Combinatorial effects of growth factors in chondrogenic differentiation of human periosteal cells: From development to tissue engineering

The repair and regeneration of skeletal tissue damage has gained much interest. The emerging field of regenerative medicine and tissue engineering (TE) is expected to reduce the bulk of this burden by creating constructs capable of regenerating damaged tissues in a faster, safer and reproducible manner. A possible to way to manufacture successful TE constructs is through the combination of concepts of embryonic tissue development with those from process design, a strategy referred as developmental engineering. In this case, the key to success is the ability to differentiate progenitor cells in a manner which mimic the process of in vivo tissue formation during development. This can be achieved by creating biomimetic in vitro culture conditions capable of activating cellular pathways leading to proper tissue morphogenesis in vivo. When considering the development of long bones, a cartilaginous template formed by aggregation and differentiation of mesenchymal stem cells precedes new bone formation. Later, the appearance of permanent articular cartilage at the ends of the bones is essential to allow locomotion of the skeleton. Therefore, this PhD dissertation aimed at achieving advanced control over the differentiation status of human periosteum-derived cells (hPDCs) by following developmental instructions, leading to the generation of either stable of transient cartilage. In the first part of this PhD dissertation, a Design of Experiments methodology was employed to screen the effect of multiple growth factors (BMP2; BMP6; GDF5; TGF-β1; FGF2) in chondrogenic differentiation of hPDCs in vitro. This strategy identified BMP2 as the strongest effector for chondrogenic differentiation of hPDCs, acting synergistically with BMP6 to enhance cartilage-like tissue formation and matrix mineralization. In line with other studies, it was found that FGF2 negatively contributes to GAG deposition and matrix mineralization, therefore limiting chondrogenic differentiation of hPDCs. When implanted ectopically in nude mice, µMasses differentiated for 7 days in chondrogenic medium supplemented with 100ng/mL BMP2; 100ng/mL BMP6; 0.1ng/mL TGFβ1; 0.2ng/mL FGF2 and 1ng/mL showed maintenance of the cartilaginous phenotype after 4 weeks in vivo. This was interpreted as a promising outcome, especially considering the short term chondrogenic induction in vitro. However, these results indicated that this GF cocktail may not be suitable for TE applications focusing on bone and osteochondral tissue repair. In the second part of this PhD dissertation, a different GF cocktail was used to generate not only cartilage but also bone tissue upon ectopic in vivo implantation. As BMP6 and FGF2 were negatively correlated to GAG deposition, mMasses of hPDCs were cultured in chondrogenic medium supplemented with high concentration of TGFb1, BMP2 and GDF5 and low concentration of BMP6 and FGF2. This combination generated osteochondral-like tissues in vitro that were able to form bone and cartilage ectopically in nude mice. When implanted into osteochondral defects in rats, these mMasses allowed subchondral bone and cartilage regeneration, with the newly formed cartilage tissue displaying GAG production, type-II collagen and lubricin expression. Noteworthy, implantation of TGF-b1 cultured μMasses resulted in fibrocartilage formation and absence of subchondral bone repair in the same osteochondral defect model, demonstrating the advantage of using advanced biomimetic medium formulations in skeletal tissue engineering. The aim of the last part of this PhD dissertation was to optimize the culture conditions towards the generation of constructs displaying a strong commitment to bone formation upon implantation. First, it was shown that the GF cocktail used to generate osteochondral tissues allowed consistent bone formation when combined with anchorage-independent culture conditions. However, this process required long periods of in vivo cartilage ECM remodeling (4-5 weeks) before bone formation. Subsequently, it was hypothesized that a more faithful recapitulation of endochondral differentiation prior to implantation could increase the speed of bone formation. By studying the sequential expression of TGF-β1, BMP2, GDF5, BMP6, FGF2 and VEGF in limb bud cells cultured in a mMass system, it was possible to create a developmental inspired sequential application of GFs that can generate cartilage tissue intermediates optimized for bone formation. This new protocol consists of 2 chondrogenic steps, the first comprising 10ng/mL TGF-β1, 100ng/mL BMP2, 100ng/mL GDF5, 1ng/mL BMP6 and 0.2ng/mL FGF2 and the second comprising 10ng/mL VEGF and 10ng/mL TGF-b1 in the presence of T4 and b-glycerolphosphate. By using fluorescent reporter murine PDCs, it was found that this protocol increased the number of hypertrophic chondrocytes, explaining at least partially the enhanced bone forming capacity after 2 weeks in vivo. In summary, this PhD dissertation revealed the potential use of hPDCs in the context of bone and cartilage TE and revealed the strength of using DoE methodologies for the design of biomimetic culture conditions.Chapter 1: General Limitations of Tissue Engineering and Aims of this PhD Dissertation 1 1.1 - General Limitations of Bone and Cartilage Tissue Engineering 1 1.2 - Main Aim and Objectives 4 1.3 - Specific Objectives 4 1.4 - References of Chapter 1 5 Chapter 2 - General Introduction 7 2.1 – A brief overview on bone and cartilage tissue engineering: the frame of this PhD dissertation 7 2.2 - Long bones: development and repair 8 2.2.1 – Endochondral bone development 8 2.2.2 – Growth plate: a model of endochondral bone formation 10 2.2.2.1 – TGF- signaling pathway in limb bud development 11 2.2.2.2 – FGF signaling pathway in limb bud development 12 2.2.3 – Bone fracture and the process of bone repair 13 2.2.3.1 – Failure of natural bone repair and clinical solutions 14 2.3 – Articular cartilage development and architecture 15 2.3.1 – GDF5: a determinant of articular cartilage differentiation and joint stability 17 2.3.2 – The osteochondral unit and the molecular aspects of osteochondral maintenance 18 2.3.3 – Osteochondral damage and clinical solutions 19 2.4 – Tissue Engineering for bone and cartilage regeneration: present and future 20 2.4.1 - Paradigm shift in tissue engineering: Developmental Engineering 22 2.4.2 – Bone periosteum as a source of progenitor cells for skeletal tissue engineering 23 2.4.3 – Cell-based scaffold-free strategies in Tissue Engineering 23 2.5 - References of chapter 2 25 Chapter 3 - Combinatorial analysis of growth factors reveals the contribution of bone morphogenetic proteins to chondrogenic differentiation of human periosteal cells 31 3.1 - Abstract 32 3.2 - Introduction 33 3.3 - Material and Methods 35 3.4 – Results 39 3.5 - Discussion 46 3.6 - Conclusion 50 3.7 - References of Chapter 3 50 Chapter 4 - Advancing osteochondral tissue engineering: TGF-β1, BMP2 and GDF5 drive ordered differentiation of periosteal cells resulting in stable cartilage and bone formation in vivo 55 4.1 - Abstract 56 4.2 - Introduction 57 4.3 - Material and Methods 59 4.4 – Results 62 4.5 - Discussion 72 4.6 - Conclusions 75 4.8 – Supplementary information 76 4.9 – References of chapter 4 77 Chapter 5 - Developmental inspired sequential application of growth factors to periosteal cells for efficient terminal chondrocyte differentiation and in vivo bone formation 81 5.1 - Abstract 82 5.2 - Introduction 83 5.3 - Material and Methods 84 5.4 - Results 88 5.5 - Discussion and Conclusion 97 5.6 – References of Chapter 5 101 Chapter 6 - General Discussion and Future Perspectives 103 6.1 – Optimization of cell culture conditions: a road towards in vitro biomimicry and developmental engineering 103 6.2 – Osteochondral tissue regeneration by micro-environment responsive scaffold-free osteochondral grafts 105 6.3 - Complete differentiation of chondrocytes in vitro: strategy for robust bone formation? 106 6.4 – Conclusions and Future Perspectives 107 6.5 – References of Chapter 6 108 Summary of the Research 109 Curriculum Vitae 111status: publishe