Due to the inability of intra-articular injuries to adequately self-heal, current therapies are largely focused on palliative care and restoration of joint function rather than true regeneration. Subsequently tissue engineering of chondral and osteochondral tissue constructs has emerged as a promising strategy for the repair of partial and full-thickness intra-articular defects. Unfortunately, the fabrication of large tissue constructs is plagued by poor nutrient transport to the interior of the tissue resulting in poor tissue growth and necrosis. Further, for the specific case of osteochondral grafts, the presence of two distinct tissue types offers additional challenges related to cell sourcing, scaffolding strategies, and bioprocessing. To overcome these constraints, this dissertation was focused on the development and validation of a microfluidic hydrogel platform which reduces nutrient transport limitations within an engineered tissue construct through a serpentine microfluidic network embedded within the developing tissue. To this end, a microfluidic hydrogel was designed to meet the nutrition requirements of a developing tissue and validated through the cultivation of chondral tissue constructs of clinically relevant thicknesses. Additionally, optimal bioprocessing conditions with respect to morphogen delivery and hydrodynamic loading were pursued for the production of bony and cartilaginous tissue from bone marrow derived mesenchymal stem cells. Finally, the optimal bioprocessing conditions were implemented within MSC laden microfluidic hydrogels to spatially engineer the matrix composition of a biphasic osteochondral graft through directed differentiation.

Formation of an intact, continuous and biological interface with proper zonal organization between mechanically dissimilar tissues is a key challenge in complex tissue engineering. The presence of a stable interface between soft and hard tissues is important. In particular, the presence of the osteochondral interface can prevent mechanical failure by reducing the shear stress across it. It also prevents vascularization and subsequent mineralization of the uncalcified cartilage, thus maintaining the normal tissue function. In this study, we demonstrated that with the use of mesenchymal stem cells, the collagen scaffold and the microencapsulation technology, an osteochondral interface with a zone of calcified cartilage could be generated in vitro in 5 weeks. Specifically, by placing an undifferentiated mesenchymal stem cell-collagen gel between an upper cartilage-like part and a lower bone-like part, cells in the middle layer were able to remodel the collagen gel into an interface similar to that found in vivo. Hypertrophic chondrocytes populated this in vitro generated interface, secreting GAGs, collagen type II and X, and calcium phosphates. Vertically running collagen fibers were found in this interface as well. We also demonstrated the importance of culture medium together with an appropriate configuration for interface formation. In particular, only with the use of both the chondrogenic medium and the three-layer configuration could we generate the osteochondral interface in vitro. Finally we conducted a pilot animal study on the efficacy of cartilage repair using constructs with a pre-formed osteochondral interface and demonstrated that cartilage re-surfacing was successful in only one month. Hyaline-like cartilage with a continuous tidemark was regenerated. This observed phenomenon could be maintained up to 3 months. Results of this study contribute to the development of better cartilage repair in future. / published_or_final_version / Mechanical Engineering / Doctoral / Doctor of Philosophy

With Athletes and individuals developing osteoarthritis and chondral defects at younger ages, long term treatments are in high demand. Total knee replacements only last for 10-15 years, so younger individuals would need to have multiple knee replacements within their lifetime. Allograft transplantation has shown to last long term and have high success rates, but the lack of donors and the possibility of damaging other areas of the knee to obtain tissue grafts has become a large concern. Xenografts derived from porcine cartilage is cost effective and the supply is abundant. Two antigen removal processes were examined: a short term antigen removal process to maintain the mechanical stability of the tissue, and a long antigen removal process to minimize the risk of triggering an immune response. The antigen removal processes were compared, and the future precautions were determined to enhance the probability of creating a viable osteochondral xenograft preparation technique.

Articular cartilage provides an almost frictionless surface for the articulating ends of the bone. Cartilage functions to lubricate and transmit compressive forces resulting from joint loading and impact. If the cartilage is damaged, through traumatic injury or disease, it lacks the ability of self-repairing as the tissue lacks vascular system. If the injuries to articular cartilage are left untreated, they may progress to Osteoarthritis. Osteoarthritis, a degenerative disease, is one of the leading disabilities in the United States. Tissue engineering has the potential to regenerate healthy hyaline cartilage, which can alleviate pain and restore the functions of normal tissue. This study explores the production of engineered cartilage on top of composite calcium phosphate scaffold. The current research is related to a biphasic approach to cartilage tissue engineering — in which one layer supports to form subchondral bone (osteogenesis) and another supports cartilage formation (chondrogenesis). Chondrocyte and bone marrow-derived stem cell attachment to chitosan will be investigated for producing a bilayered construct for osteochondral repair. The main objectives of my research include the following: attachment and proliferation of human mesenchymal stem cells on chitosan calcium phosphate scaffolds, techniques to create a biphasic construct, the effect of coating chitosan calcium phosphate scaffolds with type I collagen and determining the ideal bead size for making chitosan calcium phosphate scaffolds.

The objective was to compare the effects of three corticosteroids at various equimolar concentrations on equine articular explant co-cultures in an inflammatory environment. Synovial and osteochondral explant co-cultures from 6 equine cadavers were exposed to IL-1β (10 ng/mL) and various concentrations (10-4, 10-7, or 10-10 M) of MPA, TA, IPA. Concentrations of PGE2, MMP-13, LDH, and GAG in media were determined at 48 and 96 hours. Results indicated wells with low concentrations of MPA (10-7 and 10-10 M at 48 and 96 hours), TA (10-7 M at 48 hours and 10-7 and 10-10 M at 48 and 96 hours), and IPA (10-10 M at 48 hours) had significantly less PGE2 than positive control samples. Groups with low concentrations (10-7 and 10-10 M) of MPA and TA had significantly less PGE2 than the highest concentration (10-4 M) at 48 hours. Significantly less MMP-13 was detected for all concentrations of MPA, TA, and IPA at 96 hours. The LDH assay results indicated cytotoxicity only for samples treated with IPA at 10-4 M at 48 and 96 hours. GAG was significantly lower for samples treated with TA 10-7 M at 48 hours and MPA 10-10 M at 96 hours versus positive controls. These findings suggest corticosteroids at low concentrations mitigated the inflammatory and catabolic effects of IL-1β to a greater extent than high concentrations. Effects of IPA and MPA were similar to TA at clinically relevant low equimolar concentrations. / Master of Science / Recent data suggest that isoflupredone acetate is commonly administered intra-articularly for treatment of joint disease (osteoarthritis) in horses. Although much data has been published regarding effects of other corticosteroids on cartilage, to our knowledge there have been no similar studies of the effects of isoflupredone acetate. With increased scrutiny from the general public and more stringent control of medication use by regulatory agencies, determination of information regarding such common intra-articular therapies is imperative. In addition, prior studies have only evaluated the effects of corticosteroids on cartilage, whereas other joint tissues (subchondral bone and synovium) have been shown to be important to the biological responses of joints. Therefore, to more closely simulate the natural joint environment, this study was conducted with a co-culture model incorporating synovial tissue, articular cartilage, and subchondral bone within an inflammatory environment (via stimulation with interleukin-1β). The effects of various concentrations of methylprednisolone acetate, triamcinolone acetonide, and isoflupredone acetate on joint tissues were determined via measurement of selected biomarkers. This study provided the first data regarding biological effects of IPA on joint tissues of horses, and the first comparison of such effects with those of other corticosteroids commonly used intra-articularly for the treatment of joint disease in horses.

Background: Fresh osteochondral allograft (OCA) transplantation represents a biologic restoration technique as an alternative treatment option for larger osteochondral lesions of the talus (OLT). The purpose of this study was to evaluate midterm outcomes after OCA transplantation for the treatment of OLT. Methods: Nineteen patients (20 ankles) received partial unipolar OCA transplant for symptomatic OLT between January 1998 and October 2014. The mean age was 34.7 years, and 53% were male. The average graft size was 3.8 cm2. All patients had a minimum follow-up of 2 years. Outcomes included the American Academy of Orthopaedic Surgeons Foot and Ankle Module (AAOS-FAM), the Olerud-Molander Ankle Score (OMAS), and pain and satisfaction questionnaires. Failure of OCA was defined as conversion to arthrodesis or revision OCA transplantation. Results: Five of 20 ankles (25%) required further surgery, of which 3 (5%) were considered OCA failures (2 arthrodesis and 1 OCA revision). The mean time to failure was 3.5 (range, 0.9 to 6.7) years. Survivorship was 88.7% at 5 years and 81.3% at 10 years. The median follow-up of the 17 patients with grafts in situ was 9.7 years. The mean OMAS improved significantly from 40 points preoperatively to 71 points postoperatively (P < .05; range, 5 to 55). The mean postoperative AAOS-FAM core score was 81.5 ± 15 (range, 40.5 to 96.6). Fifteen of 17 patients responded to follow-up questions regarding their ankle; 14 patients reported less pain and better function, and 13 patients were satisfied with the results of the procedure. Conclusion: Our study of midterm results after OCA transplantations showed that this procedure was a reasonable treatment option for large OLT. Level of Evidence: Level IV, case series.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13636号 / 論医博第2327号 / 新制||医||1074(附属図書館) / (主査)教授 川上 浩司, 教授 森本 尚樹, 教授 安達 泰治 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13068号 / 論医博第2123号 / 新制||医||1019(附属図書館) / 33219 / 京都大学大学院医学研究科医学専攻 / (主査)教授 妻木 範行, 教授 開 祐司, 教授 戸口田 淳也 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Background: Collagens of marine origin are applied increasingly as alternatives to mammalian collagens in tissue engineering. The aim of the present study was to develop a biphasic scaffold from exclusively marine collagens supporting both osteogenic and chondrogenic differentiation and to find a suitable setup for in vitro chondrogenic and osteogenic differentiation of human mesenchymal stroma cells (hMSC). Methods: Biphasic scaffolds from biomimetically mineralized salmon collagen and fibrillized jellyfish collagen were fabricated by joint freeze-drying and crosslinking. Different experiments were performed to analyze the influence of cell density and TGF-β on osteogenic differentiation of the cells in the scaffolds. Gene expression analysis and analysis of cartilage extracellular matrix components were performed and activity of alkaline phosphatase was determined. Furthermore, histological sections of differentiated cells in the biphasic scaffolds were analyzed. Results: Stable biphasic scaffolds from two different marine collagens were prepared. An in vitro setup for osteochondral differentiation was developed involving (1) different seeding densities in the phases; (2) additional application of alginate hydrogel in the chondral part; (3) pre-differentiation and sequential seeding of the scaffolds and (4) osteochondral medium. Spatially separated osteogenic and chondrogenic differentiation of hMSC was achieved in this setup, while osteochondral medium in combination with the biphasic scaffolds alone was not sufficient to reach this ambition. Conclusions: Biphasic, but monolithic scaffolds from exclusively marine collagens are suitable for the development of osteochondral constructs.