met-fed rats than in sedentary rats and rats fed the other 2 diets (P < 0.05 for both). Rats fed the 0.17% Met diet had lower trabecular bone volume, bone mineralization activities, and bone mineral content (BMC; e.g., total, cortical, and spongy BMC) and bone mineral density (BMD; e.g., total and spongy BMD) indices compared with rats fed the other 2 diets (P < 0.05). Exercise-trained rats also had lower bone mineralization activity, trabecular osteoclast density, total BMC, cortical BMC, and total BMD compared with sedentary rats (P < 0.05). In total BMD, only the 0.17% Met-Ex group had values lower than the other 2 exercise groups and the 0.17% Met group (P < 0.05). Compared with rats fed the other 2 diets and sedentary rats, the femora of 0.17% Met-fed and exercise-trained rats, respectively, had smaller size and/or lower extrinsic strength but enhanced intrinsic biomechanical properties (P < 0.05). The results indicate that MR and endurance exercise caused lower whole bone mass, size, and/or strength but might enhance intrinsic bone strength.

A method to cleave target molecules in a neocartilage.

The mechanical function of many matrix molecules is unknown. A common method to determine whether a molecule is a load-carrying structural molecule is to measure the mechanical properties of a tissue, digest the tissue with an enzyme specific for cleaving that molecule, and then remeasure the mechanical properties. A limitation of this technique is that there are no specific lytic enzymes for most molecules of interest. This article introduces a method that may allow evaluation of a large number of candidate structural molecules. A translated thrombin proteolytic recognition and cleavage site is inserted in the cDNA of a target molecule, and the target molecule then expressed in a cell that produces a tissue. After growing the tissue with cells expressing the engineered target molecule, the traditional procedure of mechanical testing, digesting, and retesting is performed. This method was demonstrated using decorin and its dermatan sulfate (DS) glycosaminoglycan chain in a neocartilage. A tissue was generated with cells expressing a genetically engineered decorin with a thrombin cleavage site. The tissue was then tested in tension and compression, digested with thrombin, and mechanically retested. The decorin protein was found in the tissue, the DS glycosaminoglycan chain was removed with thrombin digestion, and there was no change in the mechanical properties of the tissue due to the thrombin digestion relative to controls. These findings were in agreement with previously reported tests on decorin, collectively supporting the proposed method. All methods involving animals were reviewed and approved by our Institutional Review Board.

Effect of decorin and dermatan sulfate on the mechanical properties of a neocartilage.

Decorin is known to influence the size of collagen fibrils in ligaments and tendons and it has been hypothesized to provide a structural link between collagen fibrils in connective tissues, including cartilage. Coincidently, mechanical properties of skin, ligament, and tendons are altered in decorin knockout mice, suggesting it may influence the structural properties of tissue or tissue matrix organization. To further examine the role of decorin in the extracellular matrix development and subsequent material properties of cartilage, tissue (neocartilage) was grown in a 3D culture model using a pure population of genetically modified chondrocytes stably overexpressing decorin (DCN) or decorin lacking dermatan sulfate (MDCN). An empty vector (CON) served as a virus control. Following generation of the cartilage-like tissues, mechanical properties in tension and compression, collagen fibril diameter, matrix organization, and biochemistry of the tissue were determined. There were no differences between CON and DCN tissues in any parameter measured. In contrast, tissue generated in MDCN cultures was thinner, had higher collagen density, and higher elastic moduli as compared to both CON and DCN tissues. Considering there was no difference in stiffness between CON and DCN tissues, the notion that decorin contributes to the mechanical properties via load transfer was refuted in this model. However, contrasts in the mechanical properties of the MDCN tissue suggest that the dermatan sulfate chains on decorin influences the organization/maturation and resultant mechanical properties of the matrix by as an yet-unidentified regulatory mechanism.

Influence of bone morphogenetic protein-2 on the extracellular matrix, material properties, and gene expression of long-term articular chondrocyte cultures: loss of chondrocyte stability.

The aim of this study was to determine the effects of bone morphogenetic protein-2 (BMP-2) on articular chondrocyte tissues grown as monolayers in vitro for up to 8 weeks. Articular chondrocytes were isolated from New Zealand White rabbits and plated in monolayer cultures. The cultures were supplemented with 100 ng/mL of BMP-2 for up to 8 weeks and the extracellular matrix (ECM) composition, material properties, and messenger RNA (mRNA) expression were analyzed. mRNA expression of cartilage-specific genes, type II collagen, and aggrecan showed that BMP-2 enhanced chondrocyte stability for up to 3 weeks. After 3 weeks in culture, there was substantially more type I collagen expression and more osteopontin and runt-related transcription factor 2 expression in 5- and 8-week cultures treated with BMP-2 than in controls. Additionally, matrix metalloproteinase-13 and ADAMTS-5 (A disintegrin-like and metalloproteinase with thrombospondin 5) were upregulated in 5- and 8-week cultures treated with BMP-2, coinciding with a loss of ECM density, collagen, and proteoglycan. Eight-week tissue stimulated with BMP-2 was more fragile and tore more easily when removed from the culture dish as compared to controls, suggesting temporal limitations to the effectiveness of BMP-2 in monolayer systems and perhaps other models to enhance the generation of a cartilage-like tissue for tissue engineering purposes.

Poroviscoelastic cartilage properties in the mouse from indentation.

A method for fitting parameters in a poroviscoelastic (PVE) model of articular cartilage in the mouse is presented. Indentation is performed using two different sized indenters and then these data are fitted using a PVE finite element program and parameter extraction algorithm. Data from a smaller indenter, a 15 mum diameter flat-ended 60 deg cone, is first used to fit the viscoelastic (VE) parameters, on the basis that for this tip size the gel diffusion time (approximate time constant of the poroelastic (PE) response) is of the order of 0.1 s, so that the PE response is negligible. These parameters are then used to fit the data from a second 170 mum diameter flat-ended 60 deg cone for the PE parameters, using the VE parameters extracted from the data from the 15 mum tip. Data from tests on five different mouse tibial plateaus are presented and fitted. Parameter variation studies for the larger indenter show that for this case the VE and PE time responses overlap in time, necessitating the use of both models.

Photo-cross-linked hybrid polymer networks consisting of poly(propylene fumarate) and poly(caprolactone fumarate): controlled physical properties and regulated bone and nerve cell responses.

Aiming to achieve suitable polymeric biomaterials with controlled physical properties for hard and soft tissue replacements, we have developed a series of blends consisting of two photo-cross-linkable polymers: polypropylene fumarate (PPF) and polycaprolactone fumarate (PCLF). Physical properties of both un-cross-linked and UV cross-linked PPF/PCLF blends with PPF composition ranging from 0% to 100% have been investigated extensively. It has been found that the physical properties such as thermal, rheological, and mechanical properties could be modulated efficiently by varying the PPF composition in the blends. Thermal properties including glass transition temperature (T g) and melting temperature (T m) have been correlated with their rheological and mechanical properties. Surface characteristics such as surface morphology, hydrophilicity, and the capability of adsorbing serum protein from culture medium have also been examined for the cross-linked polymer and blend disks. For potential applications in bone and nerve tissue engineering, in vitro cell studies including cytotoxicity, cell adhesion, and proliferation on cross-linked disks with controlled physical properties have been performed using rat bone marrow stromal cells and SPL201 cells, respectively. In addition, the role of mechanical properties such as surface stiffness in modulating cell responses has been emphasized using this model blend system.

Effect of indenter size on elastic modulus of cartilage measured by indentation.

Our preliminary indentation experiments showed that the equilibrium elastic modulus of murine tibial cartilage increased with decreasing indenter size: flat-ended 60 deg conical tips with end diameters of 15 microm and 90 microm gave 1.50+/-0.82 MPa (mean+/-standard deviation) and 0.55+/-0.11 MPa, respectively (p<0.01). The goal of this paper is to determine if the dependence on tip size is an inherent feature of the equilibrium elastic modulus of cartilage as measured by indentation. Since modulus values from nonindentation tests are not available for comparison for murine cartilage, bovine cartilage was used. Flat-ended conical or cylindrical tips with end diameters ranging from 5 microm to 4 mm were used to measure the equilibrium elastic modulus of bovine patellar cartilage. The same tips were used to test urethane rubber for comparison. The equilibrium modulus of the bovine patellar cartilage increased monotonically with decreasing tip size. The modulus obtained from the 2 mm and 4 mm tips (0.63+/-0.21 MPa) agreed with values reported in the literature; however, the modulus measured by the 90 microm tip was over two and a half times larger than the value obtained from the 1000 microm tip. In contrast, the elastic modulus of urethane rubber obtained using the same 5 microm-4 mm tips was independent of tip size. The equilibrium elastic modulus of bovine patellar cartilage measured by indentation depends on tip size. This appears to be an inherent feature of indentation of cartilage, perhaps due to its inhomogeneous structure.

Nerve growth factor sequestering therapy attenuates non-malignant skeletal pain following fracture.

Current therapies to treat skeletal fracture pain are extremely limited. Some non-steroidal anti-inflammatory drugs have been shown to inhibit bone healing and opiates induce cognitive dysfunction and respiratory depression which are especially problematic in the elderly suffering from osteoporotic fractures. In the present report, we developed a closed femur fracture pain model in the mouse where skeletal pain behaviors such as flinching and guarding of the fractured limb are reversed by 10mg/kg morphine. Using this model we showed that the administration of a monoclonal antibody against nerve growth factor (anti-NGF) reduced fracture-induced pain-related behaviors by over 50%. Treatment with anti-NGF reduced c-Fos and dynorphin up-regulation in the spinal cord at day 2 post-fracture. However, anti-NGF treatment did not reduce p-ERK and c-Fos expression at 20 and 90 min, respectively, following fracture. This suggests NGF is involved in maintenance but not the acute generation of fracture pain. Anti-NGF therapy did not inhibit bone healing as measured by callus formation, bridging of the fracture site or mechanical strength of the bone. As the anti-NGF antibody does not appreciably cross the blood-brain barrier, the present data suggest that the anti-hyperalgesic action of anti-NGF therapy results from blockade of activation and/or sensitization of the CGRP/trkA positive fibers that normally constitute the majority of sensory fibers that innervate the bone. These results demonstrate that NGF plays a significant role in driving fracture pain and that NGF sequestering therapies may be efficacious in attenuating this pain.

Effects of a monoclonal antibody raised against nerve growth factor on skeletal pain and bone healing after fracture of the C57BL/6J mouse femur.

UNLABELLED: A closed femur fracture pain model was developed in the C57BL/6J mouse. One day after fracture, a monoclonal antibody raised against nerve growth factor (anti-NGF) was delivered intraperitoneally and resulted in a reduction in fracture pain-related behaviors of approximately 50%. Anti-NGF therapy did not interfere with bone healing as assessed by mechanical testing and histomorphometric analysis. INTRODUCTION: Current therapies to treat skeletal fracture pain are limited. This is because of the side effect profile of available analgesics and the scarcity of animal models that can be used to understand the mechanisms that drive this pain. Whereas previous studies have shown that mineralized bone, marrow, and periosteum are innervated by sensory and sympathetic fibers, it is not understood how skeletal pain is generated and maintained even in common conditions such as osteoarthritis, low back pain, or fracture. MATERIALS AND METHODS: In this study, we characterized the pain-related behaviors after a closed femur fracture in the C57BL/6J mouse. Additionally, we assessed the effect of a monoclonal antibody that binds to and sequesters nerve growth factor (anti-NGF) on pain-related behaviors and bone healing (mechanical properties and histomorphometric analysis) after fracture. RESULTS: Administration of anti-NGF therapy (10 mg/kg, days 1, 6, and 11 after fracture) resulted in a reduction of fracture pain-related behaviors of approximately 50%. Attenuation of fracture pain was evident as early as 24 h after the initial dosing and remained efficacious throughout the course of fracture pain. Anti-NGF therapy did not modify biomechanical properties of the femur or histomorphometric indices of bone healing. CONCLUSIONS: These findings suggest that therapies that target NGF or its cognate receptor(s) may be effective in attenuating nonmalignant fracture pain without interfering with bone healing.

Direct measurement of the rupture force of single pair of decorin interactions.

Decorin is one important member of the family of small leucine-rich proteoglycans, which are widely distributed in connective tissues in the body such as tendon and ligament. Decorin may be responsible for collagen fibril connection in those tissues. A recent hypothesis suggests that decorin may bind to collagen with its core protein while binding to another decorin through the interaction with their glycosaminoglycan (GAG) chains. However, there is no direct evidence supporting this hypothesis to date. In this study, the interaction of decorin GAG chains was directly determined for the first time. The rupture force of single bonds between decorins (GAG chains interaction) was determined directly as 16.5+/-5.1 pN using a laser tweezers/interferometer single molecular nanomechanical testing system. This information can improve our understanding of the mechanical properties of connective tissues at the molecular level.

Werner Goldsmith: life and work (1924-2003).

Werner Goldsmith, one of the foremost authorities on the mechanics of impact and the biomechanics of head and neck injuries, died peacefully at home in Oakland, California, on August 23, 2003, at age 79 after a short, courageous battle with leukemia, ending a long and very distinguished career in mechanics, dynamics, and biomechanics, and an almost six-decades-long association with the University of California, Berkeley. He was one of the pioneering, eminent solid and fluid mechanicians who made an early transition to biomechanics, and in rising to equal distinction in their new fields, added great credibility to biomechanics as a discipline in its own right. He was also a distinguished and influential figure in bioengineering education at his own institution, and, more broadly, in the United States and abroad. An emeritus professor for over a decade, he continued to be active in research and teaching until the very last days of his life.

Determination of Poisson's ratio of articular cartilage by indentation using different-sized indenters.

Articular cartilage is often characterized as an isotropic elastic material with no interstitial fluid flow during instantaneous and equilibrium conditions, and indentation testing commonly used to deduce material properties of Young's modulus and Poisson's ratio. Since only one elastic parameter can be deduced from a single indentation test, some other test method is often used to allow separate measurement of both parameters. In this study, a new method is introduced by which the two material parameters can be obtained using indentation tests alone, without requiring a secondary different type of test. This feature makes the method more suitable for testing small samples in situ. The method takes advantages of the finite layer effect. By indenting the sample twice with different-sized indenters, a nonlinear equation with the Poisson's ratio as the only unknown can be formed and Poisson's ratio obtained by solving the nonlinear equation. The method was validated by comparing the predicted Poisson's ratio for urethane rubber with the manufacturer's supplied value, and comparing the predicted Young's modulus for urethane rubber and an elastic foam material with modulii measured by unconfined compression. Anisotropic and nonhomogeneous finite-element (FE) models of the indentation were developed to aid in data interpretation. Applying the method to bovine patellar cartilage, the tissue Young's modulus was found to be 1.79 +/- 0.59 MPa in instantaneous response and 0.45 +/- 0.26 MPa in equilibrium, and the Poisson's ratio 0.503 +/- 0.028 and 0.463 +/- 0.073 in instantaneous and equilibrium, respectively. The equilibrium Poisson's ratio obtained in our work was substantially higher than those derived from biphasic indentation theory and those optically measured in an unconfined compression test. The finite element model results and examination of viscoelastic-biphasic models suggest this could be due to viscoelastic, inhomogeneity, and anisotropy effects.

Cell death after cartilage impact occurs around matrix cracks.

The damage from rapid high energy impacts to cartilage may contribute to the development of osteoarthritis (OA). Understanding how and when cells are damaged during and after the impact may provide insight into how these lesions progress. Mature bovine articular cartilage on the intact patella was impacted with a flat impacter to 53 MPa in 250 ms. Cell viability was determined by culturing the cartilage with nitroblue tetrazolium for 18 h or for 4 days in medium containing 5% serum before labeling (5-day sample) and compared to adjacent, non-impacted tissue as viable cells per area. There was a decrease in viable cell density only in specimens with macroscopic cracks and the loss was localized primarily near matrix cracks, which were in the upper 25% of the tissue. This was confirmed using confocal microscopy with a fluorescent live/dead assay, using 5'-chloromethylfluorescein diacetate and propidium iodide. Cell viability in the impacted regions distant from visible cracks was no different than the non-impacted control. At 5 days, viable cell density decreased in the surface layer in both the control and impacted tissue, but there was no additional impact-related change. In summary, cell death after the impaction of cartilage on bone occurred around impact induced cracks, but not in impacted areas without cracks. If true in vivo, early stabilization of the damaged area may prevent late sequelae that lead to OA.

A model of fracture testing of soft viscoelastic tissues.

Fracture, or tear, toughness of soft tissues can be computed from the work of fracture divided by the area of new crack surface. For soft tissues without significant plastic deformation, total work, which can be measured experimentally, is composed of the sum of fracture and viscoelastic work. In order to deduce fracture work, a method is needed to estimate viscoelastic work. Two different methods (Ph.D. Dissertation, University of Minnesota, 2000; J. Mater. Sci.: Mater. Med. 12 (2001) 327) have been proposed to estimate viscoelastic work in a fracture test of a soft tissue. The relative merits of these methods are unknown because the true viscoelastic work in an experiment is unknown. In order to characterize the accuracy of these methods, a theoretical model of crack propagation of viscoelastic soft tissue in a tensile test is presented, from which the exact viscoelastic work is calculated. The material is assumed to obey the standard linear solid model. The "exact" solution for the viscoelastic work during the fracture is computed from the model and compared with the work estimated by the two methods. It was found that both methods tend to underestimate the viscoelastic work done, and thus overestimate the fracture work and fracture toughness, although the errors were greater with the Fedewa method. It was further found that low displacement rates can give rise to a "snap" effect, where rapid crack growth can cause a disproportionate amount of viscoelastic energy to be dissipated during unloading. This modeling approach may be useful in evaluating other experimental methods of soft tissue fracture.

The influence of the integrity of posterolateral structures on tibiofemoral orientation when an anterior cruciate ligament graft is tensioned.

BACKGROUND: The effect of injury to the posterolateral structures of the knee on the success of an anterior cruciate ligament reconstruction is not well known. HYPOTHESIS: Increasing graft tension increases the amount of external rotation of the tibia if the posterolateral structures are deficient. STUDY DESIGN: Laboratory study. METHODS: Eight cadaveric knees underwent techniques similar to a clinical reconstruction except that the distal fixation on the tibia was an external tensioning device used to apply a traction force on the graft. The knee was secured in a joint-testing machine and an instrumented spatial linkage was used to measure the motion of the tibia with respect to the femur. Measurements were taken with forces increasing from 0 to 100 N. The fibular collateral ligament, popliteofibular ligament, and the popliteus tendon were individually cut sequentially, and differences in the relative position of the tibia with respect to the femur were compared with the intact baseline. RESULTS: External rotation increased significantly when all of the posterolateral structures were cut and 60, 80, or 100 N of distal traction was applied. CONCLUSIONS: Deficiency of posterolateral structures of the knee significantly affected the relative external rotation of the tibia. CLINICAL RELEVANCE: Injured posterolateral structures should be repaired before fixation of anterior cruciate ligament grafts.

The effect of injury to the posterolateral structures of the knee on force in a posterior cruciate ligament graft: a biomechanical study.

To determine whether untreated grade 3 posterolateral knee injuries contribute to a significant increase in force on a posterior cruciate ligament reconstruction graft, we measured the force on the graft during joint loading of a posterior cruciate ligament-reconstructed knee with otherwise intact structures and then selectively cut the popliteofibular ligament, popliteus tendon, and the fibular collateral ligament. A posterior cruciate ligament reconstruction was performed in eight fresh-frozen cadaveric knees. One end of the graft was fixed to a tensioning jig with a load cell used to measure force in the graft as loads were applied to the knee. The force on the graft was significantly higher with the posterolateral structures cut during varus loading at 30 degrees, 60 degrees, and 90 degrees of flexion than it was in the same joint under the same loading conditions but with the posterolateral structures intact. Additionally, coupled loading of posterior drawer force and external tibial torque at 30 degrees, 60 degrees, and 90 degrees significantly increased force on the graft with the posterolateral structures cut. There was no significant increase in force on the graft under any condition with a posterior force, valgus force, or internal and external tibial torque applied alone. A significant increase in force occurs in a posterior cruciate ligament graft in knees with deficient posterolateral knee structures. We recommend that in knees with grade 3 posterolateral injuries and evidence of varus or coupled posterior-external rotation instability the posterolateral structures be repaired or reconstructed at the time of posterior cruciate ligament reconstruction to decrease the chance of later graft failure.

The effects of displacement rate and proteoglycan digestion on the fracture resistance of tissue grown from chondrocyte culture.

Fracture toughness of cartilage and cartilage replacement tissues is important in injury and disease. For example, cartilage is thought to weaken before it fibrillates in the disease osteoarthritis. Since both loading rate and proteoglycan content affect viscoelastic properties, they may both affect fracture toughness of cartilage and cartilage analogs. In this study, fracture toughness of tissue grown in chondrocyte culture was measured as a function of loading rate and proteoglycan digestion. Control tissue and tissue digested with chondroitinase ABC (cABC) to remove proteoglycans were tested at displacement rates of 0.1 and 0.5 mm/sec. Displacement rate had no effect on fracture toughness for either control or digested tissue. Proteoglycan digestion reduced tissue thickness by 30% and when evaluated on a material basis increased fracture toughness. There was no interaction between digestion and loading rate. When the fracture toughness was normalized to collagen content, which removed the effect of tissue shrinkage, there was no effect of proteoglycan digestion on fracture toughness. These data suggest that proteoglycans do not contribute to tissue toughness, other than by reducing thickness and increasing collagen density.

Interfibrillar collagen bonding exists in matrix produced by chondrocytes in culture: evidence by electron microscopy.

Interfibrillar bonding of collagen fibrils in tissue grown from rabbit chondrocytes in culture was examined by a variety of electron microscopy techniques. Interfibrillar bonding is expected to increase tissue strength and may be a desirable feature in engineered cartilage and other soft tissues. The apparent bonding evident by scanning electron microscopy, using standard chemical fixation processing, is suspected to be artifact due to drying. The goal of this article was to establish the existence of interfibrillar bonding, apart from any processing artifacts. Specimens prepared by transmission electron microscopy, scanning electron microscopy (SEM) after notching and fixing under load, and cryo-SEM all showed evidence of bonding, supporting the existence of bonding in the unprocessed tissue. Exclusion from the bond space of gold particles labeled to decorin further supported the existence of natural bonds. Artifactual bonding may still be occurring with some of the methods used, but interfibrillar bonds exist in natural tissue. The bond distance was estimated to be 7-14 nm. Demonstration of the existence of these bonds supports further study of their mechanism and effect on tissue properties.