ETD

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Tesi etd-02272012-125641


Tipo di tesi
Tesi di dottorato di ricerca
Autore
VERONESI, FRANCESCA
URN
etd-02272012-125641
Titolo
Polymeric and ceramic biomaterials in bone regeneration
Settore scientifico disciplinare
CHIM/05
Corso di studi
BIOMATERIALI
Relatori
tutor Prof. Giardino, Roberto
tutor Dott.ssa Fini, Milena
Parole chiave
  • polycaprolactone in bone tissue engineering
  • in vitro and in vivo biocompatibility evaluations
  • hydroxyapatite
  • extracellular matrix-like scaffold for bone tissue
  • composite scaffold
  • bone tissue engineering
Data inizio appello
23/03/2012
Consultabilità
Completa
Riassunto
Bone is a dynamic, vascular, connective tissue, that changes throughout life, characterized by different types of cells (osteoblasts, osteocytes, osteoclasts and osteoprogenitor cells) that become embedded in their own extracellular matrix (ECM). Bone is a highly specialized tissue with a complex, hierarchical structure over multiple levels. Its ability to adapt its mass and morphology to functional demands, to repair itself without leaving a scar, and to rapidly mobilize mineral stores on metabolic demand, makes bone to be considered the ultimate “smart” material and a dynamic example of “form follows function” in biological systems. The principal role of the bone is to provide structural support for the body but it also serves as the body’s mineral reservoir and producer of blood cells. Bone is composed of ECM characterized by an organic phase, composed by collagenous (collagen I, III, IV and V) and non collagenous (mainly osteocalcin, osteonectin, sialoproteins, proteoglycans, osteopontin, fibronectin, Growth Factors and bone morphogenetic proteins) proteins, reinforced with inorganic component, characterized by calcium phosphate hydroxyapatite [(Ca10(PO4)6(OH)2)] crystals (HA, TCP) with a Ca:P ratio of 5:3, sodium (Na), magnesium (Mg) and potassium (K). Bone tissue is arranged in two macroarchitectural forms-cortical and cancellous- which are employed in various proportions and geometries to form the individual bones of the body. Bone has the ability to adapt to mechanical loads through continuous bone resorption and bone formation that, in a homeostatic equilibrium, are balanced and old bone is continuously replaced by new tissue. This ensures that the mechanical integrity of the bone is maintained but it causes no global changes in morphology (remodeling).
The continuity of a bone is disrupted, when a fracture or bone defect occurs, and the healing process can be developed on 4 phases: haematoma formation, soft callus formation, hard callus formation and remodeling phases. Bone regeneration is highly efficient and tightly regulated and the majority of fractures heals well under standard conservative or surgical therapy, but when complicated fractures and large bone defects have to be bridged, the healing process fails in many cases and non-union or pseudoarthrosis can occur. Nearly 5-10% of all fractures is associated with impaired healing, resulting in delayed union or non-union.
Extended bone defects, following trauma or cancer resection, non-unions of fractures and loss of substance may require more sophisticated treatments, such as bone grafting procedures, segmental bone transport, distraction osteogenesis and scaffolds, that are applied for reconstruction.
A tissue graft is a medical procedure in which tissue from a donor is used to replace missing or damaged tissue on a patient and can be divided into 3 major categories according to the genetic relationship between the donor and the recipient: autogenous tissue graft (a tissue graft from one site to another within the same individual); allogenous tissue graft (a tissue graft between individuals of the same species); xenogenous tissue graft (donor and recipient are individuals from different species). Among them, the first is defined as the 'gold standard' for regeneration, is a safe solution for compatibility and the absence of immune response, and it is considered to be the most suitable material, because the graft has osteogenic (marrow-derived osteoblastic cells as well as preosteoblastic precursor cells), osteoinductive (noncollagenous bone matrix proteins, including growth factors) and osteoconductive (bone mineral and collagen) properties.
Nowadays, bone graft materials with completely different origins are commercially available for many applications throughout the human body. They are variable in their composition, their mechanism of action and, therefore, their indication, but problems related to the availability of graft material, donor-site morbidity, immunogenicity and biomechanical integrity represent some limitations of bone grafts and clinical success.
Bone substitute materials are generally considered to be a highly important alternative to bone grafting. Due to the numerous disadvantages present in all kinds of grafts, there is a rationale for the designing and developing of artificial supports (scaffolds) for tissue engineering applications and their demand is growing steadily. Tissue engineering is a promising strategy to overcome the need to restore or regenerate tissues and proposes an alternative to tissue grafting with the use of artificial designed scaffolds or implants, fabricated using various materials (biomaterials). Scaffolds are tissue engineered product biomaterials, which function as an ECM, defined as degradable materials used, through implantation or injection, in a host for the purpose of stimulating tissue engineering or cell therapy process. Nowadays, natural (for example, collagen, hyaluronic acid, fibrin, silk) and synthetic (metals, ceramics, polymers and composites) materials, are used clinically for implants and medical devices in many medical areas.
Biocompatibility is a fundamental requirement of scaffolds and is defined as “the ability of a material to perform with an appropriate host response in a specific application”.
Biocompatibility ensures the absence of toxicity, teratogenicity or carcinogenicity and the lack of antigenicity guarantees the avoidance of pro-inflammatory and immunogenic reactions. All such requirements serve as a basis for effective long-term tolerance and such criteria are mainly fulfilled by available synthetic materials. It can be assessed by in vitro and in vivo studies and the results of these evaluations help to provide an objective picture of the associated composite biocompatibility. Generally, the sequence of evaluations increases in complexity and scope over time, from basic in vitro cell culture cytotoxicity evaluation to in vivo large animal anatomically relevant evaluations of biocompatibility and biofunctionality.
Moreover, a scaffold, for bone tissue engineering, must be osteoinductive (to stimulate bone formation through the recruitment and differentiation of pluripotent stromal cells into osteoblasts), osteoconductive (for the bony ingrowth from local osseous tissue onto surface), osseointegrated (to achieve stable direct anchorage and contact between bone and scaffold surface), and bioactive (a phenomenon by which a biomaterial elicits or modulates biological activity).
Besides biological characteristics, the scaffolds may be bioactive because of the appropriate chemical and physical (topography and microstructure) properties, which are very important for processing and performance, as they are directly related to the mechanical and biological properties of the scaffolds.
For bone tissue engineering applications, an important aspect, in the design of scaffolds, is their required 3D porous architecture because porosity is important for the development of new tissue. A scaffold should provide an open porous network to assure an uniform cell distribution and tissue regeneration, an appropriate transport of soluble signaling molecules, as well as nutrients and oxygen, and metabolic waste removal, and this aim is reached by the combination of different pore sizes (micro- and macro-pores) and their interconnectivity.
Synthetic scaffolds, due to their non-biological origin, have no cross contamination risk, but there is the stringent need to study their biocompatibility and clinical behaviour. Synthetic polymers, such as polylactic acid (PLA), polyglycolic acid (PGA) and poly ε-caprolactone (PCL), are largely used in orthopaedic applications because they can exhibit predictable and reproducible mechanical and physical properties, such as tensile strength, elastic modulus and degradation rate, by fine controlling their chemical synthesis and processing. Compared to metal and ceramic, they are easy to be fabricated into various shapes, with easy secondary processability and reasonable cost. Synthetic polymers have the advantage to be tailored to give a wide range of properties and more predictable uniformity, and they are generally free from the problems of immunogenicity, which can be a concern for naturally derived materials. PCL is a semicrystalline polymer that degrades at a much lower rate than the other, indeed the homopolymer has a degradation time of the order of two to three years. PCL is biocompatible and has a propensity to form blends with a wide variety of polymers. For this reason, PCL is used for developing long-term implantable materials.
Among synthetic materials, in recent years, studies of scaffold composite materials have been performed as to successfully reproduce the microenvironment required to support and improve the molecular interactions which occur within tissues, between cells and within the mineralized extracellular matrix. To date, polymer matrices reinforced by ceramic fillers such as hydroxyapatite (PCL/HA) represent promising composite materials, able to mimic the collagen/hydroxyapatite micro/macromorphology of “native bone material”. The main benefit of using composite scaffolds is the ability to tailor their properties as per need, providing significant advantage over homogeneous materials. It has been demonstrated that the incorporation of biocompatible insoluble signals, such as hydroxyapatite (HA), into polymer matrix, promotes cell activity, due to the well-known osteoconductive and osteogenic potential, as well as an improvement of mechanical properties for its reinforcement action. The idea of combining bioactive ceramics and degradable polymers to produce 3D scaffolds with high porosity is a promising strategy for the design and development of composite system for hard tissue regeneration materials.
In this thesis, for the regeneration of bone defects, the “in situ tissue regeneration” approach (in vivo implant of biological scaffolds in order to stimulate tissue regeneration) was taken into consideration and two scaffolds were investigated: a PCL and a composite PCL/HA scaffolds.
PCL and PCL/HA were manufactured by researchers from the Interdisciplinary Research Centre on Biomaterials, CRIB and the Institute of Composite and Biomedical Materials, National Research Council, respectively, from the University of Naples Federico II.
The scaffolds were fabricated with two different methods: (1) gas foaming (GF) and selective polymer extraction (PE) from co-continuous blends, for PCL scaffold; (2) phase inversion/salt leaching for the composite PCL/HA 13% scaffold. These research groups have also performed chemical-physical tests on the scaffolds with microstructural (macro- and micro- porosity, pore size and pore size distribution) and mechanical (compressive mechanical properties) studies, wettability and in vitro degradation mechanisms and kinetics tests. They found that a highly controlled porosity, characterized by a µ-bimodal distribution of pore size, was achieved: an open macroporous network that assures a uniform cell distribution and tissue regeneration, appeared interconnected by micropores that provide an efficient transport of soluble signaling molecules, as well as nutrients and oxygen, and metabolic waste removal.
The aim of the thesis was to comparatively investigate the in vitro and in vivo behaviour of these two kinds of scaffolds. During my PhD, at the Preclinical and Surgical Studies laboratory of Rizzoli Orthopaedic Institute of Bologna, microtomographic, in vitro and in vivo evaluations were performed, to assess the two scaffolds in terms of porosity, cell colonization of the scaffolds, biocompatibility, bioactivity, biofunctionality, biodegradability, and new bone formation.
More precisely, before in vitro and in vivo studies, tests were conducted with a new microtomographic approach (Micro-CT, SkyScan 1172) to evaluate the overall porosity, macroporosity and microporosity of these new scaffolds, at rest and after three different compression stages (1 mm, 3mm and at maximum compression of 222N).
Moreover, a visualization and a quantification of the distribution of cells through the entire PCL scaffold were performed with Micro-CT technique, after the seeding of cells (MG63) on the scaffold, because in literature, porous scaffolds with novel porous architecture should promote and guide the in vitro and in vivo cell adhesion, proliferation and 3D colonization.
Microtomography is based on the same principles of a normal X-ray tomography used in clinical medicine but avails itself of a microscopic level resolution. Micro-CT is extremely helpful when it comes to characterize devices in the preimplant phase and evaluating possible deformations and/or degradations after the explantation phase, moreover it is useful for the analysis of both tissue engineering scaffolds and bone tissue regeneration after the preclinical application of innovative biomaterials and biocomposites. Micro-CT shows some advantages over the classical histology, such as the maintenance of the integrity of the analyzed specimen, the lower acquisition time and the higher spatial resolution and number of analyzed sections than histology and the possibility to perform 2D and 3D studies.
Subsequently, in vitro and in vivo tests, for the evaluations of cytotoxicity, biocompatibility, bioactivity, biofunctionality and biodegradability of the scaffolds, were carried out with osteoblastic-like cells (MG63), seeded at a concentration of 1X10^5 cells/ml on both scaffolds in 24-wells plates, and by implantation of the two scaffolds, without cells, in rabbit bone defects.
In in vitro tests, early evaluation of cell adhesion (at 4 hours), morphology and lactate dehydrogenase (LDH) production (both at 24 hours) were tested for the assessment of cytotoxicity; Bone-specific alkaline phosphatase (BAP) activity, Osteocalcin (OC) measurements, Type I pro-collagen (CICP) production, and Transforming growth factor β1 (TGF-β1), Tumor necrosis factor α (TNF-α) and Interleukin-6 (IL-6) release were assessed at 7 and 14 days. Moreover, at 24 hours, 7 and 14 days, cell proliferation and viability were also evaluated. For all the parameters tested, as control, the same amount of cells (1X10^5 cells/ml) was seeded in empty wells of every plates, without scaffolds.
In the subsequent in vivo study, according to the Law by Decree 116, 1992, 14 skeletally mature, adult New Zealand rabbits were used. Under general anaesthesia, bilateral confined cancellous defects were drilled in both limbs, obtaining a defect with 5 mm in diameter and 10 mm in depth. The left defect was treated with PCL/HA scaffold, while the right with PCL. Seven rabbits were euthanized after 4 weeks and the others after 12 weeks. The bone defect healing and the new bone growth were calculated with histology, static (% of bone healing rate-BHR, bone area inside-BAr/TAr.int and around-BAr/TAr the implanted scaffolds) and dynamic (mineral apposition rate-MAR and bone forming rate-BFR) histomorphometric parameters, at the end of experimental times, for the in vivo biocompatibility and biofunctionality assessment.
The Micro-CT results about the porosity (overall porosity, macroporosity and microporosity) of the two scaffolds, before and after compression, revealed that PCL/HA scaffold showed higher overall porosity and macroporosity in comparison to PCL and that both scaffolds possessed a good interconnectivity of nearly 99%. A slight improvement in porosity in the first phase of compression due to an initial expansion of pores and then a progressive loss, an improvement in the microporosity and a reduction in overall porosity, after the compression step, were observed for both scaffolds. Moreover, the scaffolds possessed good mechanical properties during the compression stages.
In this thesis a new method to detect cells, within the PCL scaffold, was established. The nominal resolution used here (2.5 µm of pixel size) allowed the presence of cells to be detected, even in Micro-CT sections far from the surface. Thus, this characteristic was used to ensure the presence of cell colonization even in deeper layers of the scaffold. The method described is good for 3D quantification of cell distribution and Micro-CT allows the results obtained by qualitative surface analysis to be combined and gives a more complete understanding of cell colonization. A good cell infiltration capacity was observed within the PCL scaffold according to the topological properties of its pore structure. The results of cell-scaffold interaction study demonstrated that high cell seeding efficiency and 3D colonization may be achieved by fine tuning the topological characteristics of the scaffold. In particular, the μ-bimodal scaffolds, used in the thesis, promoted selective 3D cell colonization into the macroporosity, and consequently ensured the presence of a separate porous network for fluid transport.
Results of the in vitro study showed that both PCL and PCL/HA possessed a good in vitro biocompatibility. The cells, grown on both scaffolds, appeared spindly, well stained with a normal morphology as cells of control, with well defined cellular membranes and without lysis or reduced proliferation, in comparison to the control. The materials did not affect cell proliferation and did not evoke cytotoxic or inflammatory effects as explained by the TNF-α, IL-6, LDH production by the materials, that did not differ from control. In addition, cells cultured on the scaffolds showed a good biofunctionality and bioactivity, as demonstrated by the amount of ECM components, at both experimental times. More precisely, proliferation increased between 24 hours and 7 days and was maintained between 7 and 14 days. In particular, at 7 days PCL/HA cells showed a lower proliferation than PCL and control cells, probably due to the starting of the differentiation process. Indeed, at 7 days, PCL/HA cells produced significantly higher level of BAP than PCL and control, and OC and CICP than PCL. At 14 days, BAP was produced significantly more by cells on PCL/HA in comparison to control. The production of OC increased between 7 and 14 days in all materials and control, in significantly manner, underlying its role as a late differentiation marker of osteoblasts, whereas the production of BAP and CICP did not show significant differences among experimental times. As about TGF-β, TNF-α and IL-6 synthesis, no differences were observed nor between scaffolds and control at both experimental times, nor between 7 and 14 days. Moreover, the production of TNF-α, was significantly lower, on PCL/HA, at 14 days in comparison to 7 days.
All in vitro results underlined that PCL/HA revealed better biocompatibility and bioactivity when compared to PCL and control.
In vivo results showed that the initial area of the defect was reduced of about 40-50%, after 4 weeks, without significant differences between the two materials and between the two experimental times, indicating that within 4 weeks the healing process started faster and then proceeded slower between 4 and 12 weeks. The formation of new bone trabeculae outside the implanted materials (BAr/TAr) and inside the scaffolds (BAr/TAr.int), and MAR and BFR parameters showed significantly higher values for PCL/HA than PCL at both experimental times. Moreover, the new bone trabeculae started to penetrate the porosity of the two scaffolds slowly, at 4 weeks till 12 weeks, when the new bone was present inside the porosity, also in the central portion of the both scaffolds, especially for PCL/HA. Due to the low degradation rate of PCL material, scaffolds were present also at 12 weeks and they were only partially degraded. For this reason, in the future, further research will be mandatory with longer experimental times for the in vivo studies. Even if further studies will be performed and refined, the composite scaffold PCL/HA showed a better behaviour in the regeneration of bone defects.
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