Current Views on Calcium Phosphate Osteogenicity and the Translation into Effective Bone Regeneration Strategies




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НазваниеCurrent Views on Calcium Phosphate Osteogenicity and the Translation into Effective Bone Regeneration Strategies
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Review Paper:


Current Views on Calcium Phosphate Osteogenicity and the Translation into Effective Bone Regeneration Strategies


Yoke Chin Chai1,3, Aurélie Carlier2,3, Johanna Bolander1,3, Scott J. Roberts1,3, Liesbet Geris3,4, Jan Schrooten3,5, Hans Van Oosterwyck2,3, Frank P. Luyten1,3


Affiliation:


1Laboratory for Skeletal Development and Joint Disorders, KU Leuven, O&N 1, Herestraat 49, PB 813, 3000 Leuven, Belgium.

2Biomechanics Section, KU Leuven, Celestijnenlaan 300 C, PB 2419, 3001 Leuven, Belgium.

3Prometheus, Division of Skeletal Tissue Engineering, KU Leuven O&N 1, Herestraat 49, PB 813, 3000 Leuven, Belgium.

4Biomechanics Research Unit, University of Liege, Chemin des Chevreuils 1 – BAT 52/3, 4000 Liege 1, Belgium.

5Department of Metallurgy and Materials Engineering, KU Leuven, Kasteelpark Arenberg 44, PB 2450, 3001 Leuven, Belgium.


Corresponding author:

Prof. Frank P. Luyten

Laboratory for Skeletal Development and Joint Disorders,

Division of Skeletal Tissue Engineering,

Katholieke Universiteit Leuven

O&N 1, Herestraat 49 – Box 7003

3000 Leuven, Belgium.

Tel: +32 16 342541

Fax: +32 16 342543

Email: frank.luyten@uz.kuleuven.ac.be


Abstract

Calcium phosphate has traditionally been used for the repair of bone defects due to 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 orthopedic 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-/micro- porosity and the dissolution kinetics. However, the exact molecular mechanism of action is unknown. This review paper first summarises the most important aspects of bone biology in relation to CaP and the mechanisms of bone matrix mineralisation. 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 material’s 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 delivery system for bone morphogenetic proteins (BMPs). 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 optimisation of CaP-based bone reparative units. Finally, limitations and possible solutions related to current experimental and computational techniques are discussed.


Keywords: calcium phosphate, bone tissue engineering, osteoinductivity, calcium ion, computational modelling


(1) Introduction

Bone is a dynamic, highly vascularised and mineralised tissue that has self-remodelling and healing capacities under normal physiological conditions, with bone loss due to disuse and upon injury. It provides structural support to the body for locomotion, serves as a protective cage for internal organs, and is a site for haematopoiesis and endocrine regulation. It also maintains the acid-base balance of blood, and serves as a storage for minerals [mainly calcium (Ca2+) and phosphate (PO43-)] and growth factors that are essential for vital physiological events, such as ions homeostasis and other intracellular signalling pathways.


In fact, bone is a biocomposite tissue consisting of an organic phase (mainly collagen type-1 fibres, ~20%) and an inorganic phase [mainly carbonated hydroxyapatite (Ca10(PO4)6(OH)2), ~60%] [1] (figure 1a) that are organised in a lamellar cylindrical osteon system (i.e. the compact bone) or present as irregular thin trabecular plates and struts (i.e. the spongy bone) [2]. This special organisation of collagen fibres and mineralised matrix (deposited by the bone forming cells, i.e. the osteoblasts) renders the bone tissue with relatively high elastic modulus and compressive strength, but low tensile and shear strength [3-4] (figure 1b). These mechanical properties are dependent on the anatomic location [5-6], and are among others influenced by the porosity and percentage of the mineral content within a bone tissue to suit a particular functionality (e.g. 80% mineral content within ossicles for sound transduction by vibration) [7]. In general, a higher mineral content increases the stiffness but decreases the toughness of the bone [8]. Recently, these stiffness and energy dissipation properties of bone were found to be attributed to calcium-mediated sacrificial bonds of a non-fibrillar organic matrix, which act as a “glue” to hold the mineralised fibrils together (through a hidden length mechanism) during bone deformation [9-10].


Indeed, calcium phosphate [11] plays a critical role in the mineralisation of collagen fibres and thus contributes to physiologically important bone tissue characteristics. Two theories of collagen fibre biomineralisation are reported: (a) direct nucleation of calcium phosphate crystals onto collagen fibrils [12], and (b) matrix vesicle (MV) mediated matrix mineralisation [13-14]. Direct nucleation involves the formation of stable mineral droplets comprised of calcium phosphate cluster-biopolymer complexes that bind to a distinct region on the collagen fibres and diffuse through the interior of the fibril where they solidify into an amorphous phase (figure 2a). This amorphous phase is then transformed into oriented apatite crystals directed by the collagen fibril arrangement. The second theory is based on the production of intracellular MV containing calcium phosphate crystals by osteoblasts. Three possible mechanisms for the initiation of MV mediated matrix mineralisation have been suggested [15] (figure 2b): (i) MV only regulates ion concentrations, leading to the formation of soluble molecular species which initiate mineral formation in collagen fibrils; (ii) MV regulates ion compositions leading to the formation of intravesicular apatite crystals, which leave the vesicle and initiate the mineralisation process; (iii) MV associate directly with collagen and cooperates to initiate matrix mineralisation.


These essential matrix mineralisation events are tightly regulated by several bone-related proteins and growth factors. For instance, alkaline phosphatase (ALP) is a periplasmic enzyme (membrane of cell and matrix vesicle) that hydrolyzes pyrophosphate (a mineralisation inhibitor), thereby providing phosphate ion (PO43-) to promote mineralisation [16-18]. PHOSPHO1 is another phosphatase highly expressed in bone [19], which is present within matrix vesicles (MV) and plays a role in the initiation of mineral formation [20]. Osteocalcin, osteonectin, osteopontin and bone sialoprotein are the four major non-collagenous bone proteins. Osteocalcin and osteonectin were reported to regulate the size and speed of crystal formation [21], bone sialoprotein was found to act as a crystal nucleator [22], whereas osteopontin influences the type of crystal formed [23-24]. On the other hand, mineralisation inhibitors such as decorin, a member of the small leucine-rich proteoglycans family, negatively interfere with mineralisation by modulating collagen assembly [25]. Another inhibitor, Matrix Gla Protein was associated with parathyroid hormone-mediated inhibition of osteoblast mineralisation [26]. Recently, bone-morphogenetic protein-2 (BMP-2), a potent osteoinductive growth factor, was found to be involved in the control of ALP expression and osteoblast mineralisation via a Wnt autocrine loop [27], as well as in the enhancement of PO43- transportation into cells for matrix mineralisation [28]. Fibroblast growth factor-2 (FGF-2), is another growth factor that was found to reduce the expression of genes associated with matrix mineralisation [29].


(2) The Effect of Calcium (Ca2+) and Phosphate (PO43-) On Osteoblastic Cell Behaviour and Its Applications

This section will describe primarily (pre)osteoblasts since these cells play a key role during osteogenesis. Notice that the extracellular calcium ion (Ca2+) can be bound to proteins (e.g. albumin). In this complex form, the ion cannot influence cellular behaviour like differentiation and proliferation [30-31]. The terms “Ca2+” and “PO43-“ refer, in the remaining part of this paper, to the active, free ions.


During in vivo bone resorption, osteoclasts release Ca2+ and PO43- derived from bone matrix. This causes a local increase in the ion concentration to supra-physiological levels, which has a significant impact on the proliferation and differentiation of osteoblasts, as well as on the subsequent bone formation process. In fact, extracellular Ca2+ gradients are present in a number of distinct micro-environments and represent potent chemical signals for cell migration (chemotaxis) and directed growth [32]. Additionally, Ca2+ is an important homing signal that brings together different cell types required for the initiation of a multicellular process like bone remodelling or wound repair [33]. For instance, high Ca2+ concentrations are shown to stimulate pre-osteoblast chemotaxis to the site of bone resorption, and their maturation into cells that produce new bone [34]. This chemotactic response to Ca2+ was also demonstrated experimentally on different cell types, including monocytes [35], osteoblasts [36], haematopoietic stem cells [37] and bone marrow progenitor cells [38]. The latter reported a dose-dependent relationship, with a maximal effect achieved at concentrations from 3-10 mM of Ca2+. These findings indicate that extracellular Ca2+ is a coupling factor between osteoclasts and osteoblasts [39] (apart from other potential coupling factors, such as mechanical coupling [40]). Mechanistically, extracellular Ca2+ regulates the migration of osteoblasts via the activation of calcium sensing receptors (CaSR) and/or by increasing the influx of Ca2+ [41]. In fact, the CaSR is reported to act as a (gradient) sensor, thus triggering chemotaxis of motile cells to critical micro-environments and transducing the Ca2+ signal to intracellular signalling pathways that regulate cell function [33]. Interestingly, studies suggest that the CaSR in osteoblasts is functionally similar to, but molecularly distinct from, the CaSR present in the parathyroid and the kidney [41-42]. Whereas, the influx of Ca2+ elevates intracellular Ca2+ level and thus the polarisation of cell membrane at the leading edge, which was reported to be critical in determining the persistent directional cell migration [43-44].


Besides the effect on cell chemotaxis, the release of extracellular Ca2+ also plays an important role in controlling the proliferation (via c-fos transcription factor expression) and differentiation (via dephosphorylation of NFAT transcription factor) of osteoblasts near the bone resorption site (Howship’s lacunae), through the calcium/calmodulin signalling [41]. These effects are revealed to be mediated by the calcium-sensing receptor (CaSR) [45], voltage-gated Ca2+ channels [41, 46] or inositol 1,4,5-triphosphate receptors (InsP(3)R) [47] present in the osteoblast, which serve to increase the intracellular Ca2+ level. In recent years, in vitro studies, based on the addition of elevated Ca2+ levels into osteoblastic cell cultures (~ 2 – 8 mM), demonstrated a profound impact on bone cell fate, which were independent of systemic calciotropic factors in a concentration-dependent way [42, 48]. These include osteoblast chemotaxis [36], DNA synthesis [49], proliferation, differentiation [50], and mineralisation of extracellular matrix [51-52]. Interestingly, Ca2+ also induced the expression of osteogenic growth factors, such as parathyroid hormone-related peptide (PTHrP) [53], BMP-2 and BMP-4 [52]. Similar effects were observed when osteoblasts were cultured on Ca2+-functionalised biomaterials, such as nanocrystalline CaP glass [54], Ca2+ implanted titanium substrate [55], Ca2+-exposed collagen gel [51] and CaP-coated implants [56-57]. In vivo osseointegration and bone formation were also improved when implants were enriched with calcium ions [58]. Encouragingly, Ca2+ has been implicated recently as an important messenger involved in the non-canonical β-catenin-independent Wnt/Calcium signalling for bone formation [59]. This pathway relies on an intracellular release of Ca2+ to activate calcium sensitive enzymes like Ca2+-CaMKII, protein kinase C (PKC) or calcineurin.


Essentially, the release of PO43- at the resorption site also plays a role in osteoblast proliferation and differentiation. In fact, PO43- has been identified as an important signalling molecule that regulates cell cycle and proliferation rate, alteration of signal transduction pathways (e.g. Fos-related antigen-1 (Fra-1) [60] and extracellular signal-regulated kinase (ERK1/2) [61]), gene expression (e.g. osteopontin) [62], as well as the secretion of bone-related proteins (e.g. matrix Gla protein (MGP) [63]). Contradictory, several in vitro studies showed that addition of high concentrations of exogenous inorganic phosphate (Pi, range from 5 to 7 mM) induced in vitro osteoblast apoptosis and non-physiological mineral deposition [64]. Nevertheless, Pi is believed to play a critical role in physiological bone matrix mineralisation [65]. This mineralisation event is mediated by the enzyme ALP and has been associated to the bone matrix calcification induced by BMP-2, where BMP-2 was found to stimulate Pi transport by osteogenic cells primarily via the sodium-dependent phosphate transporters [28]. Unfortunately, the use of Pi-functionalised biomaterials for tissue engineering applications is rarely described, possibly due to the technical limitations of existing technologies on handling Pi in an ionic form or the lack of awareness on the potential use of Pi. However, the use of polymers mixed with phosphate salts as a sustained delivery of Pi to induce in vivo mineralisation of the carrier has been reported recently [66].


Despite of the vast in vitro research findings, the influences of Ca2+ and PO43- differ from cell type to cell type [67]. This implies that there will be not one optimal Ca2+ and Pi concentration that could universally drive all cell types toward successful osteogenesis. Moreover, the optimal concentration may vary according to the cellular stage including proliferation and differentiation. Therefore, specific windows of ion concentration need to be determined for a specific cell type and its’ cellular stage, in order to initiate the desired in vitro cell behaviour effectively. For instance, our group has recently reported on the identification of specific Ca2+ and Pi concentrations that could induce higher proliferation and osteogenic differentiation of a mesenchymal stem cell-like human osteoprogenitor [68]. These findings were translated into the formulation of bio-instructive media (containing specific concentration of Ca2+ and Pi) that could optimally initiate higher proliferation, osteogenic differentiation and bone-like matrix deposition, both in two-dimensional (2D) cultures and three-dimensional (3D) constructs. This represents a novel strategy to produce 3D bone reparative units that may have predictive osteoinductivity for an effective repair of large skeletal defects [69].

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