Yoke Chin Chai

The effect of pore geometry on the in vitro biological behavior of human periosteum-derived cells seeded on selective laser-melted Ti6Al4V bone scaffolds

The specific aim of this study was to gain insight into the influence of scaffold pore size, pore shape and permeability on the in vitro proliferation and differentiation of three-dimensional (3-D) human periosteum-derived cell (hPDC) cultures. Selective laser melting (SLM) was used to produce six distinct designed geometries of Ti6Al4V scaffolds in three different pore shapes (triangular, hexagonal and rectangular) and two different pore sizes (500 μm and 1000 μm). All scaffolds were characterized by means of two-dimensional optical microscopy, 3-D microfocus X-ray computed tomography (micro-CT) image analysis, mechanical compression testing and computational fluid dynamical analysis. The results showed that SLM was capable of producing Ti6Al4V scaffolds with a broad range of morphological and mechanical properties. The in vitro study showed that scaffolds with a lower permeability gave rise to a significantly higher number of cells attached to the scaffolds after seeding. Qualitative analysis by means of live/dead staining and scanning electron micrography showed a circular cell growth pattern which was independent of the pore size and shape. This resulted in pore occlusion which was found to be the highest on scaffolds with 500 μm hexagonal pores. Interestingly, pore size but not pore shape was found to significantly influence the growth of hPDC on the scaffolds, whereas the differentiation of hPDC was dependent on both pore shape and pore size. The results showed that, for SLM-produced Ti6Al4V scaffolds with specific morphological and mechanical properties, a functional graded scaffold will contribute to enhanced cell seeding and at the same time can maintain nutrient transport throughout the whole scaffold during in vitro culturing by avoiding pore occlusion.

A calcium-induced signaling cascade leading to osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells

The response of osteoprogenitors to calcium (Ca(2+)) is of primary interest for both normal bone homeostasis and the clinical field of bone regeneration. The latter makes use of calcium phosphate-based bone void fillers to heal bone defects, but it is currently not known how Ca(2+) released from these ceramic materials influences cells in situ. Here, we have created an in vitro environment with high extracellular Ca(2+) concentration and investigated the response of human bone marrow-derived mesenchymal stromal cells (hMSCs) to it. Ca(2+) enhanced proliferation and morphological changes in hMSCs. Moreover, the expression of osteogenic genes is highly increased. A 3-fold up-regulation of BMP-2 is observed after only 6h and pharmaceutical interference with a number of proteins involved in Ca(2+) sensing showed that not the calcium sensing receptor, but rather type L voltage-gated calcium channels are involved in mediating the signaling pathway between extracellular Ca(2+) and BMP-2 expression. MEK1/2 activity is essential for the effect of Ca(2+) and using microarray analysis, we have identified c-Fos as an early Ca(2+) response gene. We have demonstrated that hMSC osteogenesis can be induced via extracellular Ca(2+), a simple and economic way of priming hMSCs for bone tissue engineering applications.

Current views on calcium phosphate osteogenicity and the translation into effective bone regeneration strategies

Calcium phosphate (CaP) has traditionally been used for the repair of bone defects because of its strong resemblance to the inorganic phase of bone matrix. Nowadays, a variety of natural or synthetic CaP-based biomaterials are produced and have been extensively used for dental and orthopaedic applications. This is justified by their biocompatibility, osteoconductivity and osteoinductivity (i.e. the intrinsic material property that initiates de novo bone formation), which are attributed to the chemical composition, surface topography, macro/microporosity and the dissolution kinetics. However, the exact molecular mechanism of action is unknown. This review paper first summarizes the most important aspects of bone biology in relation to CaP and the mechanisms of bone matrix mineralization. This is followed by the research findings on the effects of calcium (Ca²⁺) and phosphate (PO₄³⁻) ions on the migration, proliferation and differentiation of osteoblasts during in vivo bone formation and in vitro culture conditions. Further, the rationale of using CaP for bone regeneration is explained, focusing thereby specifically on the materials osteoinductive properties. Examples of different material forms and production techniques are given, with the emphasis on the state-of-the art in fine-tuning the physicochemical properties of CaP-based biomaterials for improved bone induction and the use of CaP as a delivery system for bone morphogenetic proteins. The use of computational models to simulate the CaP-driven osteogenesis is introduced as part of a bone tissue engineering strategy in order to facilitate the understanding of cell-material interactions and to gain further insight into the design and optimization of CaP-based bone reparative units. Finally, limitations and possible solutions related to current experimental and computational techniques are discussed.

Bone regeneration performance of surface-treated porous titanium

The large surface area of highly porous titanium structures produced by additive manufacturing can be modified using biofunctionalizing surface treatments to improve the bone regeneration performance of these otherwise bioinert biomaterials. In this longitudinal study, we applied and compared three types of biofunctionalizing surface treatments, namely acid-alkali (AcAl), alkali-acid-heat treatment (AlAcH), and anodizing-heat treatment (AnH). The effects of treatments on apatite forming ability, cell attachment, cell proliferation, osteogenic gene expression, bone regeneration, biomechanical stability, and bone-biomaterial contact were evaluated using apatite forming ability test, cell culture assays, and animal experiments. It was found that AcAl and AnH work through completely different routes. While AcAl improved the apatite forming ability of as-manufactured (AsM) specimens, it did not have any positive effect on cell attachment, cell proliferation, and osteogenic gene expression. In contrast, AnH did not improve the apatite forming ability of AsM specimens but showed significantly better cell attachment, cell proliferation, and expression of osteogenic markers. The performance of AlAcH in terms of apatite forming ability and cell response was in between both extremes of AnH and AsM. AcAl resulted in significantly larger volumes of newly formed bone within the pores of the scaffold as compared to AnH. Interestingly, larger volumes of regenerated bone did not translate into improved biomechanical stability as AnH exhibited significantly better biomechanical stability as compared to AcAl suggesting that the beneficial effects of cell-nanotopography modulations somehow surpassed the benefits of improved apatite forming ability. In conclusion, the applied surface treatments have considerable effects on apatite forming ability, cell attachment, cell proliferation, and bone ingrowth of the studied biomaterials. The relationship between these properties and the bone-implant biomechanics is, however, not trivial.

Mechanisms of ectopic bone formation by human osteoprogenitor cells on CaP biomaterial carriers

Stem cell-based strategies for bone regeneration, which use calcium phosphate (CaP)-based biomaterials in combination with developmentally relevant progenitor populations, have significant potential for clinical repair of skeletal defects. However, the exact mechanism of action and the stem cell-host-material interactions are still poorly understood. We studied if pre-conditioning of human periosteum-derived cells (hPDCs) in vitro could enhance, in combination with a CaP-based biomaterial carrier, ectopic bone formation in vivo. By culturing hPDCs in a biomimetic calcium (Ca(2+)) and phosphate (P(i)) enriched culture conditions, we observed an enhanced cell proliferation, decreased expression of mesenchymal stem cell (MSC) markers and upregulation of osteogenic genes including osterix, Runx2, osteocalcin, osteopontin, and BMP-2. However, the in vitro pre-conditioning protocols were non-predictive for in vivo ectopic bone formation. Surprisingly, culturing in the presence of Ca(2+) and P(i) supplements resulted in partial or complete abrogation of in vivo ectopic bone formation. Through histological, immunohistochemical and microfocus X-ray computed tomography (μCT) analysis of the explants, we found that in situ proliferation, collagen matrix deposition and the mediation of osteoclastic activity by hPDCs are associated to their ectopic bone forming capacity. These data were validated by the multivariate analysis and partial least square regression modelling confirming the non-predictability of in vitro parameters on in vivo ectopic bone formation. Our series of experiments provided further insights on the stem cell-host-material interactions that govern in vivo ectopic bone induction driven by hPDCs on CaP-based biomaterials.

Ectopic bone formation by 3D porous calcium phosphate-Ti6Al4V hybrids produced by perfusion electrodeposition.

Successful clinical repair of non-healing skeletal defects requires the use of bone substitutes with robust bone inductivity and excellent biomechanical stability. Thus, three-dimensionally functionalised porous calcium phosphate-Ti6Al4V (CaP-Ti) hybrids were produced by perfusion electrodeposition, and the in vitro and in vivo biological performances were evaluated using human periosteum derived cells (hPDCs). By applying various current densities at the optimised deposition conditions, CaP coatings with sub-micrometer to nano-scale porous crystalline structures and different ion dissolution kinetics were deposited on the porous Ti6Al4V scaffolds. These distinctive physicochemical properties caused a significant impact on in vitro proliferation, osteogenic differentiation, and matrix mineralisation of hPDCs. This includes a potential role of hPDCs in mediating osteoclastogenesis for the resorption of CaP coatings, as indicated by a significant down-regulation of osteoprotegerin (OPG) gene expression and by the histological observation of abundant multi-nucleated giant cells near to the coatings. By subcutaneous implantation, the produced hybrids induced ectopic bone formation, which was highly dependent on the physicochemical properties of the CaP coating (including the Ca(2+) dissolution kinetics and coating surface topography), in a cell density-dependent manner. This study provided further insight on stem cell-CaP biomaterial interactions, and the feasibility to produced bone reparative units that are predictively osteoinductive in vivo by perfusion electrodeposition technology.

Perfusion electrodeposition of calcium phosphate on additive manufactured titanium scaffolds for bone engineering.

A perfusion electrodeposition (P-ELD) system was reported to functionalize additive manufactured Ti6Al4V scaffolds with a calcium phosphate (CaP) coating in a controlled and reproducible manner. The effects and interactions of four main process parameters - current density (I), deposition time (t), flow rate (f) and process temperature (T) - on the properties of the CaP coating were investigated. The results showed a direct relation between the parameters and the deposited CaP mass, with a significant effect for t (P=0.001) and t-f interaction (P=0.019). Computational fluid dynamic analysis showed a relatively low electrolyte velocity within the struts and a high velocity in the open areas within the P-ELD chamber, which were not influenced by a change in f. This is beneficial for promoting a controlled CaP deposition and hydrogen gas removal. Optimization studies showed that a minimum t of 6 h was needed to obtain complete coating of the scaffold regardless of I, and the thickness was increased by increasing I and t. Energy-dispersive X-ray and X-ray diffraction analysis confirmed the deposition of highly crystalline synthetic carbonated hydroxyapatite under all conditions (Ca/P ratio=1.41). High cell viability and cell-material interactions were demonstrated by in vitro culture of human periosteum derived cells on coated scaffolds. This study showed that P-ELD provides a technological tool to functionalize complex scaffold structures with a biocompatible CaP layer that has controlled and reproducible physicochemical properties suitable for bone engineering.

In vitro cell-biological performance and structural characterization of selective laser sintered and plasma surface functionalized polycaprolactone scaffolds for bone regeneration

In the present study a structural characterization and in vitro cell-biological evaluation was performed on polycaprolactone (PCL) scaffolds that were produced by the additive manufacturing technique selective laser sintering (SLS), followed by a plasma-based surface modification technique, either non-thermal oxygen plasma or double protein coating, to functionalize the PCL scaffold surfaces. In the first part of this study pore morphology by means of 2D optical microscopy, surface chemistry by means of hydrophilicity measurement and X-ray photoelectron spectroscopy, strut surface roughness by means of 3D micro-computed tomography (CT) imaging and scaffold mechanical properties by means of compression testing were evaluated before and after the surface modifications. The results showed that both surface modifications increased the PCL scaffold hydrophilicity without altering the morphological and mechanical properties. In the second part of this study the in vitro cell proliferation and differentiation of human osteoprogenitor cells, over 14 days of culture in osteogenic and growth medium were investigated. The O2 plasma modification gave rise to a significant lower in vitro cell proliferation compared to the untreated and double protein coated scaffolds. Furthermore the double protein coating increased in vitro cell metabolic activity and cell differentiation compared to the untreated and O2 plasma PCL scaffolds when OM was used.

Gelatin functionalised porous titanium alloy implants for orthopaedic applications.

In the present work, we studied the immobilisation of the biopolymer gelatin onto the surface of three dimensional (3D) regular Ti6Al4V porous implants to improve their surface bio-activity. The successful immobilisation of the gelatin coating was made possible by a polydopamine interlayer, a polymer coating inspired by the adhesive nature of mussels. The presence of both coatings was first optimised on two dimensional titanium (2D Ti) substrates and confirmed by different techniques including X-ray photelectron spectroscopy, contact angle measurements, atomic force microscopy and fluorescence microscopy. Results showed homogeneous coatings that are stable for at least 24h in phosphate buffer at 37°C. In a next step, the coating procedure was successfully transferred to 3D Ti6Al4V porous implants, which indicates the versatility of the applied coating procedure with regard to complex surface morphologies. Furthermore, the bio-activity of these stable gelatin coatings was enhanced by applying a third and final coating using the cell-attractive protein fibronectin. The reproducible immobilisation process allowed for a controlled biomolecule presentation to the surrounding tissue. This newly developed coating procedure outperformed the previously reported silanisation procedure for immobilising gelatin. In vitro cell adhesion and culture studies with human periosteum-derived cells showed that the investigated coatings did not compromise the biocompatible nature of Ti6Al4V porous implants, but no distinct biological differences between the coatings were found.

Combining microCT-based characterization with empirical modelling as a robust screening approach for the design of optimized CaP-containing scaffolds for progenitor cell-mediated bone formation.

UNLABELLED Biomaterials are a key ingredient to the success of bone tissue engineering (TE), which focuses on the healing of bone defects by combining scaffolds with cells and/or growth factors. Due to the widely variable material characteristics and patient-specificities, however, current bone TE strategies still suffer from low repeatability and lack of robustness, which hamper clinical translation. Hence, optimal TE construct (i.e. cells and scaffold) characteristics are still under debate. This study aimed to reduce the material-specific variability for cell-based construct design, avoiding trial-and-error, by combining microCT characterization and empirical modelling as an innovative and robust screening approach. Via microCT characterization we have built a quantitative construct library of morphological and compositional properties of six CE approved CaP-based scaffolds (CopiOs®, BioOss™, Integra Mozaik™, chronOS Vivify, MBCP™ and ReproBone™), and of their bone forming capacity and in vivo scaffold degradation when combined with human periosteal derived cells (hPDCs). The empirical model, based on the construct library, allowed identification of the construct characteristics driving optimized bone formation, i.e. (a) the percentage of β-TCP and dibasic calcium phosphate, (b) the concavity of the CaP structure, (c) the average CaP structure thickness and (d) the seeded cell amount (taking into account the seeding efficiency). Additionally, the model allowed to quantitatively predict the bone forming response of different hPDC-CaP scaffold combinations, thus providing input for a more robust design of optimized constructs and avoiding trial-and error. This could improve and facilitate clinical translation. STATEMENT OF SIGNIFICANCE Biomaterials that support regenerative processes are a key ingredient for successful bone tissue engineering (TE). However, the optimal scaffold structure is still under debate. In this study, we have provided a useful innovative approach for robust screening of potential biomaterials or constructs (i.e. scaffolds seeded with cells and/or growth factors) by combining microCT characterization with empirical modelling. This novel approach leads to a better insight in the scaffold parameters influencing progenitor cell-mediated bone formation. Additionally, it serves as input for more controlled and robust design of optimized CaP-containing bone TE scaffolds. Hence, this novel approach could improve and facilitate clinical translation.