This study investigates the changes due to crosslinking treatment in stiffness, permeability, and glycosaminoglycan (GAG) content of bovine intervertebral discs. The objective of this study was to determine the mechanical and biochemical effects of crosslinking treatment on lumbar bovine tissue. Previous studies have found that crosslinking can increase stiffness and permeability in the intervertebral disc. These changes have not yet been investigated by confined compression, stress-relaxation tests of young bovine tissue. Eleven lumbar motion segments were harvested from calf spines and soaked in a saline solution or one of four crosslinking treatments (genipin, methylglyoxal, proanthrocyanidin, and EDC). Five mm diameter samples were removed from the midannulus region at anterior / anterior-lateral locations, confined in a saline bath, swelled to equilibrium, and tested in confined compression stress-relaxation to 15% strain in 5% increments. Radial samples were also harvested, treated with saline solution and EDC, and tested in the same manner. The aggregate modulus and hydraulic permeability were calculated using the nonlinear biphasic theory. Swelling pressure was calculated as the load at swelling equilibrium. GAG content was measured using the dimethylmethylene blue assay. Differences with P value < 0.05 were considered significant. In the axial orientation, all crosslinking treatments except methyglyoxal at least doubled the aggregate modulus relative to soaked controls (P less than 0.05). Genipin treatment resulted in 78% lower axial permeability, proanthrocyanidin (PA) 50% lower, and EDC treatment 84% lower relative to soaked controls (P &lt; 0.05). GAG content measured in the methyglyoxal treatment group was 25% lower than in soaked control group. Genipin (G), proanthrocyanidin (PA), and EDC treatment increased the swelling pressure by at least 65% (P less than 0.05). In the radial orientation, EDC treatment increased the stiffness by 75%, and did not significantly affect the permeability or swelling pressure. Some crosslinking treatments proved effective in increasing the stiffness and swelling pressure of the disc. The increased swelling pressure in G, PA, and EDC treatment groups relative to soaked controls suggests reduced GAG leaching during soaking treatment, further confirmed by the reduction in permeability in these groups.

<p>Articular cartilage is a connective tissue that lines the surfaces of diarthrodial joints; and functions to support and distribute loads as wells as facilitate smooth joint articulation. Unfortunately, cartilage possesses a limited capacity to self-repair. Once damaged, cartilage continues to degenerate until widespread cartilage loss results in the debilitating and painful disease of osteoarthritis. Current treatment options are limited to palliative interventions that seek to mitigate pain, and fail to recapitulate the native function. Cartilage tissue engineering offers a novel treatment option for the repair of focal defects as well as the complete resurfacing of osteoarthritic joints. Tissue engineering combines cells, growth factors, and biomaterials in order to synthesize new cartilage tissue that recapitulates the native structure, mechanical properties, and function of the native tissue. In this endeavor, there has been a growing interest in the use of scaffolds derived from the native extracellular matrix of cartilage. These cartilage-derived matrix (CDM) scaffolds have been show to recapitulate the native epitopes for cell-matrix interactions as well as provide entrapped growth factors; and have been shown to stimulate chondrogenic differentiation of a variety of cell types. Despite the potent chondroinductive properties of CDM scaffolds, they possess very weak mechanical properties that are several orders of magnitude lower than the native tissue. These poor mechanical properties lead to CDM scaffolds succumbing to cell-mediated contraction, which dramatically and unpredictably alters the size and shape of CDM constructs. Cell-mediated contraction not only prevents the fabrication of CDM constructs with specific, pre-determined dimensions, but also limits cellular proliferation and metabolic synthesis of cartilage proteins. This dissertation utilized collagen crosslinking techniques as well as ice-templating in order to enhance the mechanical properties of CDM scaffolds and prevent cell-mediated contraction. Furthermore, the decellularization of CDM was investigated in order to remove possible sources of immunogenicity. This work found that both physical and chemical crosslinking techniques were capable of preventing cell-mediated contraction in CDM scaffolds; however, the crosslinking techniques produced distinct effects on the chondroinductive capacity of CDM. Furthermore, the mechanical properties of CDM scaffolds were able to be enhanced by increasing the CDM concentration; however, this led to a concomitant decrease in pore size, which limited cellular infiltration. The pore size was able to be rescued through the use of an ice-templating technique that led to the formation of large aligned grooves, which enabled cellular infiltration. Additionally, a decellularization protocol was developed that successfully removed foreign DNA to the same order of magnitude as clinically approved materials, while preserving the native GAG content of the CDM, which has been shown to be critical in preserving the mechanical properties of the CDM. Altogether, this body of work demonstrated that dehydrothermal crosslinking was best suited for maintaining the chondroinductive capacity of the CDM, and given the appropriate scaffold fabrication parameters, such as CDM concentration and ice-templating technique, dehydrothermal treatment was able to confer mechanical properties that prevented cell-mediated contraction. To emphasize this finding, this work culminated in the fabrication of an anatomically-relevant hemispherical scaffold entirely from CDM alone. The CDM hemispheres not only supported chondrogenic differentiation, but also retained the original scaffold dimensions and shape throughout chondrogenic culture. These findings illustrate that CDM is a promising material for the fabrication of tailor-made scaffolds for cartilage tissue engineering.</p> / Dissertation

OBEJTIVOS: Analisar a segurança e eficácia da cirurgia de crosslinking (CXL) de córnea em pacientes com ceratocone em estágios avançados após um seguimento de 4 anos. MÉTODOS: Trata-se de um estudo retrospectivo de pacientes com ceratocone avançado em progressão (Estágio 3 e 4 da classificação de Amsler-Krumeich) que foram submetidos a cirurgia de CXL seguindo o protocolo padrão. Os parâmetros examinados foram acuidade visual com (AVCC) e sem (AVSC) correção, valores ceratométricos (média, plana, curva e apical), paquimetria, e contagem de células endoteliais no préoperatório e após 12, 24 e 48 meses do procedimento. RESULTADOS: Este estudo abrange quarenta olhos de 40 pacientes que foram submetidos a cirurgia de CXL. A média de idade foi 22,5 anos (Intervalo:15 a 37 anos). Tanto a AVSC quanto a AVCC permaneceram estáveis durante o período de seguimento, sem mudanças estatisticamente significativas. Apesar de todos os valores ceratométricos sofrerem uma leve diminuição, apenas a ceratometria apical atingiu uma mudança com significado estatístico (P=0,037) após 4 anos de seguimento. Uma redução significativa da espessura corneana foi também observada (paquimetria ultrassônica era de 388 ± 49 e passou para 379 ± 48 ?m, P < 0,0001; paquimetria através de tomografia de imagem em fenda era de 362 ± 48 e foi para 353 ± 51 um, P < 0,0001); embora essa diferença não seja clinicamente significativa. A contagem de células endoteliais não sofreu alterações significativas durante o seguimento. A taxa de falha do tratamento foi de 5% (dois pacientes) durante o seguimento. CONCLUSA?O: A cirurgia de CXL corneano em pacientes com ceratocone avançado se mostrou segura e capaz de manter os parâmetros visuais e topográficos pelo menos durante 4 anos / PURPOSE: To analyze the safety and efficacy of standard corneal collagen crosslinking (CXL) in advanced cases of progressive keratoconus (KC) after four years of follow-up. METHODS: A retrospective case series of patients with advanced progressive KC (stage 3 and 4 of Amsler-Krumeich classification) underwent standard CXL treatment. The parameters examined were changes in uncorrected visual acuity (UDVA), corrected visual acuity (CDVA), keratometry values (mean K, flattest K, steepest K, and apical K), pachymetry, and endothelial cell count at the baseline and at 12-, 24- and 48-months postoperatively. RESULTS: Forty eyes of 40 patients were enrolled into the study. The mean patient age was 22.5 years (range: 15 to 37 years). Both mean UCVA and CDVA remained stable during the time points; no statistically significant change was noted. Although a slightly reduction was observed in all keratometric readings, a statistically significant reduction was only reached in the apical K (p=0.037) at four years after CXL. A significant reduction in the pachymetry was also found (from 388±49 to 379±48 um, P < 0,0001 and from 362±48 to 353±51 um, P < 0,0001, ultrasonic and slit-scanning readings, respectively) ; however this change is not likely clinically meaningful. Endothelial cell count was not significantly modified at the end of the study. Treatment failure or progression was noted in two patients (5%) over the followup period. CONCLUSION: Standard CXL treatment seems to be safe and able to stabilize both visual acuity and topographic parameters at four-year follow-up in advanced keratoconic eyes

OBEJTIVOS: Analisar a segurança e eficácia da cirurgia de crosslinking (CXL) de córnea em pacientes com ceratocone em estágios avançados após um seguimento de 4 anos. MÉTODOS: Trata-se de um estudo retrospectivo de pacientes com ceratocone avançado em progressão (Estágio 3 e 4 da classificação de Amsler-Krumeich) que foram submetidos a cirurgia de CXL seguindo o protocolo padrão. Os parâmetros examinados foram acuidade visual com (AVCC) e sem (AVSC) correção, valores ceratométricos (média, plana, curva e apical), paquimetria, e contagem de células endoteliais no préoperatório e após 12, 24 e 48 meses do procedimento. RESULTADOS: Este estudo abrange quarenta olhos de 40 pacientes que foram submetidos a cirurgia de CXL. A média de idade foi 22,5 anos (Intervalo:15 a 37 anos). Tanto a AVSC quanto a AVCC permaneceram estáveis durante o período de seguimento, sem mudanças estatisticamente significativas. Apesar de todos os valores ceratométricos sofrerem uma leve diminuição, apenas a ceratometria apical atingiu uma mudança com significado estatístico (P=0,037) após 4 anos de seguimento. Uma redução significativa da espessura corneana foi também observada (paquimetria ultrassônica era de 388 ± 49 e passou para 379 ± 48 ?m, P < 0,0001; paquimetria através de tomografia de imagem em fenda era de 362 ± 48 e foi para 353 ± 51 um, P < 0,0001); embora essa diferença não seja clinicamente significativa. A contagem de células endoteliais não sofreu alterações significativas durante o seguimento. A taxa de falha do tratamento foi de 5% (dois pacientes) durante o seguimento. CONCLUSA?O: A cirurgia de CXL corneano em pacientes com ceratocone avançado se mostrou segura e capaz de manter os parâmetros visuais e topográficos pelo menos durante 4 anos / PURPOSE: To analyze the safety and efficacy of standard corneal collagen crosslinking (CXL) in advanced cases of progressive keratoconus (KC) after four years of follow-up. METHODS: A retrospective case series of patients with advanced progressive KC (stage 3 and 4 of Amsler-Krumeich classification) underwent standard CXL treatment. The parameters examined were changes in uncorrected visual acuity (UDVA), corrected visual acuity (CDVA), keratometry values (mean K, flattest K, steepest K, and apical K), pachymetry, and endothelial cell count at the baseline and at 12-, 24- and 48-months postoperatively. RESULTS: Forty eyes of 40 patients were enrolled into the study. The mean patient age was 22.5 years (range: 15 to 37 years). Both mean UCVA and CDVA remained stable during the time points; no statistically significant change was noted. Although a slightly reduction was observed in all keratometric readings, a statistically significant reduction was only reached in the apical K (p=0.037) at four years after CXL. A significant reduction in the pachymetry was also found (from 388±49 to 379±48 um, P < 0,0001 and from 362±48 to 353±51 um, P < 0,0001, ultrasonic and slit-scanning readings, respectively) ; however this change is not likely clinically meaningful. Endothelial cell count was not significantly modified at the end of the study. Treatment failure or progression was noted in two patients (5%) over the followup period. CONCLUSION: Standard CXL treatment seems to be safe and able to stabilize both visual acuity and topographic parameters at four-year follow-up in advanced keratoconic eyes

Hereditary equine regional dermal asthenia (HERDA) is a recessive connective tissue disorder of Quarter Horse lineages. This study correlates previously identified decreases in skin tensile strength in HERDA with abnormal dermal collagen cross linking patterns that are also identified in urine from HERDA horses. Dermal collagen from HERDA horses has significantly less pyridinoline and significantly more deoxypyridinoline than control or carriers. Concentrations of hydroxylysine, the rate limiting substrate for these crosslinks were significantly lower in HERDA versus control and carriers. These characteristics of HERDA skin parallel humans with a similar syndrome of skin fragility, Ehlers Danlos Syndrome TypeVIA. This is the first biochemical evidence explaining the clinical skin fragility that characterizes HERDA and suggests that altered collagen lysine metabolism may be physiologically relevant to the clinical manifestation of HERDA. Evaluations of mature scars indicate that lesion and nonlesioned skin should not be viewed as biologically equivalent in HERDA investigations.