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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(3) The Rationale of Using Calcium Phosphate For Bone Regeneration

Bone is a remarkable organ as it has impressive self-healing capacity without scar formation (when the defect size is not critical). However, delayed healing and non-unions still often develop and will occur more frequently due the aging of the population. In the United States, approximately 6 million fractures occur yearly, of which 5-10 % develop into a delayed union or non-union. An extrapolation of these numbers to the Indian population results in 240 million fractures a year, of which 12 million non-unions [70]. Therefore, the need for bone tissue regeneration is continously increasing and the emergence of combined engineering and life sciences technologies, such as tissue engineering, may lead to more effective bone healing therapeutic modalities.


Bone tissue engineering aims at offering a better solution for the healing of large bone defects and non-unions. This interdisciplinary research field applies principles of engineering and life sciences to create an in vivo micro-environment that promotes local bone repair or regeneration [71-72]. In this context, CaP bioceramics appear to be interesting candidates for bone tissue engineering applications, due to their biomimetic properties, supported by the following findings. Firstly, in the event of endochondral ossification, mineralised cartilaginous matrix is reported to induce osteoprogenitor differentiation and thereafter bone matrix deposition [73]. This phenomenon has been further investigated in order to elucidate the necessary physicochemical properties of CaP that may effectively trigger cellular signalling cascades for bone formation [74]. Secondly, the bone matrix calcification or mineralisation process is a critical stage of bone formation, either through direct mineralisation of bone matrix or the pre-formation of cartilaginous tissue template that is mineralised at a later stage. Thirdly, the dissolution of CaP-based biomaterials, either physicochemically or cell mediated upon implantation, may also resemble the physiological bone resorption process [75]. Finally, the discussed data in the previous section show that the effects of Ca2+ and Pi on osteogenic cell behaviour are appreciable. Therefore, the rationale of using CaP-based biomaterials for bone engineering strategies is clear.


(4) Historical and Hypothetical Mechanisms of Calcium Phosphate Osteoinductivity

Carbonated hydroxyapatite (HA) is the prevalent form of CaP mineral found in the bone. It provides mechanical strength to the bone and plays a critical role in the mineralisation of the bone matrix. Due to chemical and biological similarities, HA derived from natural sources (e.g. bone allograft, autograft, or coral) or synthetic HA, is widely used as bone filler for treating skeletal defects. However, HA is a highly stable CaP mineral and has therefore a lower solubility at the physiological pH (7.2 – 7.6) as compared to other types of CaP that have higher solubility [such as tricalcium phosphate (TCP) and octacalcium phosphate (OCP)]. Because the dissolution behaviour has been associated to the osteogenicity as well as the osteoinductivity of CaP [75], the search for an improved CaP-based biomaterial with higher osteoinductivity, also targets CaP with an appropriately high solubility. This may be more effective in stimulating osteogenic differentiation of stem cells and initiating bone formation. However, the exact mechanism of osteoinduction by CaP is currently unknown[67, 71, 76].

In the mid 60’s, osteoinduction was described as “a process which supports the mitogenesis of undifferentiated perivascular mesenchymal cells leading to the formation of osteoprogenitor cells with the capacity to form new bone” [77]. Since the discovery of Bone Morphogenetic Proteins (BMPs) as potent inducers of ectopic bone formation [78], the term “osteoinduction” has generally been used to describe an observation of heterotopic bone formation in association with the in vivo ectopic bone inductivity of a substance, such as growth factors, chemical compounds or biomaterials. In pathophysiological conditions, ectopic bone formation was induced upon tissue calcification in vivo, such as in tendons and arteries [79]. This phenomenon was associated to the osteogenic differentiation of the calcified tissue [80], including the expression of BMP-2 by the calcifying cells [81]. In the context of osteoinduction by synthetic biomaterials, investigations over the past decades suggest that: (i) the presence of a CaP component within a biomaterial (e.g. CaP-based ceramics [82], composite [83], coating [84], coral-derived ceramic [85], and bioactive glass [86]), or (ii) the ability of a non-CaP containing biomaterial to induce in vivo calcification (e.g. poly-hydroxyethylmethacrylate (poly-HEMA) sponge [87], and chemical-treated oxidised titanium substrate [88]), are the pre-requisite for heterotopic direct bone formation.


Recently, the osteoinductive effect of CaP-based biomaterials and the in vivo host-biomaterial interactions were reviewed to gain insight into the physicochemical properties that may govern osteoinduction[72, 89]. These include the effects of the macrostructures (e.g. dimension, geometry and porosity), the micro/nano structures [90] (e.g. microporosity [91], grain size, surface topography), and the chemical composition and characteristics of the biomaterials (e.g. active chemical surface for apatite formation, and dissolution kinetics of CaP biomaterials). Additionally, the in vivo osteoinductivity by CaP was reported to be dependent on the animal model used (higher incidence in large animals, possibly due to higher osteoclastic activity [92]), implantation site [93] and the duration of implantation [85] (higher incidence and faster in intramuscular than subcutaneous implantation). At present, the hypothetical mechanisms of CaP-driven osteoinduction from a material perspective can be classified into four categories:


  1. Direct effect of the biomaterial

The physical properties of a CaP-based biomaterial, such as geometry, macroporosity, microporosity, surface topography and grain size, have, in combination with its chemical properties, a significant impact on: (i) the nutrient, oxygen and waste exchange for cells within a biomaterial, (ii) the host blood vessel in-growth, (iii) the total volume of open pores available for cell growth and bone tissue formation, (iv) the effect on osteochondrogenic differentiation of stem cells [94], (v) the total surface area available for protein adsorption (e.g. endogenous BMPs), cell attachment (which activates a BMP-2 autocrine loop via a2β1 integrin [95] ) and growth, and (vi) ion dissolution and re-precipitation [93] which contributes to the formation of a biological apatite layer that in turn stimulates the osteogenic differentiation of stem cells.


  1. Indirect effect of the biomaterial

Osteoinductive proteins such as BMPs and transforming growth factor-beta (TGF-beta) are known to have a high affinity to CaP [96]. It has been hypothesised that CaP-based biomaterials may act as an in vivo affinity column/concentrator of the endogenous osteoinductive molecules upon implantation, which then renders the biomaterial osteoinductive. This is supported by a study which showed ectopic bone induction by CaP ceramics implanted intramuscularly (without cells) in proximity to the fractured fibula, where osteogenic events were actively ongoing [97].


  1. Inflammatory response

Upon implantation, CaP-based biomaterials elicit an inflammatory response that attracts the infiltration of mono- and multinucleated cells, and subsequently activates osteoclastogenesis which results in CaP degradation and resorption [98]. Subsequently, the released Ca2+ and PO43- stimulate osteoprogenitor differentiation and bone matrix deposition [99-100].


  1. Pathological ossification/bone formation

Osteoinduction by CaP-based biomaterials may resemble a pathological condition of heterotopic bone formation due to tissue calcification in vivo, resembling atherosclerosis or calcified tendonitis. The bone tissue formed often lacks functionality and eventually may resolve over the time. This resorption process may be related to a foreign body reaction or due to the depletion of biochemical cues upon implantation.


(5) The Translation of Calcium Phosphate Osteogenicity for Bone Tissue Engineering

In bone tissue engineering, two main forms of CaP materials are used: (i) bulk (fully dense or open porous) CaP biomaterials (eg. bone fillers, carriers, cement) [101], either as pure CaP or as part of a composite [102], and (ii) CaP coatings to functionalise biomaterial surfaces [103]. The use of CaP as growth factor delivery system [104] is also discussed, specifically BMPs-incorporated CaP biomaterials for bone formation [105].


    1. Bulk CaP

The physicochemical properties of bulk CaP are often modified during the in vitro production process, aiming at finding the optimal construct geometry for CaP scaffolds that could enhance osteoinductivity and hence improve the clinical outcome [106]. This includes manipulation of the architecture of biomaterials, such as the geometry, macro- and micro-porosity and surface topography [107]. The aim is to adjust the surface area for the entrapment of endogenous osteoinductive biomolecules and to accomodate optimum osteogenic cell density, as well as to fine-tune the dissolution kinetics that optimally elicit osteogenic differentiation and bone matrix deposition. For instance, production of cylindrical hydroxyapatite (HA) scaffolds with a hollow center (2 - 4 mm) were reported to enhance ectopic bone formation [108]. Also, incorporating HA scaffolds with cylindrical tunnels of 90 – 120 μm and 350 μm induced endochondral and intramembranous ossification [109]. The use of biphasic CaP (BCP) scaffolds (HA:TCP = 65:35 wt%) with cubical pores of 500 μm resulted in the highest bone formation as compared to the scaffold with lower (100 μm) or higher (1000 μm) pore sizes [110]. Besides that, the surface topography (i.e. surface roughness) has also been reported to influence the pattern of bone formation within CaP-based biomaterials [111]. Moreover, the surface topography is beneficial for the formation of bone-like apatite and provides a surface area that increases the dissolution of Ca2+ and PO43- ions [112]. This surface area was later on found to be associated to the microporosity within the macropores of CaP, which affected the material-fluid interface dynamics and triggered osteogenic differentiation [91]. Indeed, CaP discs with larger surface area induce higher in vitro ALP activity and are capable to concentrate more osteoinductive molecules that differentiate cells into the osteogenic lineage [113]. A similar phenomenon was observed in an in vivo study, where CaP carriers with lower CaP grain size and higher microporosity (and thus higher ionic dissolution kinetics) were found to be osteoinductive without using stem cell technology [75]. The surface area can also be increased by the production of CaP microparticles, as their osteoinductive properties are highly correlated to their size. It was shown that microparticles with size ranges from 80 – 300 μm demonstrated ectopic bone formation, whereas particle sizes above 500 μm were not osteoinductive [114-115]. Indeed, several important in vivo studies have shown that a CaP-based biomaterial with specific surface area above a threshold level of 1.0 m2/g was critical for osteoinduction (figure 3). Unfortunately, the availability of a technology to enable a high controllability and reproducibility on these desired features is currently lacking.


Also biphasic CaP (BCP) composites, being mixtures of HA with CaP with a higher solubility (such as β-TCP) in a specific ratio, appear to be an effective alternative to enhance the osteoinductivity of CaP [116]. The introduction of a more soluble phase of CaP may stimulate osteogenic differentiation of osteoprogenitors [117-118] and meanwhile provide a stable phase of CaP (i.e. the HA) that may act as a homing site for the implanted cells and promote bone ingrowth [119]. Indeed, implantation of pure HA or TCP was shown to be not osteoinductive, either due to a too high stability or a too high dissolution rate [120], whereas implantation of BCP induced bone formation with a low inflammatory response with the newly formed bone being sustainable in vivo during a long term animal study [121]. Unfortunately, no optimised HA:TCP ratio has been reported until now [122]. This is mainly due to the large variations in the bone forming capacity by studies using BCP with different HA:TCP ratio, which is also species and material specific [76]. Due to the lack of mechanical strength, these biphasic CaP biomaterials are more suitable for the treatment of skeletal defects at non-load bearing sites, such as bone fillers for cranial defects. Nevertheless, BCP alone was reported recently to successfully heal large bone defects in the clinic together with the use of an external fixator [123].


    1. CaP coatings on biomaterials

Due to the brittleness of CaP, CaP is often combined with a mechanically superior biomaterial like titanium. This is necessary especially in designing an osteoinductive composite for the repair of skeletal defects that receive high mechanical loading in situ [124]. To date, various methods have been proposed to deposit CaP onto the surface of Ti-based biomaterials [125], including a recent method that is based on the immobilisation of chemical moieties to induce mineralisation on the surface of biomaterials both in vitro [69] and in vivo [126]. These methods aim at producing composites containing mineralised components that are highly mimicking the hierarchies and bioactivity of the mineralised compositions of the bone, in order to provide an inductive environment that is capable of triggering bone formation. This directs us again to the principle question regarding what type of CaP has to be deposited onto the biomaterial of choice to ensure sufficient and predictive osteoinductivity. Therefore, the controllability and reproducibility of a technique that deposits CaP with desirable physicochemical properties, in the context of achieving optimum osteoinductivity, is highly demanded. A second consideration is the applicability of a deposition technique to three-dimensional porous structures. This represents a major drawback for many of the existing methods, where deposition of CaP onto complex 3D structures is technically challenging [127]. For instance, as an effort to overcome this major drawback, our research group has developed the perfusion electrodeposition (P-ELD) technology to deposit CaP coating homogenously onto a complex 3D Ti-scaffold porous structure, which offers high controllability and reproducibility over the physicochemical properties of the deposited CaP coatings [127] and displayed a promising osteoinductive effect [128].


5.3 BMP-incorporated CaP carriers

As an alternative to existing bone grafting techniques, CaP-carriers have been investigated to enhance bone formation through mediated delivery of bone-inducing factors (including biomolecules and metallic ions [129]), with some promising results reported up to date [130]. An example of such factors are the bone morphogenetic proteins (BMPs), discovered by Urist et al., 1965 as potent proteins with the ability to induce heterotopic bone formation [77]. These proteins are members of the TGF-β family which are secreted by chondrocytes, osteoblasts and osteoclasts [131-134], and play pivotal roles throughout embryonic skeletogenesis and in postnatal bone formation and endogenous repair mechanisms [130]. Indeed, BMPs are attractive for treatments of critical bone defects such as spinal fusions and non-unions, and have been successfully combined with substrates in order to induce bone formation by recapitulating the molecular cascades during skeletal development [135-137]. For instance, BMP-7 (also called osteogenic protein-1 or OP-1) and BMP-2, have been approved for clinical use and are delivered for spinal fusion and open tibial fractures via bone derived collagen particles or an absorbable collagen sponge [138]. Recently, these proteins have been extensively studied within the field of tissue engineering in order to mimic natural bone formation from a tissue engineering point of view. When implanted in animal models, these growth factors mainly induce bone formation via the endochondral pathway, through the formation of an intermediate cartilage template [135, 139]. Unfortunately, the currently used delivery system for BMPs causes rapid protein release and diffusion, which besides inducing bone formation, also causes inflammation and excessive bone formation. Consequently, the uncontrollable or improper release kinetics resulted in undesirable side effects such as male sterility, cancer and brain injury [140]. An additional reason for this could be receptor saturation at higher doses or limited avaliability of responding cells, which can lead to stimulation by these BMPs at undesirable locations. Moreover, the carrier composition may affect the regenerative response by BMPs as it may modulate protein stability and variate release kinetics differently. Nonetheless, Langer et al., showed as early as 1976 that protein release could be sustained when BMPs were encapsulated in biocompatible biopolymers [141]. Therefore, an attractive approach is to investigate carriers which may favour spatiotemporal physiological protein release but yet promote recruitment of endogenous progenitor cells. These carriers also need to provide a suitable microenvironment which promote efficient cell proliferation and differentiation, as this optimally may lead to bone formation and turnover [109, 136]. In addition to the collagen sponge used in clinical settings today, studies have shown promising results by the use of osteoconductive and osteoinductive CaP-carriers [11, 68, 142-143]. These carriers possess suitable characteristics (eg. the interconnected microporosity and the variation in CaP ratio) that are critical for the release kinetics of BMPs, as the electrostatic energy possibly plays a dominant role in carrier-to-BMP interactions (high binding affinity of BMPs to CaP), as well as in up-scaled constructs where multiple carriers may be combined [144-145].


Even though CaP carriers in combination with BMPs sounds like a promising approach, an impediment may be variation in activated bone forming pathways. The osteoinductive effect of CaP-carriers have displayed to form bone mainly via an intramembranous pathway, whereas BMPs mainly induce bone formation via the endochondral pathway. However, Eyckmans et al., showed in 2010 a close connection between CaP-induced bone formation and BMP-signalling [71]. In this study, removal of CaP-granules resulted in loss of bone formation in combination with rescinded BMP-signalling as well as abrogated osteoinduction in the construct after inhibition of endogenous BMPs [146-147]. This study displayed that CaP-carriers affect BMP-signalling in a model similar to physiological intramembranous bone formation in a BMP and CaP-dependent manner.


Another hurdle could possibly be that CaP may inhibit the osteoinductive effect of BMPs, already displayed by Urist et al., 1965, where partially demineralised bone induced inflammation and inhibited osteogenesis [77]. Additional complications regarding these issues were shown by Wehof et al., 2002 where the osteoinductive ability in recombinant human BMP-2 (rhBMP-2) coated porous particles of HA (PPHA) were investigated [148]. Prior to the study, PPHA had displayed some ability to induce de novo formed bone through the intramembranous pathway [149-150]. Remarkably, PPHA in combination with large amounts of rhBMP-2 in this study displayed an ability to attract a greater amount of inflammatory cells, which increased in time, together with multinucleated cells. Absence of collagen type II prior to hypertrophic chondrocyte differentiation and mineralization of the formed cartilage tissue, together with lack of bone marrow formation was seen. This suggests that the de novo formed bone in this construct was not following the usual BMP-induced endochondral pathway, therefore indicating an intermediate pathway that may have been activated [148].


In conclusion, BMPs mainly affect bone formation via an endochondral process whereas CaP-carriers mainly induce an intramembranous pathway. Possibly, the combination of appropriate concentrations of specific BMPs and well defined characteristics in CaP-carriers may function through an intermediate bone forming pathway.
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