Thermal analysis reveals differential effects of various crosslinkers on bovine annulus fibrosis.

Treatment of a pathological spinal disc in vivo by injection of protein crosslinking reagents to restore the disc's mechanical properties is a new approach to the treatment of degenerative disc disease. In this study, the thermal stability of the collagen in disc annulus was measured by differential scanning calorimetry following treatment with six different crosslinking agents. The crosslinkers used were; L-threose (LT), genipin (GP), methylglyoxal (MG), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), glutaraldehyde (GA), and proanthrocyanidin (PA). Untreated tissue displayed a prominent peak at about 66-68°C. Comparison of endothermal patterns of untreated and crosslinker-treated disc annulus tissue samples showed that a new peak appeared at a higher temperature following treatment. The temperature of the new peak qualitatively depended on the crosslinker in the following order GA > MG > GP > PA = EDC > LT, suggesting that the enhanced thermal stability of collagen in the annulus tissue was related to the nature of the crosslinker. Also, the enthalpic ratios of the lower temperature (noncrosslinked) peaks in the treated and untreated tissue, and of the higher and lower temperature peaks in the treated tissue, both indicated that the various agents crosslinked the tissue with different efficiencies. Our data suggest that the ability of GP to penetrate into the disc and form long- and short-range crosslinks may make it the most suitable candidate for clinical development. In addition, binary combinations of long- and short-range crosslinkers, such as PA with LT, may also provide synergistic effects due to their substantially different physicochemical properties.

Optimization of protein crosslinking formulations for the treatment of degenerative disc disease.

STUDY DESIGN: Biochemical studies aimed at optimization of protein crosslinking formulations for the treatment of degenerative disc disease and subsequent biomechanical testing of tissues treated with these formulations. OBJECTIVE: To optimize protein crosslinking formulations for treatment of degenerating spinal discs. SUMMARY OF BACKGROUND DATA: Nonsurgical exogenous crosslinking therapy is a potential new, noninvasive technology for the treatment of degenerative disc disease. The technology is based on the injection of protein crosslinking reagents into the pathologic disc to restore its mechanical properties and also to potentially increase the permeability of the tissue and so facilitate the exchange of waste products and nutrients. METHODS: Diffusion of genipin (GP) was monitored following injection into spinal discs and the effects of surfactants on diffusion studied. Formulations for GP and methylglyoxal (MG) were biochemically optimized and used to treat bovine spinal discs. Their effects on bovine anulus tissue were evaluated using a circumferential tensile test, while the GP formulation was also tested with respect to its ability to reduce disc bulge under load. RESULTS: GP exhibited a distinct time-dependent diffusion and sodium-dodecyl-sulfate, but not Tween-20, enhanced diffusion by 30%. Two crosslinkers, GP and MG, were inhibited by amines but enhanced by phosphate ions. Both formulations could enhance a number of physical parameters of bovine anulus tissue, while the GP formulation could reduce disc bulge following injections into spinal discs. CONCLUSION: Formulations lacking amines and containing phosphate ions appear to be promising candidates for clinical use of the crosslinkers GP and MG.

Kinetic characterization and comparison of various protein crosslinking reagents for matrix modification.

We have characterized the relative efficacies of a number of protein crosslinking agents that have the potential for use in the crosslinking of proteinaceous matrices both in vitro and in vivo. The crosslinkers tested were; L: -threose (LT), Genipin (GP), Methylglyoxal (MG), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), proanthrocyanidin (PA) and glutaraldehyde (GA). The relative effectiveness of the crosslinkers with regard to their saturating concentrations was: GA > PA > EDC > MG = GP >> LT. Most of the crosslinkers displayed a pH dependence and were more effective at more alkaline pH. At optimal pH and saturating conditions, the relative reaction rates of the crosslinkers were: PA = GA > EDC > GP > MG >> LT.

Kinetic analysis of genipin degradation in aqueous solution.

Degradation of genipin (GP), a low toxicity natural protein crosslinking agent, in aqueous solution was monitored by HPLC at various pH levels. Degradation of GP was consistent with a mechanism consisting of a first order reaction with a reversible first step. Formation of the intermediate was slowest at more neutral pHs while formation of the irreversible product was correlated to increasing alkalinity. Degradation at all pHs was enhanced by the presence of phosphate ions. Degradation of GP most likely proceeds via the reversible opening of the dihydropyran ring by water followed by irreversible polymerization of the intermediate. Degraded solutions containing no detectable GP or intermediate, however, are still capable of crosslinking proteins.

Reconstruction of large rotator cuff tendon defects with porcine small intestinal submucosa in an animal model.

Large rotator cuff tears represent a challenging problem. The purpose of our study was to evaluate the small intestinal submucosa (SIS) extracellular matrix in reconstruction of such defects in an animal model. Forty rats were equally divided into an SIS group (reconstruction of a large supraspinatus tendon defect by use of the SIS) and a defect group (no repair). The operative and contralateral normal shoulders underwent histologic evaluation and biomechanical testing at 6 and 16 weeks. Neovascularization and fibroblastic ingrowth were present in SIS-regenerated tendons, which had an ultimate force to failure that was 78% of normal at 16 weeks. This was higher than in the defect group, which demonstrated an ultimate force to failure that was 34% of normal (P = .008). The ultimate force to failure of the SIS-regenerated tendons approached that of the normal tendon at 16 weeks. The SIS extracellular matrix served as a scaffold promoting host tissue ingrowth and appears promising in the management of large rotator cuff defects in a rat model. However, human beings sustain tears at the bone-tendon interface and demonstrate decreased healing potential relative to rats. Therefore, the findings of this preliminary study should not be extrapolated to human beings without further investigation.

Changes in abduction and rotation range of motion in response to simulated dorsal and ventral translational mobilization of the glenohumeral joint.

BACKGROUND AND PURPOSE: Translational mobilization techniques are frequently used by physical therapists as an intervention for patients with limited ranges of motion (ROMs). However, concrete experimental support for such practice is lacking. The purpose of the study was to evaluate the effect of simulated dorsal and ventral translational mobilization (DTM and VTM) of the glenohumeral joint on abduction and rotational ROMs. METHODS: Fourteen fresh frozen shoulder specimens from 5 men and 3 women (mean age=77.3 years, SD=10.1, range=62-91) were used for this study. Each specimen underwent 5 repetitions of DTM and VTM in the plane of scapula simulated by a material testing system (MTS) in the resting position (40 of abduction in neutral rotation) and at the end range of abduction with 100 N of force. Abduction and rotation were assessed as the main outcome measures before and after each mobilization procedure performed and monitored by the MTS (abduction, 4 N m) and by a servomotor attached to the piston of the actuator of the MTS (medial and lateral rotation, 2 N m). RESULTS: There were increases in abduction ROM for both DTM (mean=2.10 , SD=1.76 ) and VTM (mean=2.06 , SD=1.96 ) at the end-range position. No changes were found in the resting position following the same procedure. Small increases were also found in lateral rotation ROM after VTM in the resting position (mean=0.90 , SD=0.92 , t=3.65, P=.003) and in medial rotation ROM after DTM (mean=0.97 , SD=1.45 , t=2.51, P=.026) at the end range of abduction. DISCUSSION AND CONCLUSION: The results indicate that both DTM and VTM procedures applied at the end range of abduction improved glenohumeral abduction range of motion. Whether these changes would result in improved function could not be determined because of the use of a cadaver model.

Characterization of tissue resistance during a dorsally directed translational mobilization of the glenohumeral joint.

OBJECTIVES: To quantify forces applied by therapists during dorsal glide translational mobilization of the glenohumeral joint, to determine the relationship of tissue resistance to the load-displacement relation of the glenohumeral joint, and to determine the safety of the forces applied by the therapists during dorsal glide translational mobilization. DESIGN: A fresh cadaver shoulder specimen mounted on a 6-axis load cell was used to register forces applied by therapists during dorsal glide translational mobilization of the glenohumeral joint in a test-retest pattern. SETTING: Biomechanics laboratory. PARTICIPANTS: Twelve experienced orthopedic physical therapists. INTERVENTION: Not applicable. MAIN OUTCOME MEASURES: Forces exerted by therapists during passive dorsal glide translational mobilization in the loose-packed position and in the end range of abduction, with different grades of movements. The movements did not include any manipulation or thrust-type procedures. Simulated dorsal glide procedures were performed by the material testing system to construct the load-displacement curve of the glenohumeral specimen. The corresponding locations of the forces applied by therapists were interpolated and plotted on the load-displacement curve. RESULTS: The peak force values measured during mobilization were characterized by large intertherapist variability: coefficients of variation ranged from 40.97% to 77.49%. Test-retest reliability for intrasession measures was high (ICC(2,1) range,.90-.94); intersession reliability was poor (ICC(2,1) range,.01-.54). The mean forces ranged from 18.36 to 38.76N. When interpolated to the load-displacement curve, the mean peak forces obtained fell mostly in the toe and the linear elastic regions of the load-displacement curve. CONCLUSION: Force parameters measured during dorsal glide mobilization were characterized by large intertherapist variability with high intrasession and poor intersession test-retest reliability. The mobilization forces applied by experienced orthopedic physical therapists fall safely in the toe and the linear elastic regions of the load-displacement curve.