| Summary: | 博士 === 國立臺北科技大學 === 機電科技研究所 === 104 === Collagen crosslinks act as essential bonds in providing the mechanical strength of load-supporting tissues. Collagens in human discs have a higher concentration of the mature crosslinks than in other connective tissues, which may indicate an extremely demanding mechanical environment the disc exposed. With disc aging or degeneration, the contents of the mature crosslinks were slightly decreased or unchanged, while the quantities of the glycation crosslinks were increased. This suggests that a natural, endogenous crosslink augmentation of the disc exists, perhaps in order to prevent the tissue’s structural deterioration caused by aging or degeneration. Moreover, an absence of the glycation crosslinks in the severely degenerated disc was also found. This again implies the importance of these age-related crosslinks in maintaining disc integrity.
Genipin, a naturally occurring crosslinker extracted from gardenia fruit, has been widely utilized for modification of the biomaterials. In the previous in vitro studies, collagen crosslink augmentation by genipin treatment have shown the benefits to the disc biomechanical behavior, including to improve instability of the degenerated motion segments, and to augment the annular modulus, strength, and toughness in the principal stress directions. According to these biomechanical findings, the exogenous crosslinking treatment may have potential to be a promising therapy for treating degenerative disc disease. Therefore, the objectives of the present study were to further explore the in vivo effects of genipin crosslinking on the disc mechanical function, and also to evaluate its feasibility and efficacy in treating living disc tissues.
Two animal studies were involved in this dissertation. In the first animal study, a porcine lumbar disc model was used to test the hypothesis that genipin crosslinking could restore the disc structural integrity following a stab injury. The porcine lumbar discs were randomly divided into four experimental groups: intact, injured, untreated, and genipin crosslinked. The disc injury was produced by a 16 gauge needle puncture into the nucleus. Genipin solution was delivered into the annulus tissue around the disc injury by 28 gauge needle injection as a treatment. Quantitative discomanometry was performed to evaluate the disc integrity for the intact and injured groups at the time of the first surgery, and for the untreated and ginipin crosslinked groups after 1-week recovery. Four quantitative parameters: leakage pressure and volume, and saturation pressure and volume, were analyzed and compared from the evaluations of the four groups. The results showed that the injury can immediately reduce the disc leakage and saturation pressures. The intradiscal pressures of the stab-injured discs cannot be restored without treatment. Significantly higher leakage and saturation pressures were found in the genipin crosslinked discs relative to the values of the untreated group. These results suggested that genipin can react with the living disc tissues during 1-week treatment, and the disc matrix modification by genipin crosslinking may produce a “seal” effect on the annular damage, therefore to restore the intradiscal pressure of the injured discs.
In the second animal study, a rat tail disc model was used to test the hypothesis that genipin crosslinking could maintain the mechanical function of injured discs under a given physiological loading environment. The rat tail motion segments C7-C8 or C8-C9 were obtained and randomly divided into six experimental groups: intact, injury, injury-load stimulus sham, load stimulus, injury-load stimulus, and genipin treatment-injury-load stimulus. The disc injury was produced by a 20 gauge needle puncture into the nucleus. Two rings were installed to the adjacent caudal bones for preparation of the load stimulus. A daily dynamic compressive loading for 1 week was applied to the disc using a custom-made loading apparatus. For exogenous crosslinking treatment, genipin solution was delivered into the nucleus by 25 gauge needle injection. Following animal euthanasia, the rat tail motion segments were dissected and then mechanically tested by compression-tension ramp cycles. Individual stiffness in the tension, compression, and neutral zones, and length in the neutral zone were analyzed according to the force-displacement curve obtained from the mechanical tests. The results showed that the injury can significantly lower disc neutral zone stiffness and increase the neutral zone length. After the short-term dynamic load stimulus, the stimulated discs behaved a higher stiffness in tension zone than the intact level. The given mechanical stimulation can further lower the compression and neutral zone stiffness, and enhance the neutral zone length for the injured discs. The stab-injured disc with genipin crosslinking treatment, despite subjected to the short-term mechanical stimulation, had a higher stiffness in each zone than the injured discs without load stimulus. Notably, as compared to the injury-load stimulus group, the genipin crosslinked discs had a better stiffness in every zone and a decreased neutral zone length. These results suggested that the injured discs have an insufficient capacity in mechanical load support that could facilitate the damage accumulation when the disc is exposure to the daily dynamic load. Genipin crosslinking has previously been demonstrated to recover the integrity of the injured disc, and to increase the mechanical strength of the annulus fibrosus, both of that are beneficial for the disc mechanical function. A better mechanical function is helpful for the disc to resist the injury induced either by over loading or fatigue loading.
The drug optimization, delivery efficiency, toxicity, and dose effects of genipin crosslinking to disc tissues were excluded in the present animal studies. However, the biomechanical findings obtained from these animal studies give proof that supports the feasibility of genipin injection in treating living disc tissues. The exogenous crosslink augmentation of the stab-injured disc by genipin treatment could be advantageous in restoring the tissue’s structural integrity and maintaining the tissue’s mechanical function in vivo.
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