Development and assessment of modified-honeycomb-structure scaffold for bone tissue engineering

The potential of melt-electrowriting (MEW) to fabricate highly porous 3D composite scaffolds for bone tissue engineering (BTE) with antibacterial properties was explored. Zinc oxide (ZnO) nanoparticles (NPs) were loaded into biodegradable medical grade Polycaprolactone (PCL) loaded with 10 wt% hydroxyapatite (HA) NPs to create antibacterial constructs by the MEW technique. The constructs’ morphology, chemical, thermal and mechanical properties, osteoblasts cell viability and bacterial attachment were investigated. Our results demonstrate a unique homogeneous dispersion of ZnO-HA NPs in the outer shell of the fabricated composite fibres by MEW, leading to significant inhibition of bacterial attachment against microorganisms while retaining osteoiblast viability. This novel composite MEW 3D scaffolds yielded promising multifunctional constructs with high potential in BTE.

Three-dimensional scaffolds play important roles in scaffold guided tissue engineering because they provide critical functions as extra-cellular matrices onto which cells can attach, grow, and form new tissues. To design scaffolds for load bearing tissue replacement, researchers often needs to address multiple biological, mechanical and geometrical design constraints. Therefore a novel fabrication system, Precision Extrusion Deposition (PED), has been developed allowing for the precise control of the scaffold external and internal geometry, porosity, pore size and interconnectivity. The system consists of a screw driven heated mini-extruder mounted on a 3-axis positioning system. This allows for bulk agglomerates of thermoplastic material to be fabricated into structures of a specified geometry. A computer-aided tissue engineering approach (CATE), combined with the PED process, Polycaprolactone and composite Polycaprolactone/ Hydroxyapatite scaffolds for bone tissue engineering applications were fabricated. Characterizations through SEM, Micro-CT and mechanical testing demonstrate the viability of the PED process. The results show good mechanical property, structural integrity, controlled pore size, and pore interconnectivity. The biological compatibility of the fabricated scaffolds was proved through both In-vitro cell culture and In-vivo nude mice studies. This major accomplishments reported in this thesis are:· The development of a novel fabrication technology to manufacture load bearing tissue scaffolds with controlled and reproducible microarchitecture· Demonstrated the compatibility of the structures, through a variety of characterization techniques, with biological systems and the potential in Tissue Engineering applicationsThe PED technique offers a unique opportunity to study the influence of the microarchitecture upon cell proliferation and ECM generation allowing for the needed structural integrity, strength, and transport properties, for and an ideal micro-environment for cell and tissue in-growth and healing.

Currently available bone grafts present certain limitations such as donor-site morbidity for autografts and risk of disease transmission for allografts. These limitations suggest a need for alternative strategies. The use of polymer/ceramic composites as scaffolds for trabecular bone tissue engineering capitalizes on the benefits of both materials. Polymers are easily formed and shaped and add structural rigidity while calcium phosphates impart osteoconductivity and osteointegration. We have developed a biodegradable, microsphere-based scaffold for bone tissue engineering based on poly(lactide-co-glycolide)/calcium phosphate composite microspheres in which a nanocrystalline hydroxyapatite is synthesized within the forming microspheres.

In the past 20 years, mesoporous materials have been attracted great attention due to their significant feature of large surface area, ordered mesoporous structure, tunable pore size and volume, and well-defined surface property. They have many potential applications, such as catalysis, adsorption/separation, biomedicine, etc. [1]. Recently, the studies of the applications of mesoporous materials have been expanded into the field of biomaterials science. A new class of bioactive glass, referred to as mesoporous bioactive glass (MBG), was first developed in 2004. This material has a highly ordered mesopore channel structure with a pore size ranging from 5–20 nm [1]. Compared to non-mesopore bioactive glass (BG), MBG possesses a more optimal surface area, pore volume and improved in vitro apatite mineralization in simulated body fluids [1,2]. Vallet-Regi et al. has systematically investigated the in vitro apatite formation of different types of mesoporous materials, and they demonstrated that an apatite-like layer can be formed on the surfaces of Mobil Composition of Matters (MCM)-48, hexagonal mesoporous silica (SBA-15), phosphorous-doped MCM-41, bioglass-containing MCM-41 and ordered mesoporous MBG, allowing their use in biomedical engineering for tissue regeneration [2-4]. Chang et al. has found that MBG particles can be used for a bioactive drug-delivery system [5,6]. Our study has shown that MBG powders, when incorporated into a poly (lactide-co-glycolide) (PLGA) film, significantly enhance the apatite-mineralization ability and cell response of PLGA films. compared to BG [7]. These studies suggest that MBG is a very promising bioactive material with respect to bone regeneration. It is known that for bone defect repair, tissue engineering represents an optional method by creating three-dimensional (3D) porous scaffolds which will have more advantages than powders or granules as 3D scaffolds will provide an interconnected macroporous network to allow cell migration, nutrient delivery, bone ingrowth, and eventually vascularization [8]. For this reason, we try to apply MBG for bone tissue engineering by developing MBG scaffolds. However, one of the main disadvantages of MBG scaffolds is their low mechanical strength and high brittleness; the other issue is that they have very quick degradation, which leads to an unstable surface for bone cell growth limiting their applications. Silk fibroin, as a new family of native biomaterials, has been widely studied for bone and cartilage repair applications in the form of pure silk or its composite scaffolds [9-14]. Compared to traditional synthetic polymer materials, such as PLGA and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), the chief advantage of silk fibroin is its water-soluble nature, which eliminates the need for organic solvents, that tend to be highly cytotoxic in the process of scaffold preparation [15]. Other advantages of silk scaffolds are their excellent mechanical properties, controllable biodegradability and cytocompatibility [15-17]. However, for the purposes of bone tissue engineering, the osteoconductivity of pure silk scaffolds is suboptimal. It is expected that combining MBG with silk to produce MBG/silk composite scaffolds would greatly improve their physiochemical and osteogenic properties for bone tissue engineering application. Therefore, in this chapter, we will introduce the research development of MBG/silk scaffolds for bone tissue engineering.

A wide variety of composite scaffolds with unique geometry, porosity and pore size can be fabricated with versatile 3D printing techniques. In this work, we fabricated 3D-printed composite scaffolds of polycaprolactone (PCL) incorporating bioactive glass (BG) particles (13-93 and 13-93B3 compositions) by using fused deposition modeling (FDM). The scaffolds were modified with a "mussel-inspired surface coating" to regulate biological properties. The chemical and surface properties of scaffolds were analyzed by Fourier transform infrared spectroscopy (FTIR), contact angle and scanning electron microscopy (SEM). Polydopamine (PDA) surface-modified composite scaffolds exhibited attractive properties. Firstly, after the surface modification, the adhesion of a composite coating based on gelatin incorporated with strontium-doped mesoporous bioactive glass (Sr-MBGNs/gelatin) was significantly improved. In addition, cell attachment and differentiation were promoted, and the antibacterial properties of the scaffolds were increased. Moreover, the bioactivity of these scaffolds was also significantly influenced: a hydroxyapatite layer formed on the scaffold surface after 3 days of immersion in SBF. Our results suggest that the promoting effect of PDA coating on PCL-BG scaffolds leads to improved scaffolds for bone tissue engineering.

Polylactic acid fibre-reinforced polycaprolactone scaffolds for bone tissue engineering

Electrospun fibers show great potential as scaffolds for bone tissue engineering due to their architectural biomimicry to the extracellular matrix (ECM). Cation substitution of strontium for calcium in hydroxyapatite (HAp) positively influences the mechanism of bone remodeling including enhancing bone regeneration and reducing bone resorption. The objective of this study was to attach strontium-doped HAp (SrHAp) to electrospun poly(ɛ-caprolactone) (PCL) fibers for creation of novel composite scaffolds that can not only mimic the architecture and composition of ECM but also affect bone remodeling favorably. We demonstrated for the first time the highly controlled SrHAp coatings on electrospun PCL fibers. We showed the reproducible manufacturing of composite fiber scaffolds with controllable thickness, composition, and morphology of SrHAp coatings. We further showed that the released strontium and calcium cations from coatings could reach effective concentrations within 1 day and endure more than 28 days. Additionally, the Young's modulus of the SrHAp-coated PCL fibers was up to around six times higher than that of raw fibers dependent on the coating thickness and composition. Together, this novel class of composite fiber scaffolds may hold great promise for bone regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 753-763, 2017.

Computer-aided tissue engineering approach was used to develop a novel Precision Extrusion Deposition (PED) process to directly fabricate Polycaprolactone (PCL) and composite PCL/Hydroxyapatite (PCL-HA) tissue scaffolds. The process optimization was carried out to fabricate both PCL and PCL-HA (25% concentration by weight of HA) with a controlled pore size and internal pore structure of the 0°/90° pattern. Two groups of scaffolds having 60 and 70% porosity and with pore sizes of 450 and 750 microns, respectively, were evaluated for their morphology and compressive properties using Scanning Electron Microscopy (SEM) and mechanical testing. The surface modification with plasma was conducted on PCL scaffold to increase the cellular attachment and proliferation. Our results suggested that inclusion of HA significantly increased the compressive modulus from 59 to 84 MPa for 60% porous scaffolds and from 30 to 76 MPa for 70% porous scaffolds. In vitro cell–scaffolds interaction study was carried out using primary fetal bovine osteoblasts to assess the feasibility of scaffolds for bone tissue engineering application. In addition, the results in surface hydrophilicity and roughness show that plasma surface modification can increase the hydrophilicity while introducing the nano-scale surface roughness on PCL surface. The cell proliferation and differentiation were calculated by Alamar Blue assay and by determining alkaline phosphatase activity. The osteoblasts were able to migrate and proliferate over the cultured time for both PCL as well as PCL-HA scaffolds. Our study demonstrated the viability of the PED process to the fabricate PCL and PCL-HA composite scaffolds having necessary mechanical property, structural integrity, controlled pore size and pore interconnectivity desired for bone tissue engineering.

Bone tissue engineering is one of the vital approaches used to tackle bone defects in the past decades. This approach is a risk-free alternative to conventional bone graft substitutes, such as autografts, allografts, and xenografts. Silicate bioceramics have improved mechanical properties and biological performances compared with phosphate-based ceramics. However, their brittleness limits their application in load-bearing sites. Therefore, silicate-based ceramics have been used in combination with several polymers in the form of a composite scaffold. This review article summarizes the influence of silicate ceramics on the mechanical, cytocompatibility, and bioactivity of polymeric-based scaffold for bone tissue engineering application.

In order to induce bone regeneration several natural and synthetic materials have been proposed. However, single-phase scaffolds present some insurmountable disadvantages such as poor mechanical strength or brittleness and too low or too high degradation rate. In order to overcome these drawbacks, composite systems can be an interesting and promising option. In the present work a novel hybrid porous scaffold for bone tissue engineering is proposed. Chitosan/Forsterite (Ch/FS) composite scaffolds were prepared by freeze-drying method using a chitosan/forsterite ratio of 90/10. The FS nanopowder (Mg2SiO4) is synthesized using a simple solgel based method. The FS composition was checked by XRD analysis. The macrostructure of the Ch/FS scaffolds were analyzed by SEM, the FS distribution within the chitosan matrix observed by EDS, the mechanical strength measured by compression test in PBS and the biocompatibility of the composite on human osteosarcoma cell line (MG-63) verified by MTT assay after 48 hours. The porosity appears interconnected and with a pore size ranging from 1 to 100 μm. The FS is overall distributed within the chitosan matrix. The compression strength of composite scaffolds increased with respect to the pure chitosan scaffolds of more than two times (from 0.8 to 1.9 KPa) and the composites did not show any toxicity effect on human osteosarcoma cells.

A series of highly interconnected porous poly(D,L-lactide acid) (PDLLA)/pyrite (Zi-Ran-Tong, FeS2) scaffold containing 5–20% of pyrite was fabricated by particle leaching combined with the thermal-induced phase separation method. Pyrite (FeS2, named as Zi-Ran-Tong in Chinese medicine), as a traditional Chinese medicine, has been used in the Chinese population to treat bone diseases and to promote bone healing. The mechanical properties of the PDLLA scaffold were significantly enhanced after the addition of pyrite. The osteoblastic ROS17/2.8 cell line was used and seeded on the PDLLA/pyrite scaffold to study its potential to support the growth of osteoblastic cells and to estimate the optimal dose of pyrite for bone tissue engineering. The effects of pyrite on cell proliferation and differentiation were evaluated by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide and alkaline phosphatase activity assay. The cells on the porous composite scaffold formed a continuous layer on the outer and inner surface observed by scanning electron microscopy and fluorescence microscope. The results strongly suggested that the PDLLA/pyrite composite scaffold could stimulate the growth of ROS17/2.8 cells in vitro and it could be potentially used as a scaffold for bone tissue engineering.

Heat-treated polyacrylonitrile (HT-PAN), also referred to as black orlon (BO), is a promising carbon-based material used for applications in tissue engineering and regenerative medicine. To the best of our knowledge, no such complex bone morphology-mimicking three-dimensional (3D) BO structure has been reported to date. We report that BO can be easily made into 3D cryogel scaffolds with porous structures, using succinonitrile as a porogen. The cryogels possess a porous morphology, similar to bone tissue. The prepared scaffolds showed strong osteoconductive activity, providing excellent support for the adhesion, proliferation, and mitochondrial activity of human bone-derived cells. This effect was more apparent in scaffolds prepared from a matrix with a higher content of PAN (i.e., 10% rather than 5%). The scaffolds with 10% of PAN also showed enhanced mechanical properties, as revealed by higher compressive modulus and higher compressive strength. Therefore, these scaffolds have a robust potential for use in bone tissue engineering.

Fabrication of a three-dimensional (3D) scaffold with increased mechanical strength may be an essential requirement for more advanced bone tissue engineering scaffolds. Various material- and chemical-based approaches have been explored to enhance the mechanical properties of engineered bone tissue scaffolds. In this study, the effects of pore architecture and stacking direction on the mechanical and cell proliferation properties of a scaffold were investigated. The 3D scaffold was prepared using solid freeform fabrication technology with a multihead deposition system. Various types of scaffolds with different pore architectures (lattice, stagger, and triangle types) and stacking directions (horizontal and vertical directions) were fabricated with a blend of polycaprolactone and poly lactic-co-glycolic acid. In compression tests, the triangle-type scaffold was the strongest among the experimental groups. Stacking direction affected the mechanical properties of scaffolds. An in vitro cell counting kit-8 assay showed no significant differences in optical density depending on the different pore architectures and stacking directions. In conclusion, mechanical properties of scaffolds can be enhanced by controlling pore architecture and stacking direction.

Preparation and characterization of nano-sized hydroxyapatite/alginate/chitosan composite scaffolds for bone tissue engineering.

The synergetic effect of bioactive ceramic and nanoclay on the properties of chitosan–gelatin/nanohydroxyapatite–montmorillonite scaffold for bone tissue engineering