Some considerations on biomaterials and bone

Micron 36 (2005) 583–592 www.elsevier.com/locate/micron

Review

Some considerations on biomaterials and bone Davide Zaffe* Department of Anatomy and Histology, Section of Human Anatomy, University of Modena and Reggio Emilia, 41100 Modena, Italy

Abstract Osteoinduction is a property not traditionally attributed to Calcium Phosphate ceramics. Histologic, SEM and X-ray microanalyses of a biopsy of pulmonary alveolar microlithiasis allow to discredit this opinion. Bone, even lamellar type, was ectopically formed on microliths undergoing osteoclastic erosion. The SEM and X-ray microanalyses of coral granules implanted in humans indicate an osteoconductive property for both Calcium and Phosphorus. Analysis of in vitro allows to propose an enhancement of the osteocapability of coral. Lamellar bone formation in the near absence of loads undermines the opinion which sees a correlation between lamellar bone and mechanical loads. Analysis of the bone surrounding an uncemented titanium hip prosthesis highlights that both remodeled and newly formed bone have lamellae oriented parallel to prosthesis surfaces, i.e. orthogonal to loads, as opposed to that of lamellar bone of osteons which are oriented parallel to loads. Analysis of longitudinal sections of cortical bone under polarized light points out that lamellae are displaced parallel to the cement line surface both in the conic end of osteons and in Volkman’s canals with thick wall, i.e. undergoing sloped load directions. In conclusion, there may be a relationship between lamellae formation and gravity. q 2005 Elsevier Ltd. All rights reserved. Keywords: Amorphous calcium phosphate; Autologous bone; Bone regeneration; Bone remodeling; Ceramics; Coral; Dense lamella; Ectopic bone; Hydroxyapatite; Lamellar bone; Loose lamella; Mechanical properties; Osteoblasts; Osteoclasts; Osteocytes; Osteons; Osteoinduction; Pulmonary alveolar microlithiasis; X-ray microanalysis; Woven bone

Contents 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Osteoinductive capability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bone structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Material and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Osteoinduction (or sometimes osteoinduction) is a rather common used term in biomaterial articles concerning bone (834 out of 2491 articles of the past 15 years—Medline).

* Address: Dipartimento di Anatomia e Istologia, Sezione di Anatomia Umana Normale, Va Del Pozzo, 71, 41100 Modena, Italy. Tel.: C39 594224800; fax: C39 594224861. E-mail address: [email protected].

0968-4328/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2005.05.008

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Nonetheless, the term is sometimes misused (Young et al., 1991; Turk and Parhiscar, 2000; Kamakura et al., 2001; Yuan et al., 2001; Bauer and Smith, 2002; Beruto et al., 2002; Kim et al., 2002; Papavero et al., 2002; Bahar et al., 2003; Moreira-Gonzalez et al., 2005) since it is intended as the ability of a material to stimulate or promoting (osteostimulation and osteopromotion, respectively) bone formation at the site where the material is lodged. Osteoinduction is not only ‘the process by which immature cells are recruited and then stimulated to form new bone’ (Albrektsson and Johansson, 2001) but also the capability to induce formation of bone ubiquitously. It is

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highly improper to speak of osteoinduction when implanting a material in a skeletal site such as tendon, ligament or muscle (Boyan et al., 2000; Liao et al., 2000; Yuan et al., 2000, 2001; Habibovic et al., 2005), or when testing it in culture media of osteoblasts, fibroblasts or, worse yet, immortalized cell lines media (Adnet et al., 1998; Fini et al., 2002; Chouteau et al., 2003). An osteopromotive capability of a material may be misinterpreted as an osteinductive one. Only the criterion of bone formation on ectopic extraskeletal sites of living tissue heralds a valid osteoinductive capability of a material. On the contrary, some authors (Misch and Dietsh, 1993; Heymann et al., 2001; Durrieu et al., 2004; Zerbo et al., 2004) exclude the osteoinductive property of some

ceramics. All solid and inorganic materials are named ceramics in Material Science (Muster, 1987). Aluminumor Zirconium-based ceramics definitely lack osteoinductive capability. However, though a robust demonstration is lacking, Calcium- or Calcium-Phosphate-based ceramics may arguably have some osteoinductive potential, which may explain the good outcomes obtained with their use. A first-time evidence of this capability may derive from my unpublished data. Pulmonary alveolar microlithiasis (PAM) is a rare disease (Castellana and Lamorgese, 2003) characterized by the appearance of mineralized structures in pulmonary alveoli (Fig. 1). The lung presents interstitial fibrosis and intra-alveolar and interstitial formations of concentrically

Fig. 1. Histology (A and D—Trichrome Gomori stain), microradiograph (B) and back-scattered SEM (C, E and F) images of a biopsy of pulmonary alveolar microlithiasis embedded in PMMA. Black arrows point to microliths having smooth surface; white arrows point to microliths having indented surface. Note in A the relatively great amount of fibrous tissue (fibrosis). The correct appearance of microliths may be appreciated in microradiograph B because they completely crack under tungsten carbide blade sectioning. Note in C that the outer surface of microliths appears indented (white arrows), owing to osteoclastic resorption (Howship’s lacunae). C (lower part of the image) and D (in green) display ectopic bone formed in apposition with microliths. Woven bone of E, containing typical irregular-shaped osteocytes, is formed in apposition to microliths with indented surface (white arrows) or not (black arrows). The bone trabecula of F shows signs of remodeling and, above all, the presence of lamellar bone, containing typical ellipsoid-shaped osteocytes. Field width: AZ1560 mm; BZ1700 mm; CZ530 mm; DZ450 mm; EZ135 mm; FZ210 mm (For interpretation of the reference to colour in this legend, the reader is referred to the web version of this article).

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(lamellar bone) containing typical ellipsoidal-shaped osteocytes (Fig. 1(F)). These results prompt two remarks on osteoinductive capability and bone structure.

2. Osteoinductive capability

Fig. 2. Graphs of EDS (full area at !5;000 SEM magnification) of a microlith (A-dark grey) and woven bone (B-light grey), plotted on the same scale. Note in A how the microlith contains a small amount of Mg as opposed to bone. Copper (Cu) rather then gold was used for sputtering to avoid Phosphorus overlapping.

laminated calcified bodies (microliths). Microliths (measuring 50–750 mm in diameter) are nearly spherical in shape, mostly surrounded by fibrous tissue, but sometimes engulfed by bone (Fig. 1(C)–(E)). Due to macrophage (lithoclast) resorption (Howship’s lacunae), a few microliths show a smooth surface (Fig. 1(C) and (E)) but most of them have a highly indented profile (Fig. 1(C) and (E)). Most microliths, surrounded by soft tissue, have multinucleated osteoclast-like cells adhering to their surface. At X-ray semi-quantitative microanalysis (E.D.S. system) microliths (Fig. 2(A)) are composed of Calcium Phosphate with a small amount (about 1.25 wt%) of Magnesium. X-ray quantitative analysis, performed using a CaHPO4$2H2O tablet on the same stub as reference, shows that Calcium content (25–29%) of microliths is equal to or higher than that of woven bone (27%), the most mineralized type of bone. On the contrary, the Phosphorus content of microliths is always higher (12–14%) than that of woven bone (11.8%). The most mineralized parts of the microliths (lighter in backscattered SEM images) show not only high Calcium content (28.6%) but also the highest Phosphorus content (13.8%) and the lowest Ca/P ratio (2.07). The Ca/P ratio (range 2.07O2.28 wt/wt) indicates that all parts of the microlith have hydroxyapatite-like composition as bone (theoretical Ca/PZ2.15 wt/wt), but the more mineralized parts of microliths have the lowest Ca/P values, indicating enrichment of amorphous Calcium Phosphate. Bone forms in apposition to granules having both indented (Fig. 1(E), white arrows) or smooth surface (Fig. 1(E), black arrows). The new bone has a disordered spatial disposition, forming fragments or very irregularly displaced thick trabeculae, and generally appears as a woven structure containing typical irregular-shaped osteocytes. Some bone trabeculae show signs of remodeling and bone with more regular structure

Microliths act like a Calcium Phosphate (hydroxyapatitelike) ceramic, though of autologous origin, grafted into the lung where it is able to induce the formation of new bone by recruiting and inducting the differentiation of osteoprogenitor cells. These activities can probably be ascribed to ion released after clast (osteoclast-like cells) resorption of microliths. Though several authors have documented resorption of hydroxyapatite (Gomi et al., 1993; Piattelli et al., 1993; Overgaard et al., 1996, 1997, 1998; Goto et al., 2001), most studies refer to the results of implants of nonresorbable hydroxyapatite (Zeller et al., 1986; Klinge et al., 1992; Hoad-Reddick et al., 1994; Nemcovsky and Serfaty, 1996; Piattelli et al., 1998; Rafter et al., 2002). Particularly in dentistry, the use of this term generates confusion for the end users, who is led to believe that there are two types of hydroxyapatites: non-resorbable and resorbable. If pure, there is only one type of hydroxyapatite, Ca5(PO4)3OH, having a Ca/P ratio of 1.667 at/at (2.15 wt/wt) and solubility-product constant (Ksp) 2.34!10K59 (McDowell et al., 1977). Hydroxyapatite has a very low solubilityproduct constant, but not largely different from that of Octacalcium Phosphate (Ca4H(PO4)3KKspZ2!10K49), the first mineral form of bone, or that of Tricalcium Phosphate (Ca3(PO4)2KKspZ2.83!10K30) (McDowell et al., 1977). Hydroxyapatite may be manipulated by the manufacturing process (plasma-spray, sintering, ceramication etc.) which may influence the degree of cristallinity or, it may differ in composition, depending on the prime materials, but in this case the hydroxyapatite can produce noxious results (Zaffe et al., 2004). Ion release seems to be an important factor on osteoinductive capability. One of aims of biomaterial manufacturers is to achieve a greater superficial reactivity of hydroxyapatite, and more studies should address to this issue. Accordingly, ion release seems to be responsible for osteoinduction. But which ions are the inducers: Calcium, Phosphorus or both? An answer to this question may come from previous studies on coral implants (Zaffe et al., 1994). Apulian coral, roughly reduced to 0.5–1 mm granules, has been used to fill post-extractive cavities in man. After 6 months the first result was a marked size reduction of granules. All granules in contact with bone showed a peripheral layer of amorphous Calcium Phosphate (Fig. 3(B) and (C), arrows). This layer was particularly evident in those granules partially surrounded by bone, not to be misinterpretation as a low mineralized bone layer at the interface (Fig. 3(E) and (F)). Two behaviors turned up when analyzing the granules not in contact with bone.

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Fig. 3. Secondary electron SEM images (A, D and G) and X-ray maps for Phosphorus (B, E and H) and Calcium (C, F and I) showing the topographic composition of a 6-month biopsy performed in a post-extractive cavity in a human filled by Apulian coral granules. The arrows in B and C point to a part of the coral granule not in contact with bone but surrounded by a Calcium Phosphate layer. Note in D how the coral granule, located far from bone, shows an indented surface (due to osteoclastic resorption) and is surrounded by a Calcium Phosphate layer (E and F). Note in G how this coral granule has an almost smooth outer surface and does not appear to be in contact with bone notwithstanding the proximity of the granule to the bone. Field width AZ270 mm; DZ135 mm; GZ270 mm.

Rare granules surrounded by soft tissue showed an indented surface (due to clast aggression, Fig. 3(D)) and a peripheral layer of amorphous Calcium Phosphate (Fig. 3(E) and (F)), most likely due to the reactivity of phosphate released by the ATP/ADP reaction. These granules probably undergo bone formation by apposition on their surface in time. Several granules, enveloped by soft tissue and at a variable distance from the bone, showed a nearly smooth surface (Fig. 3(G)) with the same composition as the bulk (Fig. 3(H) and (I)).

Their shrinking in size is probably due to the activity of biological fluids and to the relatively high reactivity of coral (CaCo3–Aragonite–KspZ6!10K9). The absence of a Phosphate layer is probably due to the lack of energy consuming activities of cells near the granule. Thus these granules are probably destined to disappear, without osteoinductive activity. This interpretation may explain some of the results of aragonite implants (Bahar et al., 2003) which probably do not produce the Calcium Phosphate layer

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Fig. 4. Secondary electron SEM images (A, B and C) and X-ray maps for Phosphorus (D) and Calcium (E) showing the topographic composition of a coral granule before (A) and after (BOD) treatment in an 0.1 M solution of NaH2PO4CNa2HPO4 (pH 7.0) for 3 days. Note in B how the coral granule surface differs greatly from the smooth surface before treatment (A). Compare images C, D and E (sections of treated granules embedded in PMMA) with the corresponding D, E and F images of Fig. 3. Field width AZBZ900 mm; CZ270 mm.

highlighted not only by studies in humans (Zaffe et al., 1994), but also by animal research on coral (Damien et al., 1994) or nacre (Atlan et al., 1999; Liao et al., 2000) implants. To avoid this event and to increase the osteoinductive potential of coral or nacre, treatment of the material with a phosphate solution is suggested (Fig. 4). Some years ago (Zaffe and Cantoni, 1994) we found that a Calcium Phosphate layer was formed when coral granules were treated with a pH 7 Sodium Phosphate solution for 3 days (Fig. 4(B)). The coral surface (Fig. 4(A)) reacted with the solution, thus forming an outer layer of Calcium Phosphate (Fig. 4(B)–(E)). This capability, recently confirmed by Ni and Ratner (2003), warrants further investigation to enhance and optimize the osteoinductive potential of aragonite biomaterials.

3. Bone structure Lamellation of bone is currently correlated to loading (Portigliatti-Barbos et al., 1984; Ascenzi, 1988; Ascenzi et al., 1990, 1997; Goodwin and Sharkey, 2002). Mechanical properties of bone are correlated with both circumferential, osteonal and interstitial bone and to bone tissue structure (longitudinal and transverse lamellae). The correlation between loads and collagen arrangement (longitudinal and transverse) of lamellae seems partially

unfit after Marotti’s innovative studies on lamellar bone structure (Marotti and Muglia, 1988; Marotti, 1993, 1996; Marotti et al., 1994a, 1995). The controversial finding of these works is that only two types of tissue (or osteons made by) exist: woven and parallel-fibered bone. Lamellar bone is made up of alternating collagen-rich (dense lamellae) and collagen-poor (loose lamellae) layers, all having an interwoven arrangement of fibers (Marotti and Muglia, 1988; Marotti, 1993, 1996; Marotti et al., 1994a, 1995; Ardizzoni, 2001). Loose lamella is also named cellular lamella because it contains osteocyte lacunae, whereas dense lamella is also named acellular lamella due to the absence of lacunae. Dense lamella is thinner (about 1.5– 2 mm) and the loose lamella is broader (about 3–6 and up mm—Marotti, 1996; Ardizzoni, 2001). It has been hypothesized that the alternation of dense and loose lamellae depends on osteocyte recruitment from osteogenic laminae in successive layers (Marotti, 1996). Loose lamella contains fewer fibers but more bone matrix; dense lamella contains more fibers but less bone matrix. Due to the high affinity of the bone matrix for calcium salts, the loose lamella is more mineralized whereas the dense lamella has the same apatitic structure (Ca/PZ2.15 wt/wt) but is less mineralized (about 2–4% in Ca content) (Marotti et al., 1994b). The correlation between mechanical properties and lamellae breaks down when considering lamellar bone formation in the unloaded condition. Lamellar bone is

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Fig. 5. Behavior of bone surrounding an uncemented titanium hip prosthesis harvested from a woman who died 3 years later due to vascular problems. A displays the cadaveric bone (threaded in the same way as in living) fitting to the prostheses. Note the longitudinal groove of the titanium shaft. B shows perimplant bone after dissection. The black arrow points to the newly formed bone crest inside the longitudinal groove. C, E and G (microradiographs) and D, F and H (polarized light) are corresponding images of sections of perimplantar bone embedded in PMMA. The arrows in C (microradiograph of D) mark the direction of loads. Note in D (longitudinal section of the cortex) how the bone inside the titanium threads (after remodeling) has a different spatial displacement of lamellae from the original one (the same as the remaining cortical bone). The arrow in E (microradiograph of F) marks the direction of loads coming from the prosthesis. Loads acting on the cortex (orthogonal to the section) are not marked. Note in G (transverse section of the cortex, taken at the level of the dotted line overlapped to C) the very different appearance of the structural displacement of the remodeled bone: contrary to the cortical bone (where lamellae are parallel to loads), lamellae are orthogonally displaced to loads in remodeled bone. The arrows in G (microradiograph of H) mark the direction of loading from the prosthesis. The loads acting on the cortex (orthogonal to the section) are not marked. Note in H (transverse section of the cortex and of newly formed bone inside the longitudinal groove of the prosthesis) that new bone has a different lamellae displacement than bone of the cortex, i.e. parallel to the titanium surface and orthogonal to loads. Field width AZ12.8 mm; BZ38;8 mm; CZDZEZFZ3;16 mm; GZHZ5;2 mm.

formed on autologous rabbit bone fragments inserted in an extracorporeal hydroxyapatite chamber (Zaffe et al., 2002a), i.e. without loading. Another confirmation comes from the abovementioned lamellar bone formation in pulmonary alveolar microlithiasis. Moreover, when the direction of loading changes abruptly, lamellar bone formation does not seem to be associated to loading. When loads change direction and perhaps intensity, such as in bone surrounding titanium prostheses, lamellae are formed in a peculiar way (Fig. 5(D), (F) and (H)). For example, bone surrounding the shaft of an uncemented (threaded titanium) hip prostheses (SCL, type, Lima-Lto, Italy), implanted in a woman for 3 years shows complete remodeling of the tissue in contact with the surface of the screw thread (Fig. 5(B)–(F)). The lamellae, at first longitudinally oriented like the remaining bone tissue of the cortex, assume an orientation parallel to the titanium

surface (Fig. 5(D) and (F)), i.e. orthogonal to the direction of loads, and not with the same direction as the original lamellae (as showed by the cortex). This becomes particularly evident (Fig. 5(B), (G) and (H)) in the newly formed bone sampled from inside a longitudinal groove of the titanium shaft (infibulum). Here the lamellae were parallel to the surface, i.e. orthogonal to the direction of loading (Fig. 5(H)). Similar results can be found by accurately analyzing the lamellae of cortical bone. There are two sites where loads (which are coaxial to the long axis of osteons, i.e. tangent to their cement line) are oriented differently to bone: the terminal parts of osteons and Volkman’s canals with a thick wall (Fig. 6(A) and (D)). Longitudinally oriented lamellae (i.e. parallel to the cement line and also to loads) along the cylindrical part of the osteon (Fig. 6(B)), assume a conical direction in the terminal part of the osteon, remaining

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Fig. 6. Longitudinal sections of dry, non-embedded, mid-diaphyseal cortical bone of human tibia. A and D microradiographs of sections before decalcification. B, C and E images under polarized of sections decalcified with HCl. The boxed-in areas of A and D correspond to the higher magnification images reported in B, C and E. Note in B how lamellae are parallel to the cement line and to mechanical loads (which have a longitudinal direction on tibial cortex). Note in C how the lamellae have a conic displacement at the end of the osteon: i.e. parallel to the cement line but sloped with respect to load direction. Image E displays the initial part of an oblique Volkman’s canal having a thick wall. The white arrow marks (overlapped to the osteonal wall) the loading on the tibial cortex. Note how the bone, formed in apposition to the wall of the sloped canal produced by osteoclastic erosion, has the lamellae parallely displaced to the cement line (pointed to the black arrow), i.e. sloped with respect to load direction. Field width: AZ1050 mm; BZCZ400 mm; DZ2700 mm; EZ400 mm.

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parallel to the cement line (Fig. 6(C)) but following the sloped directions of loads (i.e. different from the main part of the osteonZnot correlated to loads). An almost similar behavior was shown by bone surrounding Volkman’s canals (Fig. 6(D) and (E)). If these are not only thin channels but the result of great horizontal or oblique osteoclast excavations, the osteoblasts form lamellae parallel to the cement line which are oblique to the direction of loads (Fig. 6(E)). In summary, lamellae orientation does not seem to be correlated with loads. Lamellae differ in size and fibrousness, but both have a woven structure (Marotti and Muglia, 1988; Marotti, 1993, 1996; Marotti et al., 1994a, 1995). The loose lamella (thicker, with size correlated to that of osteocytes—Ardizzoni, 2001) contains bone cell protoplasm, inside a lacuno-canalicular network of appreciable extension (12–20% of femoral cortex volume—Marotti et al., 1992), surrounded by a calcified matrix made up of minerals, collagen fibers and large amounts of amorphous matrix. The dense lamella (thinner, with an almost constant size—Ardizzoni, 2001) contains only a part of canaliculi surrounded by a calcified matrix made up of minerals, many collagen fibers and a low amount of amorphous matrix. As a result of aluminium accumulation in the dense lamella (Zaffe et al., 2004), it is suggested that osteoblast (over fixed span of time) always work in the same way producing collagen fibers and amorphous matrix, as observed in the dense lamella. When osteoblasts are committed to become pre-osteocytes and then osteocytes, a great amount of amorphous matrix is added that rarefies the collagen fibers and enlarges the thickness of the lamella. Moreover, considering that bone only acquires its proper mechanical property when the lamellae are completely calcified and instead bone is formed inside cavities (osteoclastic cutting cones) that have almost uniform loads, the following speculation is proposed. First, the cement line (even if it does not have the same mechanical properties as dense lamella) constitutes a valid support for the first lamella, most likely a loose lamella. The loose lamella contains lacunae and canaliculi filled by fluids and protoplasm and has a lower mechanical strength, so osteocytes are subject to mechanical instability due to gravity. Osteocytes induce osteoblasts for dense lamella production. The mechanical properties of dense lamella allow a valid mechanical support, so osteocytes stop induction on osteoblasts and a new loose lamella can be produced. When loose lamella is formed osteocyte induction restarts. This hypothesis may explain why osteocytes near the osteonal cement line have a different orientation with respect that of the remaining osteocytes (Marotti et al., 1985) and agree with the reduced lamellar bone formation found in rats during spaceflight (Kitajima et al., 1996), under zero gravity conditions.

4. Material and methods Material and methods are reported in Zaffe et al., 2002b, 2003, 2004.

Acknowledgements I wish to thank Dr John Pradelli, M.D. for assistance in manuscript draft and revision, and Centro Interdiapartimentale Grandi Strumenti (C.I.G.S.) of the University of Modena and Reggio Emilia for software, SEM and EDS availability and assistance.

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