No description available.

No description available.

No description available.

Articular cartilage is a highly specialized connective tissue in the body responsible for protecting and cushioning bony ends in diarthrodial joints. Despite the unique ability of this tough, spongy matrix to absorb repetitive stress and loading, cartilage damage is a common occurrence, and as cartilage possesses poor self-repair capabilities, tissue-engineered cartilage replacement is under development as a viable method of repair. Tissue-engineered constructs have thus far been unable to replicate the matrix composition of native cartilage satisfactorily enough to produce usable mechanical properties; in particular, collagen content is very low. One means of improving engineered construct composition may be pulsed low-intensity ultrasound (PLIUS), which is used clinically to stimulate healing of chronic bone lesions, and has been shown to affect chondrocytes in cartilage explants and engineered constructs. We believe it may be of use specifically in improving collagen quantity and quality in engineered constructs. FT-IR spectroscopy shows promise as a valuable tool in collagen crosslink maturity analysis, replacing the current expensive, complicated standard of HPLC and allowing for high-resolution spatial mapping of components. A spectral parameter has been established in literature as being related to collagen maturity in bone, which we explore as a potential means of assessing collagen quality in our engineered cartilage. The specific aims of this research are twofold: first, to assess whether PLIUS improves primary bovine chondrocyte-seeded poly-glycolic acid (PGA) mesh scaffold composition by culturing groups with and without PLIUS stimulation, and second, to correlate FT-IR parameters (including the aforementioned maturity parameter) from engineered cartilage specimens and pure crosslink peptides to mechanical testing in unconfined compression. / Bioengineering

Articular cartilage has limited intrinsic healing capacity due to its dense and avascular structure. Clinical approaches have been developed to address the limitations associated with the poor ability of articular cartilage to regenerate. Current clinically approved techniques, however, can result in repair tissue that lacks appropriate hyaline cartilage biochemical and biomechanical properties, which lead to uncertain long-term clinical outcomes. Using tissue engineering strategies and a range of scaffolding materials, cell types, growth factors, culture conditions, and culture times, engineered tissues have been produced with compositional and biomechanical properties that approximate that of native tissue. In these studies, a considerable number of samples are typically sacrificed to evaluate compositional and mechanical properties, such as the amount of deposited collagen and sulfated glycosaminoglycan (sGAG) in the constructs. The number of sacrificed samples, as well as the amount of time and resources spent to evaluate the sacrificed samples using current gold standards, motivates an alternative method for evaluation of compositional properties. Vibrational spectroscopy, including infrared, has been considered as an alternative technique for assessment of tissues over the last 15-20 years. Infrared spectroscopy is based on absorbance of infrared light by tissue functional groups at specific vibrational frequencies, and thus, no external contrast is required. Vibrational spectroscopy is typically performed in two frequency regions, the mid infrared region (750-4000 cm-1), where penetration depth is limited to approximately 10 microns, and the near infrared (NIR) region (4000-12000 cm-1). In the NIR region, penetration of light is on the order of millimeters or centimeters, which makes it ideal for obtaining data through the full depth of engineered constructs. Here we employ NIR spectroscopy to nondestructively monitor the development of tissue-engineered constructs over culture period. / Bioengineering

Articular cartilage lesions are often caused by joint trauma and can progress to osteoarthritis (OA) if left untreated. Cartilage tissue engineering is a promising approach for chondral lesion repair, involving the cultivation of cell-seeded scaffolds to generate neocartilage tissues recapitulating composition, structure, and function of native cartilage. Transforming growth factor beta (TGF-β) is widely utilized in cartilage tissue engineering for its ability to promote chondrogenesis and extracellular matrix (ECM) biosynthesis. Conventionally, TGF-β is supplemented in culture medium at supraphysiologic doses (10-100 ng/mL) during in vitro cultivation to regenerate neocartilage with native-matched sGAG content and mechanical properties. However, these doses are 10-1000-fold higher than the physiologic range, promoting undesirable tissue features that are detrimental to the functional behavior of hyaline cartilage. Additionally, TGF-β gradients from media supplementation can induce pronounced heterogeneities in ECM distribution, potentially compromising the survival of engineered cartilage under physiologic loading. The dissertation aims to enhance cartilage regeneration quality using bio-inspired latent TGF-β (LTGF-β) conjugated scaffolds. We hypothesize that LTGF-β scaffolds can achieve uniform delivery of moderated, near-physiologic doses of TGF-β through cell-mediated activation, inducing homogeneous and more hyaline cartilage-like tissue growth. We first evaluated the impact of physiologic TGF-β doses on tissue growth. To address issues related to TGF-β concentration gradients and tissue heterogeneities, we employed a reduced-size construct model. Our findings demonstrate that physiologic doses of TGF-β promote significant enhancements in tissue properties for reduced-size tissues, while also mitigating undesirable outcomes associated with excessive TGF-β. Subsequently, we developed bio-inspired LTGF-β-conjugated scaffolds to deliver physiologic doses of TGF-β. We established a quantification platform based on TGF-β autoinduction to accurately measure the bioactivity level of delivered TGF-β, confirming conjugated LTGF-β can be activated in physiologic range. Further, this quantification platform exhibits versatility for applications in native tissue studies and other TE platforms. Lastly, we determined that LTGF-β conjugation led to enhancements in tissue functional properties comparable to native tissue, while mitigating the abnormal features of neocartilage associated with TGF-β excesses. Moreover, LTGF-β conjugation significantly improves tissue spatial homogeneities in composition and mechanical properties, offering promising implications for enhancing clinical regeneration outcomes.

Rib and costal cartilage fractures are the most common injuries resulting from blunt thoracic loading scenarios, including motor vehicle collisions. The costal cartilage is a cylindrical hyaline cartilage composed of two layers: a core interstitial matrix enveloped by the perichondrium. The perichondrium itself has an inner chondrogenic layer and an outer fibrous layer. The objective of this study was to evaluate the tensile material properties of human costal cartilage perichondrium at two loading rates for a range of subject demographics. Fifty-six (n=56) samples containing the fibrous layer and chondrogenic layer (i.e., two-layered samples) were fabricated from thirty-three (n=33) donors aged from 11 to 69 years of age (19 M, 14 F). Thirteen (n=13) samples without the fibrous layer (i.e., one-layered samples) were fabricated from eight (n=8) donors aged from 11 to 54 years of age (5 M, 3 F). The perichondrium was isolated from the interstitial matrix for all samples and the fibrous layer was removed for one-layered samples to assess the effect of the absence of the fibrous layer. The tissue was then stamped into a dog bone-shaped coupon and sanded down to a uniform thickness of ~1.3 mm for two-layered samples and ~1 mm for one-layered samples. The gage length of the completed coupons was marked with a black ink dot pattern to facilitate strain calculations via video tracking. The coupons were loaded axially in tension to failure at either a slow (0.005 s⁻¹) or fast (0.5 s⁻¹) target loading rate using a material testing system. The elastic modulus, ultimate stress, ultimate strain, failure stress, failure strain, and strain energy density (SED) were then calculated for each test. Material property data were compared by sample type and loading rate. Since there was no significant influence of sex on any material properties, the data were grouped together for the analysis. Modulus, ultimate stress, failure stress, and SED were found to significantly decrease with donor age at both loading rates and ultimate and failure strain also significantly decreased with donor age at the 0.5 s⁻¹ target loading rate. Failure stress in the two-layered samples was found to be greater than that of the one-layered samples at both loading rates. One-layered samples had a greater failure strain than two-layered samples at both loading rates. Perichondrium data were compared to interstitial matrix data from a previous study to further investigate the role of cartilage layer on material properties. The modulus, ultimate stress, and failure stress of costal cartilage decreased moving radially inward (greatest in two-layered perichondrium samples, least in interstitial matrix samples). The opposite was true for ultimate and failure strain, with the greatest failure strain values occurring in the interstitial matrix and the least in the two-layered perichondrium samples. The sample size of one-layered samples was too small to draw any substantial conclusions regarding age trends. This was the first study to analyze the material property trends in costal cartilage perichondrium. The results of this study can be incorporated into virtual human body models to improve the accuracy of thoracic injury prediction in the context of motor vehicle safety. / Master of Science / Motor vehicle collisions are the second leading cause of death due to unintentional injury in the United States, with rib and costal cartilage fractures being the most commonly observed injuries. The cylindrical costal cartilage connects the front of the ribs to the sternum and is composed of two layers: a core interstitial matrix enveloped by the perichondrium. The perichondrium itself has an inner chondrogenic layer and an outer fibrous layer. Virtual human body models incorporate material property data to improve their ability to predict injury risk and are frequently used among vehicle manufacturers to evaluate safety during vehicle development. Currently, models have to make simplifications and assumptions regarding the perichondrium properties, since there are no material property studies on the isolated perichondrium to date. Therefore, the purpose of this study was to quantify the tensile material properties of human costal cartilage perichondrium at two loading rates for a range of subject demographics. Dog-bone shaped coupons with either both perichondrium layers (i.e., two-layered samples) or just the chondrogenic layer (i.e., one-layered samples) were loaded to failure under tension at either a slow (0.005 s⁻¹) or fast (0.5 s⁻¹) target loading rate using a material testing system. Data were obtained for fifty-six (n=56) two-layered samples from thirty-three (n=33) donors aged from 11 to 69 years old. Data were collected for thirteen (n=13) one-layered samples from eight (n=8) donors aged from 11 to 54 years old. The elastic modulus, ultimate stress, ultimate strain, failure stress, failure strain, and strain energy density (SED) were quantified for each test. Material properties of two-layered samples decreased with increasing donor age. No trends were found with regard to donor sex. Only ultimate and failure stress of two-layered samples were significantly affected by loading rate. Perichondrium material property data were compared to interstitial matrix data from a previous study to investigate the effect of cartilage layer on costal cartilage material properties. Elastic modulus, ultimate stress, and failure stress decreased when moving inward in cartilage layers, while ultimate and failure strain increased. Overall, this is the first study to evaluate the material properties of the perichondrium and the change in material properties with cartilage layer. These data can be used to improve the accuracy of human tolerance to thoracic injury in human body models.

Insulin-like growth factor binding proteins (IGFBP) are modulators of insulin-like growth factor I (IGF-I), which functions as a regulator of cartilage and bone development. Rapid growth and high starch diets have been associated with increased circulating concentrations of IGF-I, which lead to developmental orthopedic disorders in foals. The objective of this study was to assess the effects of age, diet, growth and season on plasma IGFBP and IGF-I concentrations from birth to 16 mo of age in Thoroughbred foals. Twenty-two mares maintained on mixed grass/legume pasture were randomly divided into two dietary groups and fed either a high starch and sugar supplement (SS) or a starch-restricted fiber and fat supplement (FF) for 3 mo prior to and after foaling. Monthly blood samples were obtained from SS and FF foals up to 16 mo of age and analyzed for IGF-I using an RIA and IGFBP using western ligand blot analysis. Auxilogical measurements of foals were also obtained each month. The effect of diet, month, and diet*month interactions upon the subject horse (diet) were analyzed using a mixed model with repeated measures, and correlations of normally distributed data were calculated using Pearson's correlation. Six IGFBP bands of molecular weights 109, 39, 36, 35, 34, and 33 kDa were identified in foal plasma. Doublet bands were recognized at 109, 39, and 35 kDa, however they were not all believed to be singular pure IGFBP. A band with a molecular weight of 213 kDa was observed and presumed to be a ternary complex of IGFBP-3, IGF-I, and an acid labile subunit. The IGFBP 109 kDa has been previously recognized as a band unique to the equine, it was not a singular pure IGFBP because of its high molecular weight. No effect of diet on plasma IGFBP was found in individual sampling of yearlings, but an effect of month was noted when testing May - August 2001 against May - August 2002 in pooled plasma samples with concentrations of the IGFBP 39 kDa increasing (P < 0.0003). In contrast, concentrations of the IGFBP's 33, 34 and 36 kDa decreased (P < 0.003, P < 0.0002, and P < 0.0003 respectively). Environmental effects were noted upon IGFBP's 33, 36, 39, and 109 kDa (P < 0.003, P < 0.001, P < 0.04, and P < 0.01) with a temperature*daylength interaction. Correlations existed between ADG and IGFBP 33 (r = 0.64; P < 0.0001), 34 (r = 0.40; P < 0.0001), 35 (r = 0.33; P < 0.0006), 36 (r = 0.47; P < 0.0001), and 39 kDa (r = - 0.18, P < 0.02). A correlation was also found between IGF-I and ADG (r = 0.11; P < 0.04), confirming the previously reported relationship of IGF-I in growth rate of foals. These results underline the importance of characterizing the activity of IGFBP's in relation to growth, age and season when interpreting changes of the somatotropic axis. Further, the increase in certain IGFBP's and simultaneous decrease in others stress the need for further research on the tissue specific modulating effects that IGFBP's have on IGF-I. / Master of Science

El objetivo principal de esta tesis doctoral es el diseño de un andamio polimérico bicapa macroporoso para la regeneración del complejo osteocondral. El material empleado para la fabricación del constructo ha sido el ácido poli(L-láctico), un polímero biodegradable de la familia de los poliésteres. Una de las capas del andamio ha sido diseñada para asistir la regeneración del cartílago articular. La otra capa sirve de anclaje al hueso subcondral, y se diferencia de la anterior en sus propiedades mecánicas y bioactividad. Este comportamiento ha sido logrado por combinación del ácido poli(L-láctico) con nanopartículas inorgánicas. Ambas capas están unidas entre sí por una fina capa de material no poroso que evita el flujo de células de una parte a otra del constructo. Para lograr este objetivo se realizó un primer estudio de diseño variando la morfología de los andamios hasta obtener aquella arquitectura más adecuada para la regeneración de ambos tejidos. Se varió parámetros de síntesis tales como la concentración de polímero y el ratio entre polímero y porógeno. Los andamios fueron evaluados mecánica y fisicoquímicamente y se seleccionó los parámetros de síntesis del ácido poli(L-láctico) que dieron mejores resultados. En la regeneración del tejido es esencial conocer cómo variarán las propiedades del material una vez sea implantado y comience su degradación. Por lo tanto, fue considerado oportuno realizar un estudio de degradación del material in vitro en diversas condiciones. El estudio de la degradación fue realizado en condiciones estáticas durante 6, 12, 18, 24 semanas y 1 año y en condiciones dinámicas durante 1, 2, 4 y 6 semanas. Se evaluó tanto las características mecánicas como las fisicoquímicas tras los diversos tiempos de la degradación. Posteriormente, y para aumentar las características mecánicas y la bioactividad del anclaje óseo, se incorporó distintas cantidades de nanopartículas inorgánicas de hidroxiapatita y sílice a los andamios. / Deplaine ., H. (2012). Scaffold design and characterisation for osteochondral tissue regeneration [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/14638

This thesis is focused on the development and evaluation of different hybrid scaffolds for the treatment of injuries in cartilage or bone. These hybrid materials were three-dimensional polycaprolactone macroporous scaffolds obtained through freeze extraction and particle leaching combined method and modified with hyaluronic acid or mineral particles. In order to facilitate the description of the obtained results, the thesis is divided in two sections dedicated to bone and cartilage tissue engineering respectively. In the case of bone tissue engineering we addressed the treatment of disorders associated with the spine that require spinal immobilization. This Thesis proposes the development of a synthetic macroporous support for intervertebral fusion as an alternative to commercial bone substitutes. Macroporous scaffolds were developed with bare polycaprolactone or its blends with polylactic acid in order to increase its mechanical properties and degradation rate. Furthermore, the scaffolds obtained were reinforced with hydroxyapatite or Bioglass®45S5 to improve their mechanical properties and turn them in bioactive scaffolds. The supports were characterized physicochemically and biologically to determine if they met the requirements of the project. Finally, materials were tested in vivo in a bone critical size defect preformed in a rabbit model against a commercial support. Cartilage engineering has been extensively studied in the last years due to the inherent limited self repair ability of this tissue. The second part of the thesis was focused in developing a construct composed by in vitro differentiated chondrocyte like cells in a hybrid scaffold for cartilage tissue engineering. Polycaprolactone hybrid substrates coated with hyaluronic acid scaffold were developed obtaining a substrate with positive influence over the development of chondrocyte phenotype in culture and able to protect the cells from excessive mechanical loading in the joint. Cell-scaffolds constructs were obtained combining hybrid scaffolds with mesenchymal stem cells and differentiating them to chondrocytes using chondrogenic culture medium combined with hypoxia, mechanical stimulus or co-culture. Finally the cellularized scaffolds were mechanically, biochemically and histologically characterized to determine the production of extracellular matrix and expression of chondrogenic markers. / Ródenas Rochina, J. (2015). Scaffold surface modifications and culture conditions as key parameters to develop cartilage and bone tissue engineering implants [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48526