biomineralization: some complex crystallite-oriented skeletal structures

Biomineralization: Some complex crystallite-orientedskeletal structures

ASHOK SAHNI98 Mahatma Gandhi Marg, Lucknow 226 001, India

(Email, [email protected])

The present review focuses on some specific aspects of biomineralization with regard to the evolution of the firstfocused visioning systems in trilobites, the formation of molluscan shell architecture, dental enamel and itsbiomechanical properties and the structure of the calcified amniote egg, both fossil and recent. As an interdisciplinaryfield, biomineralization deals with the formation, structure and mechanical strength of mineralized skeletonized tissuesecreted by organisms. Mineral matter formed in this way occurs in all three domains of life and consists of severalmineral varieties, of which carbonates, phosphates and opaline silica are the most common. Animals and plants needmechanical support to counteract gravitational forces on land and hydrostatic pressure in the deep ocean, which isprovided by a skeletonized framework. Skeleton architecture mainly consists of basic elements represented by smallusually micrometer- to nanometer-sized crystallites of calcite and aragonite for carbonate systems and apatitecrystallites for phosphatic ones, and then these building blocks develop into structured more complex frameworks.As selective pressures work towards optimizing stress and response, the orientation, morphology and structuralarrangement of the crystallites indicates the distribution of the stress field of the biomineralized tissue. Large animalssuch as the dinosaurs have to deal with large gravitational forces, but in much smaller skeletonized organism such asthe coccoliths, a few micrometer in diameter made up of even smaller individual crystallites, van der Waals forces playan increasingly important role and are at present poorly understood. Skeleton formation is dependent upon manyfactors including ambient water chemistry, temperature and environment. Ocean chemistry has played a vital role inthe origins of skeletonization, 500 to 600 million years (ma) ago with the dominance of calcium carbonate as theprincipal skeleton-forming tissue and with phosphates and silica as important but secondary materials. Thepreservation of calcareous skeletons in deep time has resulted in providing interesting information: for example, thenumber of days in the Devonian year has been established on the basis of well-preserved lunar (annual) cycles, andisotope chemistry has led to an elaborate protocol for using O18/O16 stable isotopes for palaeotemperaturemeasurements in the geological past. Stable isotopes of dental apatite have helped to establish ecological shifts(terrestrial to wholly marine) during the evolution of the Cetacea. Biomineralization as a field of specialization is stillsearching for its own independent identity, but gradually, its importance is being realized as a model for engineeringapplications especially at the nanometer scale.

[Sahni A 2013 Biomineralization: Some complex crystallite-oriented skeletal structures. J. Biosci. 38 925–935] DOI 10.1007/s12038-013-9390-z

1. Introduction

Biomineralization is an interdisciplinary field that deals withthe formation, structure and function of mineral mattersecreted by organisms. The biomineral phase represents thefundamental building block of all calcified tissue, both fossiland recent, and all three domains of life possess biomineralsin some form or the other, but it is only the eukaryotes that

form mineralized skeletons. This field is therefore a greatunifying and integrating force in studying organicallymineralized tissue (Lowenstam and Weiner 1989) and haswitnessed a strong resurgence of interest with the advent ofscanning electron microscopy (Boyde 1964; Koenigswaldand Sander 1997; Sahni 1988). In general terms, mineralizedtissue usually retains its integrity and identity through deeptime in the context of chemistry and physical structure so that

http://www.ias.ac.in/jbiosci J. Biosci. 38(5), December 2013, 925–935, * Indian Academy of Sciences 925

Keywords. Oriented crystallite; biomineral; skeletal structures

Review

Published online: 6 November 2013

anatomical details can be easily observed. Occasionally, fossilmineral tissue is altered by ‘diagenesis’, which is a process thatoccurs post burial and usually involves chemical alteration bothat ambient and high temperatures. However, whenever thishappens, enough evidence is left in the form of crystalovergrowth, dissolution and distortion for this process to beidentified as such. In contrast to mineralized tissue, soft cellularmaterial on fossilization may undergo severe modification andalteration. Silicified fossil wood is a case in point.

Starting with the ‘Big Bang’ of complex multicellular lifesome 600 million years ago, skeletons originated to serveseveral functions: to provide mechanical support for organismsin various environments, to protect organisms against increasedpredation, to act as attachment surfaces for muscles and lastly toserve as a chemical reservoir for essential elements and forremoval of metabolic wastes (Kazmieraczak et al. 1985).

The present review focuses on four specific anddiversified aspects of skeletal structure to illustrate howcrystallite orientations are fundamental in the building ofbiomineralized complexes ranging from vision in trilobites,molluscan and amniote shell architecture, and the formationof dental enamel. One of the aims of this overview is to bringinto sharper focus the present status of the discipline whichforesees a brighter future for all those who wish to study theorigins, structure and functionality of mineral matter secretedby organisms. Biomineralization is usually associated withthe skeletal frame of an organism whether internal(endoskeletons) or external (exoskeletons) but also gives riseto special structures such as teeth, eggs and ear bones (suchas the otoliths of fish). Although several biominerals havebeen identified, in this review, only the major skeletonforming minerals such as calcite and aragonite (calciumcarbonate) found in invertebrates, or hydroxyapatite (calciumphosphate) occurring in vertebrates and opaline silica commonin mainly marine organisms such as sponges, radiolarians anddiatoms, will be considered. Most protistan clades possessingskeletons are silica-based. Microscopic opaline silica nodulescalled phytoliths are also found in many terrestrial plants suchas grasses and serve as mechanical support. For invertebrateswith calcareous skeletons, the mineral calcite is dominant, butin some cases, aragonite is also present as, for example, incorals, many mollusks and in turtle eggshells (Bajpai et al.1997). In order to understand functionality of the skeleton or ofa biomineralized tissue such as dental enamel, mollusk shellsor calcified eggshells, it is essential to look at the structure as awhole but at different scales of observation, starting with theprimary building block, the oriented crystallite, and thenexamine the more complex and structured framework in thelight of growth and the biological functions that need to beperformed.

This process is a complicated task and selection pressureshave found innovative ways to accomplish this for diverseforms of life through deep time, ranging from the gravity-

defying giant dinosaurs weighing some 70 to 80 tons tomicrometer-sized coccoliths (Young et al. 1999) and othereven smaller microbiota influenced by yet poorlycharacterized van der Waals forces.

Several biomineralized systems show a high degree of self-organization starting with basic components such as crystallitesand then enlarging to increasing complexity encompassing theentire skeleton. At present it is unclear (and beyond the scope ofthis work) to resolve issues concerned with the mechanismleading to a self-ordered system of skeletal architecture andwhether natural selection controls self organization or not(Kaufmann 1993). At present it appears that a Darwiniangradualistic adaptive evolutionary approach would be enoughto explain the observed structures. In cases where the temporalconstraints are good and species diversification is fairly rapid,as, for example, in whale evolution (Roe et al. 1998), it is easierto ascribe this process to adaptive evolution rather than tosystems of self organization.

2. Skeletonization: Origins and special features

Multicellular life began with a ‘Big Bang’ over 600 millionyears ago after nearly 2.8 billion years of the earth wasinhabited by unicellular organisms, and soon after, severalorganisms belonging to diverse phyla developed skeletons.Murdock and Donoghue (2011) and Dove (2013) give acomprehensive review on the advent of skeletonization.Initially around 600 million years or so only some eukaryotephyla developed skeletons, but 50 million years latercalcareous skeletonization became much more common,and by the Ordovician, it was quite prevalent (Pruss et al.2010). An interesting event is recorded by Bengtson andZhao (1992) that no sooner had some organisms developedskeletons, opportunistic borers developed the capability ofpenetrating them. The process of skeletonization has to beseen in the light of early ocean chemistry and what were themost easily available skeleton building materials: then, asnow, carbonates were abundant whereas phosphates weremore limited. Donoghue and Sansom (2002) have discussedthe origin and early evolution of vertebrate skeletonization.Apatite is a hard biomaterial forming the phosphatizedskeletons of some early invertebrates such as brachiopodsand was retained by the vertebrates but the cost (in termsmetabolic trade-off) for apatite skeletons was rather high. Apossible reason may lie in the fact that the solubility,availability and precipitation differs for phosphate andcarbonate skeletal systems. Carbonates are by far the mostcommon skeleton builders. Silica, the hardest of thebiomineralized materials, is a distant third as a skeletonbuilder and forms the framework for siliceous sponges anda variety of microorganisms such as radiolaria and diatoms.

Biomineralized skeletons of fossil organisms have beenused to obtain interesting data in deep time, ranging from the

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J. Biosci. 38(5), December 2013

number of days in a Devonian year, to palaeotemperaturemeasurements including the recognition of ancientenvironments and the ecological shifts that took place duringphyletic evolution: The fact that some animals withcalcareous skeletons such as corals retain a faithful recordof their growth has been used in exceptionally well-preserved specimens to calculate the number of days in aDevonian year (Wells 1963; Scrutton 1964). On the basis ofdiurnal, monthly and annual cycles recorded in thecalcareous exoskeleton of a fossil coral, Scrutton (1964)suggests a periodicity probably connected to the lunarbreeding cycles corresponding to 13 lunar months of 30.5days each for the Middle Devonian, which has more days inthe year than at present.

Urey (1947) was one of the first to give a theoreticalmodel for establishing a palaeotemperature scale. Later, heand his co-workers using oxygen isotopes from molluscanshells were able to calculate temperatures at which the shellformed in the ocean (Epstein et al. 1953; Goodwin et al.2003). Present day studies have built on these foundationsand made palaeotemperature measurements a sophisticatedscience (Goodwin et al. 2003).

In a pioneering study DeNiro and Epstein (1978)established how carbon isotopes could be used for inferringthe diets of mammals. Using fossil dental enamel(= bioapatite) Thewissen et al. (1996) and Roe et al.(1998) demonstrated how cetacean (whale) evolution tookplace from a four legged terrestrial ancestor to typicallymarine forms. They based their studies on stable isotopesof carbon and oxygen of tooth enamel using threecomparative groups: modern whales living in the openocean, recent freshwater delphinids and an evolving groupof primitive whales in India and Pakistan that showed atransition from freshwater to marine conditions. Dentitionsof early whales including enamel structure also confirms this(Sahni 1981; Sahni and Koenigswald 1997). Bera et al.(2010) have shown ecological and environmental shiftsusing fossil dental enamel material.

3. Trilobites and the first vision apparatus

Of the many remarkable ways in which selective pressureshave shaped the diversity of organs and organismsthemselves, the evolution of vision must rank as one of themost outstanding. The evolution of focused vision inmulticellular organisms had a remarkable origin over 542million years ago and is exemplified by a group of extinctarthropods called trilobites. In fact an exceptionally well-preserved enigmatic fossil Anomalocaris from the BurgessShale suggests that focused vision may have arisen evenearlier (Paterson et al. 2011) than that of the trilobitesthemselves. The trilobites ruled the earth for more than 300million years and occupied most of the available niches in

the ocean from tidal sand to open sea. As one of the mostcomplex swimming animals of their time, eye sight (andlocomotion) was an important component in theirevolutionary process both for those trilobites who werevicious predators and others who were their poor prey!However, many bottom dwelling, sand-burrowing andmud-grubbing trilobites lost their vision and were blind.

Using the birefringent mineral calcite, trilobites were ableto evolve sophisticated visioning systems finely focused inaqueous medium some 500 million years ago. A birefringentmineral is one in which light splits into two pathways, givingrise to two distinct and usually overlapping images, and thereis only one crystallographic axis in which a single imageforms, that is along the C-Axis. The basic lens unit of thisearly visioning system is therefore a transparent calcitecrystallite (variety Iceland Spar) oriented specifically alongthe C-axis of the crystallite. Lenses in some trilobites weregrouped into compound arrays with prominent crescent-shaped to bulbous eyes (figure 1D).

Clarkson (Clarkson 1979; Clarkson et al. 2006) pioneeredstudies on diverse trilobite vision systems and describedthree distinct types based on lens shape, spacing and thedegree of coverage by the corneal membrane. The earliestand most generalized is known as abathochroal, found in theearliest known eodiscid trilobites from China (Xi-Guang andClarkson 1990) and consists of a corneal membrane limitedto the edge of the lens with thin sclera comprising ofexoskeleton cuticle. This type is similar to schizochroal eyeswhich have fewer and larger lenses each separated by acorneal membrane and thick sclera. In contrast, holochroaleyes are the commonest vision systems in trilobites andconsist of several hundred lenses (about 30–100 μm indiameter) and are covered by a single corneal membrane.

The usual problem of spherical aberration andmaintaining focused vision in the ocean is a difficult taskbecause of turbidity, salinity and light intensity differencesin general, but trilobites managed to overcome thesedifficulties and evolved the ‘intralensar bowl’ (Lee et al.2007; Torney et al. 2008) having a different refractive indexthan the couplet lens and therefore ‘upstaged’ the work ofDes Cartes and Hugyens, who devised the same type oflenses in the era of human intelligence, a few hundred yearsago and several million years after the extinction of the lasttrilobite (Clarkson and Levisetti 1975)! There has been muchdiscussion on the properties of the intralensar bowl and itappears that at least for some trilobite species the lenses hadan interface with an undulating intralensar surface whichcorrected for spherical aberration (Horvath 1996). Torneyet al. (2008) using the electron backscatter technique havedemonstrated that some species had calcite lenses with achemically different intralensar bowl to correct aberration;other species appear to have a more complex structurewhereby light was fed to the mineral calcite through an outer

Complex crystallite-oriented skeletal structures 927


unit having radially arranged C-axes, a novel crystallographicsolution to a problem in functional morphology.

It should be borne in mind that directional sight requireseither a spherical apparatus for the lens or an array of lensessuch as those found in a compound eye. The trilobitesevolved diversely shaped eyes which consist of large arraysranging from globular, crescent to high convex dishes(Clarkson et al. 2006). Some eyes were located on stalksso that the animal could lie concealed in the water with onlythe eyes scanning the water horizon above.

Through time, diverse animals groups evolved their ownvisioning systems with materials other than calcite. Recently,however, calcite microlenses serving as photoreceptors havebeen discovered in the brittle stars (Aizenberg et al. 2001). Itis therefore interesting to observe how the birefringentcalcite biomineral was used for over 300 million years toform arthropod eye lenses and is still used as a form ofphotoreceptor by marine echinoderms.

4. Molluscan shell structures: Some insights

Molluscan shells have played an important role in thedevelopment of the field of biomineralization simplybecause of their ready availability, ease of study and the factthat mollusks are found in a variety of environments andhave extremely varied shell structures adapted toecologically specific environments (Carter 1980).Additionally, the discipline has an obvious application inthe pearl industry where oysters belonging to the familyPteriidae and other Bivalvia have been cultured to producecommercially viable products (Dakin 1913; Simkiss andWada 1980). In this regard studies on the formation of nacrehad an early start (Wada 1972) and it has recently beenshown (Fryda et al. 2009) that the controlling mechanismsfor nacre formation have been stable in deep time since theTriassic. Taylor and Layman (1972) in a comprehensivereview have discussed the mechanical properties of bivalveshells, giving a detailed account of their varied shellstructure and the adaptation to specific habitats andenvironments. The last four decades have seen a quantumincrease in the number of articles dealing with form, function

�Figure 1. Generalized schematic diagram showing general featuresand the structure of the trilobite eye. (A) Oriented transparent crystalliteof calcite (var. Iceland Spar) aligned along C-axis. Light entering thecrystallite (a few micrometers in length) in any other direction than theC-axis will split into two components causing ‘double imaging’. (B)Calcite lens with intralensar bowl, refractive index (R.I.) of theintralensar bowl is 1.63 slightly less than that of the rest of the lens(R.I.= 1.66). (C) Compound lenses arranged in symmetrical array, somemm across. (D) The bulbous schizochroal eyes of Phacops ( width ofcephalon about a cm across) as seen from the anterior. This personalspecimen is ‘rolled’, i.e. head and tail are juxtaposed in an effort toprotect the soft ventral area.

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and structural aspects of invertebrate shell structure usingadvanced imaging and theoretical modeling techniques andinstrumentation (Aizenberg et al. 2001; Checa 2002;Ubukata 2005).

In this brief overview, issues dealing with modernapproaches to understand structural complexities of shellarchitecture and the general control of molluscan neuralsystems on form, shape and ornamentation patterns of shellswill be touched upon. Chen et al. (2004) have shown theapplication of bivalve structures to the ceramic industry andhow biomimetics can help to solve engineering problems.Chateignera et al. (2000) used X-ray diffraction tocharacterize the crystallite architecture of many molluscangroups including the monoplacophorans, bivalves,gastropods and cephalopods, and came to the conclusionthat the textural patterns in the forms studied can bequantitatively determined based on a few parameters witharea specific parts of the shell having different strengthcapabilities and with diverse crystallographic orientations.Boettigera et al. (2009), in an important study, integrateddata from the neural system of the mollusks that secretes theshell, namely, the tongue-like mantel, and showed how thenerve fibres control the shell making processes for each ofthe different segments. Checa (2000) discussed thecrystallographic orientations of the calcite crystallites insome unionids and showed how the texture and the structureof the shell can be characterized using X-ray diffractometerin terms of crystal orientation of the Bragg diffraction planes001 and 112 across long shell distances.

Calcite is one of the most common building blocks of theskeleton and produces a variety of robust and stress resistantstructures both in thin and thick-shelled molluscs.

5. Dental enamel

Tooth enamel has been a focus of attention since the 19thcentury as relevance to evolution and dental anatomynecessitated a comprehensive approach to the formation,structure and architecture of individual teeth and completedentitions in the context of human and other mammalianenamel diversity (Boyde 1964; Koenigswald and Sander1997). In dental systems as well, selective pressures workat all levels of complexity as they do in Mollusks, discussedearlier. Analogous to the calcite crystallites used by mollusksand other marine organisms to build their shell structure, themineral bioapatite is the fundamental building block of allvertebrate skeletal structures (Selvig 1970) with a fewnotable exceptions, namely, fish ear bones (= otoliths) and,of course, amniote eggshells. The bio-apatite crystallite isnow a focus of attention and it has been characterized usingRaman spectroscopy (Thomas et al. 2011) and represents thefundamental building block of teeth and bones giving basic

information on structure and biomechanics. Dumont et al.(2011) have examined whether the sizes of the apatitecrystallites is the same in large and small animals such asthe giant sauropod dinosaurs and the small mammal.Similarly, Mishra and Knothe-Tate (2008) conducted studieson several fossil forms to find out the relationships betweenosteons (bone cells) and Haversian Canal diameters withreference to body weights. They have demonstrated that withincreasing weight or size of the animal, the osteon andHaversian Canal size diameter decreases per unit bodyweight. These are fundamental issues in skeletonarchitecture which are only now being examined.

One of the hardest biomaterials known, the study of dentalenamel has been influenced greatly by individual contributions(Korvenkontio 1934; Shobusawa 1952; Boyde 1964) as wellas some dedicated work at the institutional level, for example,the Steinmann Institute of the University at Bonn, where forover four decades, research on biomineralization andmammalian dental enamel diversity has been undertaken(Koenigswald 1997a, b). As a result, there is a high level ofunderstanding of the biomechanics, the functional gross andfine architectural framework of the teeth of variousmammalian group (Rensberger and Koenigswald 1980;Koenigswald et al. 1987; Koenigswald 2004; Koenigswald2012; and Koenigswald and Sander 1997 and articles therein)at various observational scales of study and at different levelsof complexity (Koenigswald and Clemens 1992). Thestructure and function of teeth and the dentition, in fact thewhole skull, is specifically optimized for diet. It is interestingto note that the diet dictates the way the enamel is strengthenedat all levels of complexity even in phyletically unrelatedgroups. For example, the response of selective pressures formammals eating grass is normally by the possession of ever-growing incisors and hypsodonty (increase in tooth height)and a complex characteristic internal crystallite structure(Sahni 1986; Koenigswald 2004). This fact is illustrated bythe similarity of architectural plans for building efficientdentitions for eating grasses which have a high silica contentin the case of rodents (Koenigswald 2004) and an unrelatedextinct group of primitive mammals (gondwanatheres) in theIndian latest Cretaceous (Prasad et al. 2005; Krause et al.1997; Patnaik et al. 2001).

Of the vast literature encompassing the field the currentsection, in this brief review, I will focus on two aspects inwhich I have been interested (Sahni 1979, 1988) : theevolution of mammalian dental enamel (Sahni 1987) andthe characteristics of rodent dental enamel (Sahni 1986)which by structural design is one of the most complex andsophisticated systems known. This evolutionary complexityhas come about because, unlike reptiles that continuouslygrow and shed their teeth, the origin of enamel structure inmammals is a result of the fact that we have only one set ofdeciduous teeth. In the permanent dentition, teeth come in



contact through life (occlude) and therefore must last untilthe life of the animal. There is need therefore forreinforcement and support of these structures (Sander1997; Sahni 1987) against fracture and continued occlusionstress during mastication. The teeth in mammals in both theupper and lower dentitions have evolved special structuresknown as ‘prisms’ in order to counteract the stresses. Prismsare basically ‘ bundles’ of similarly oriented bio-apatitecrystallites (some nanometer in size) of varying shapesranging from circular, elongate to keyhole shaped. Theprisms are surrounded by the interprismatic layer orientedusually at an angle to the plane of the prismatic layer, acondition that is technically known as decussation, leadingto what appears to be a closely knit interwoven architecture(figure 2B, C and D).

Rodent enamels are one of the hardest biomaterialsknown and their mechanical properties have been studied

in some detail. Rodents are known from the early Tertiaryonwards (Sahni 1980; Kotlia and Sahni 1993; Koenigswald2004) and have the same basic body form since their earlyevolution and this includes the shape of the skull and the factthat since the teeth are ever-growing, the skull and lower jaware suitably modified with the incisors having a groovedspace to accommodate them (figure 2A).

The pointed apex of the incisor is maintained as it formsthe interface between the harder enamel which is made up ofstructured apatite crystallites and the dentine which is moreporous and more prone to wear. In any abrasive action theenamel will erode less than the dentine and therefore thepoint will remain ever-sharp. Rodent enamel has beenstudied by several workers mainly because it is easy to doso as the incisors grow throughout life. It also providesinsights into the mechanism by which apatite crystallitesare secreted from cells known as ameloblasts. These prisms

Figure 2. (A) Skull structure of the common rat illustrating the structure of the dentitions and the pointed upper and lower incisors.Cranial components, jaw structure, attachment areas for jaw musculature, the diastema between the incisors and the molars make aneffective grass-eating machine. (B) Schematic section of the apex of a rodent incisor in black penciled outline showing the orientations ofthe apatite crystallites at the tip and internally in the basal enamel. Enamel thickness is variable, typically between 100–200 μm. The dentinewhich is non-crystalline provides a softer cutting edge in contrast to the enamel which leads to an ever-sharpened apex. (C) Intertwined(=decussating) prisms form a hardened structure. (D) Prisms and interprisms in a primitive multituberculate mammal showing bundles ofcrystallites occurring as rounded prisms with intervening rows of crystallites at an angle to the circular prisms (Sahni 1979). (E) Typicalbioapatite crystallite of enamel, bone crystallites are relatively smaller.

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and interprismatic layers form opposing rows either one row,a few rows or several rows thick giving the enamel its greatstrength (figure 2B and C).

The biomineral apatite is considerably more load-resistantthan biocalcite. It forms not only the delicate structuresassociated with mammalian enamel but also is able tosupport considerable weight in limbs of large animals suchas elephants and dinosaurs.

6. Eggshell structure

The calcareous shell-covered amniote egg is perhaps theacme of the evolutionary process, a self-contained livingentity that allows reproduction of life. Recent studies havefocused on the crystallographic structure of the avian anddinosaurian eggshell (Sakae et al. 1995; Dalbeck and Cusack2006). My interest in eggshells in general was sparked byfinds of dinosaur eggshells during field work in Montana formy doctoral dissertation (Sahni 1972). This experience wasuseful when, a decade later, we are able to find complete

nests of sauropods in India (Jain and Sahni 1985; Srivastavaet al. 1986; Sahni et al. 1994). Additional studies showedthat the nesting sites were spread over an area of exceeding10000 km2 extending from Kutch in the far west, through thecentral peninsular regions, to Andhra Pradesh and TamilNadu (Kohring et al. 1996). It is beyond the scope of thisbrief overview to discuss the various types of structures incalcified eggshells, which are characteristic for crocodiles,lizards, turtles, dinosaurs and birds, all of which have a fossilrecords in India (Sahni et al. 1994). In fact, the finds of thinshelled 65 ma eggshells from Kutch and Maharashtrabelonging to ornithoid dinosaurs, possibly to primitive birdsand to gekkonid lizards (Sahni et al. 1984) resulted in aresurgence of interest in eggshell structure. Within thedinosaurs themselves there are several eggshell types, inparticular the ‘ornithoid’, with a structure that isindistinguishable from that of birds. It is sometimes possibleusing scanning electron microscopy to find out if a preserveddinosaur nest had viable embryos as the inner (mammillarysurface) of the shell tends to get ‘cratered’; that is, thecalcium is leached out as the bones of the embryo grow

Figure 3. Sauropod eggshell structure of the parataxonMegaloolithus. (A) Complete egg from Balasinor, Kheda district in Gujarat, about16 cm in diameter. (B) Enlarged view of one egg in a nest to illustrate the shell structure occurring as a raised rim. (C) Microstructure of theeggshell, external surface to top of page showing spheroliths, incremental arched striations terminating in an external node. (D) shows abouttwo spheroliths composed of biocalcite crystallites arranged with their C-axis oriented radially outwards. The spheroliths impart strength tothe egg supported by the C-axis orientations of the biocalcite which has the highest compressive strength. The calcite crystallites act as onelarge crystal crystallographically and produce a uniaxial interference figure indicating that this plane is perpendicular to the c-axis. (E) Thesame view in polarized light showing a tangentially-sectioned spherolith(s) with a rounded air canal, a few micrometers in diameter abuttingit at the top right. Air canal diameters are quite variable in different species.



larger (Srivastava et al. 1986; Sahni et al. 1994). Thisinference calls for caution as other chemical reactionsthrough time (diagenesis) may result in the same condition.As the growing embryo within the shell lives and breathes,the structure, density and location of the air canals is acharacteristic feature of eggshell structure (figure 3E).

Younger bird eggshells have also been studied: Fossilostrich eggs in India and their fine structure suggested thatthese ratite birds had an origin in Gondwanaland and Indiawas one of the centres of diversification some 10 millionyears ago (Patnaik et al. 2009). Ostriches persisted in India atleast till the Late Pleistocene (Kumar et al. 1990; Sahni et al.1990).

In India, two type of eggs and nests are most common:one with spherical eggs, Megaloolithus (Sahni et al. 1994)and the other with elongated eggs, Ellipsoolithus (Loyalet al. 2000). The load stress response for the spherical eggswas worked out using finite element analysis, demonstratingthat the size of the eggs, and in particular the shape of thespheroliths, and the degree of fusion with neighbouringspheroliths impart mechanical strength (Srivastava et al.2005). The oval to ellipsoidal eggs are interesting for at leasttwo reasons: they represent meat-eating dinosaurs and showhow nest-laying behaviour incorporates biomechanicalprinciples. It is known from nests of the Mongolian dinosaurProtoceratops and other dinosaurs having elongated eggs thatthese are laid vertically so that their polar axis is perpendicularto the ground surface. This gives them added support againstcrushing loads.

The biomechanical support for eggshells comes largelyfrom the C-axis orientation of biocalcite. Air canals andporous air-filled spaces are critical for providing oxygenfor the growing embryo, and this is provided for incalcareous eggshells by channels or porous tissue withinthe calcareous biomineralized tissue.

7. Concluding remarks

In this short overview, I have merely touched upon certaintopics in which I have been interested. My own perspectiveon biomineralization as a palaeontologist stems from myinterest in fossils which are usually preserved because theyare biomineralized hard tissues (Kobayashi et al. 1993). Ihave tried to stress the fact that in order to get a overall ideaof the functional importance of a specific element, it isnecessary to look at all observational scales and all levelsof complexity to understand the way in which selectivepressures operate with crystallite orientations being the mostbasic and important; and furthermore, I have attempted toshow that interest in this interdisciplinary area is picking up(Bouligand 2004) withmore workers using modern techniquesto characterize the crystallographic, biomechanical form–

function relationships with regard to engineering applicationsand the control of neural systems on biomineral formation andcrystallite orientation under stress-field conditions. Theintroduction of mathematical models and cellular automata(Fowler et al. 1992; Ermentrout et al. 1986; Boettiger et al.2009) are providing new insights into studies of shell growth,ornamentation, and mechanical support.

In our pigeonholed world, many fields on the periphery ofmajor disciplines suffer as they do not get the same recognitionand support as mainstream subjects; biomineralizationhappens to be a case in point. However, this field has muchto offer as organisms large and small (particularly the latter)provide insights that cannot be obtained from any othersources. Biomineralization is currently facing a crisis ofidentity: it has the potential of generating interest acrossdisciplines, but for the time being, is generally neglected andmarginalized except for the efforts by some dedicatedindividuals, laboratories and institutions.

Acknowledgements

I wish to thank Prof Rajeev Patnaik (Chandigarh) for all hishelp. It was the support of many other students of mine in the1980s and early 1990s who helped to develop the field inthis country. I would like to record my deep appreciation toProfessors Alan Boyde and Wighart von Koenigswald andthe late Professor H K Erben for enduring interactions, andto Prof Duncan Murdock for his considered suggestions.Unknown reviewers have provided insights that have addedmaterially to this review and I thank them for theirpainstaking efforts.

References

Aizenberg J, Tkachenko A, Weiner S, Addadi L and Hendler G2001 Calcitic microlenses as part of the photoreceptor system inbrittlestars. Nature 412 819–822

Bajpai S, Srinivasan S and Sahni A 1997 Fossil turtle eggshellsfrom intertrappean beds of Duddukuru, Andhra Pradesh.J. Geol. Soc. India 49 209–213

Bengtson S and Zhao Y 1992 Predatorial borings in LatePrecambrian mineralized Exoskeletons Science 257 267–369

Bera MK, Bhattacharya K, Sarkar A, Samanta A, Kumar K andSahni A 2010 Oxygen isotope analysis of bone and tooth enamelphosphate from palaeogene sediments: experimental; techniquesand initial results. J. Geol. Soc. India 76 275–282

Boettiger A, Ermentrout B and Ostera G 2009 The neural origins ofshell structure and pattern in aquatic mollusks. Proc. Natl. Acad.Sci. USA 106 6837–6842

Bouligand Y 2004 The renewal of ideas about biomineralisations.C.R. Palevol. 3 617–628

Boyde A 1964 The structure and development of mammalianenamel. PhD thesis, University of London

932 Ashok Sahni


Carter JG 1980 Guide to bivalve shell microstructures; in: Skeletalgrowth of aquatic organisms. Biological records ofenvironmental change (eds) DC Rhoads and RA Lutz (NewYork: Plenum Press) pp 645–673

Checa A 2000 A new model for periostracum and shell formation inUnionidae (Bivalvia, Mollusca). Tissue Cell 32 405–416

Checa A 2002 Fabricational morphology of oblique ribs inbivalves. J. Morphol. 254 195–209

Chen B, Peng X, Wang JG and Wua X 2004 Laminatedmicrostructure of Bivalva shell and research of biomimeticceramic/polymer composite. Ceramics Int. 30 2011–2014

Chateignera D, Hedegaard C and Wenk H-R 2000 Mollusc shellmicrostructures and crystallographic textures. J. Struct. Geol. 221723–1735

Clarkson ENK 1979 The visual system of trilobites. Palaeontology22 1–22

Clarkson ENK and Levisetti R 1975 Trilobite eyes and optics ofDescartes and Huygens. Nature 254 663–667

Clarkson ENK, Levi-Setti R and Horvath G 2006 The eyes oftrilobites: The oldest preserved visual system. Arthropod Struct.Dev. 35 247–259

DeNiro M and Epstein S 1978 Influence of diet on distribution ofcarbon isotopes in animals. Geochimica et Cosmochimica Acta42 495–506

Dakin WJ 1913 Pearls (London: Cambridge University Press)Dalbeck P and Cusack M 2006 Crystallography (electron

backscatter diffraction) and chemistry (electron probemicroanalysis) of the avian eggshell. Crystal Growth Des. 62558–2562

Dumont MA, Kostka A, Sander PM, Borbely A and Kaysser-Pyzalla A 2011 Size and size distribution of apatite crystals insauropod fossil bones. Palaeogeogr. Palaeoclimatol.Palaeoecol. 310 108–116

Donoghue PCJ and Sansom IJ 2002 Origin and early evolution ofvertebrate skeletonization. Microsc. Res. Tech. 59 352–372

Dove PM 2013 The rise of skeletal biominerals. Elements 637–42

Ermentrout B, Campbell JG and Ostera G 1986 A model for shellpatterns based on neural activity. The Veliger 28 369–388

Epstein S, Buchbaum HA and Lowenstam HA 1953 Revisedcarbonate-water isotopic temperature scale Bull. Geol. Soc.Am. 64 1315–1326

Fowler DR, Meinhard H and Prusinkiewicz P 1992 Modelingseashells. Comput. Graph. 26 379–387

Fryda J, Bandel K and Frydov B 2009 Crystallographic texture ofLate Triassic gastropod nacre: evidence of the long-termstability of the mechanism controlling its formation. Bull.Geosci. 84 745–754

Goodwin DH, Schoene BR and Dettman DL 2003 Resolution andfidelity of oxygen isotopes as paleotemperature proxies inbivalve mollusk shells: models and observations. PALAIOS 18110–125

Horvath G 1996 Lower lens unit in Schizochroal trilobite eyesreduces reflectivity: on the possible optical function of theintralensar bowl. Hist. Biol. 12 83–93

Jain SL and Sahni A 1985 Dinosaurian eggshell fragments from theLameta Formation of Pisdura, Chandrapur District, Maharashtra.Geosc. J. 6 211–220

Kaufmann SA 1993 The origins of order: self organizationand selection in evolution (New York: Oxford UniversityPress) pp 1–709

Kazmieraczak J, Ittekkot V and Degens ET 1985 Biocalcificationthrough time: environmental challenge and cellular responsePalaeont. Zeitshrift 59 15–33

Kobayashi I, Mutvei H and Sahni A 1993 Fossil hard tissues:structure. formation and evolution; in Symposium Volume 29thIGC, Kyoto (Tokyo: Tokai University Press) pp 1–214

Koenigswald Wv 1997a Brief survey of the enamel diversity at theschmelzmuster level in Cenozoic placental mammals; in Toothenamel microstructure (eds) Wv Koenigswald and PM Sander(Rotterdam: Balkema) pp 137–161

Koenigswald Wv 1997b Evolutionary trends in the differentiationof mammalian enamel ultrastructure; in Tooth enamelmicrostructure (eds) Wv Koenigswald and PM Sander(Rotterdam: Balkema) pp 203–235

Koenigswald Wv 2004 Enamel microstructure of rodent molars,classification, and parallelisms, with a note on the systematicaffiliation of the enigmatic Eocene rodent. Protoptychus J.Mammal. Evol. 11 30–36

Koenigswald Wv 2012 Unique differentiation of radial enamel inArsinoitherium (Embrithopoda, Tethytheria). Hist. Biol. 25 1–10

Koenigswald Wv and Clemens WA 1992 Levels of Complexity inthe microstructure of mammalian enamel and their application inthe study of systematic. Scan. Microscopy 6 195–218

Koenigswald Wv and Sander PM 1997 Tooth enamelmicrostructure (Rotterdam: Balkema) pp 1–280

Koenigswald Wv, Rensberger JM and Pretzschner HM 1987Changes in the tooth enamel of Paleocene mammals allowingincreased diet diversity. Nature 328 150–152

Kohring R, Bandel K, Kortum D and Parthasarthy S 1996 Shellstructure of a dinosaur egg from the Maastrichtian of Ariyalur(Southern India). Neues Jahrbuch Geologie und Palaeontologie1 48–64

Korvenkontio VA 1934 Mikroskopische Untersuchungen anNagerincisiven unter Hinweis auf die Schmelzstruktur derBackenzahne. Ann. Zool. Soc. Zool. Bot. Fennicae Vanamo. 21–274

Kotlia BS and Sahni A 1993 Enamel ultrastructure of fossil andextant. Arvicolids Proc. 29 166

Krause DW, Prasad GVR, Koenigswald Wv, Sahni A and Grine FG1997 Cosmopolitanism amongst Gondwana Late Cretaceousmammals. Nature 390 504–507

Kumar G, Sahni A, Pancholi RK and Narvare G 1990Archaeological discoveries and a study of Late Pleistoceneostrich eggshells and egg shell objects in India. Man Environ.16 29–40

Lee MR, Torney C and Owen AW 2007 Magnesium-richintralensar structures in schizochroal trilobite eyes.Palaeontology 50 1031–1037

Loyal RS, Mohabey DM, Khosla A and Sahni A 2000 Status andpalaeobiology of the Late Cretaceous Indian theropods withdescription of a new theropod eggshell oospecies, Ellipsoolithuskhedaensis, from the Lameta Formation (District Kheda,Gujarat), Western India. GAIA Geosci. J. (Portugal) 15 1–9

Lowenstam HA and Weiner S 1989 On biomineralization (NewYork: Oxford University Press) 324 p



Mishra S and Knothe-Tate ML 2008 Comparative study of bonemicro- architecture of some mammalian bones. ThePalaeobotanist 57 299–302

Murdock DJE and Donoghue PCJ 2011 Evolutionary origins ofanimal skeletal biomineralization. Cells Tissues Organs 194 98–102

Patnaik R, Sahni A, Cameron D, Pillans B, Chatrath P, Simons E,Williams M and Bibi F 2009 Ostrich-like eggshells from a 10.1million year old Miocene ape locality, Haritalyangar (HimachalPradesh) India. Curr. Sci. 96 1485–1495

Patnaik R, Sahni A and Prasad GVR 2001 Tooth Enamelmicrostructure of a Late Cretaceous Gondwanatherian mammalfrom India. J. Palaeont. Soc. India 46 15–23

Paterson JR, Garcia-Belido DC, Lee MSY, Brock GA, Iago JB andEdgecombe GE 2011 Acute vision in the giant Cambrianpredator Anomalocaris and the origin of compound eyes. Nature480 237–240

Prasad V, Strömberg CAE, Alimohammadian H and Sahni A(2005) Dinosaur coprolites shed light on the early evolution ofgrasses and grazers. Science 310 :1177–1180

Pruss S, Finnegan S, Fischer WW and Knoll AH 2010 Carbonatesin skeleton-poor seas: New insights from Cambrian andOrdovician strata of Laurentia. Palaios 25 73–84

Rensberger JM and Koenigswald Wv 1980 Functional andphylogenetic interpretation of enamel microstructure inrhinoceroses. Paleobiology 6 477–495

Roe LJ, Thewissen JGM, Quade J, O’Neil JR, Bajpai S, Sahni Aand Hussain TS 1998 Isotopic approaches to understanding theterrestrial-to-marine transition of the earliest Cetaceans; inEmergence of whales (ed) JGM Thewissen (New York: PlenumPress) pp 399–422

Sakae T, Mishima H, Suzuki K, Kozawa Y and Sahni A 1995Crystallographic and chemical analyses of eggshell of Dinosaur(Sauropod Titanosaurids sp.). J. Fossil Res. Jpn. 27 50–54

Sahni A 1972 The vertebrate fauna of the Judith river formation.Bull. Am. Museum Natural Hist. 147 323–412

Sahni A 1979 Enamel Ultrastructure of certain North AmericanCretaceous mammals. Palaeontographica A. Stuttgart 166 7–4 9.

Sahni A 1980 SEM studies of Indian Eocene and Siwalik rodentenamels. Geosci. J. 1 21–30

Sahni A 1981 Enamel ultrastructure of fossil mammalia:Eocene archaeocete from Kutch. J. Palaeont. Soc. India25 33–37

Sahni A 1986 Enamel structure of early mammals and its role inevolutionary relationships among rodents. Proc. Int. Symp.Analysis Evol. Relation. Rodents PARIS NATO-ASI Series 92133–150

Sahni A 1987 Evolutionary Aspects of reptilian and mammalianenamel structure. Scan. Microsc. Int. 1 1903–1912

Sahni A 1988 Application of electron microscopy to problems inPalaeontology and biomineralisation; in Micropalaeontology ofshelf sequences of India (ed) P Kalia (Papyrus PublishingHouse: New Delhi) pp 1–11

Sahni A and Koenigswald Wv 1997 The enamel structure of somefossil and recent whales from the Indian subcontinent; in Toothenamel microstructure (eds) Wv Koenigswald and PM Sander(Rotterdam: Balkema) pp 177–191

Sahni A, Rana RS and Prasad GVR 1984 SEM studies of thin eggshell fragments, from the intertrappeans (Cretaceous-Tertiarytransition) at Nagpur and Asifabad, Peninsular India.J. Palaeont. Soc. India 29 26–33

Sahni A, Kumar G, Bajpai S and Srinivasan S 1990 A review ofLate Pleistocene ostriches (Struthio sp) in India. Man Environ.15 41–47

Sahni A, Tandon K, Jolly A, Bajpai S, Sood A and Srinivasan S1994 Upper Cretaceous dinosaur eggs and nesting sites fromthe Deccan Volcano - Sedimentary province of PeninsularIndia; in Dinosaur eggs and babies (eds) K Carpenter, KFHirsch and JR Horner (Cambridge University Press) pp 204–228

Sander MP 1997 Non-mammalian synapsid enamel and the originof mammalian enamel prisms: the bottom-up perspective; inTooth enamel microstructure (eds) Wv Koenigswald and PMSander (Rotterdam: Balkema) pp 41–62

Selvig KA 1970 Periodic lattice images of hydroxyapatitecrystals in bone and dental hard tissue, Calcif. Tissue Res.6 227–236

Shobusawa M 1952 Vergleichende Untersuchungen ueber die Formder Schmelzprismen der Sa¨ugetiere. Okajimas Folia anat Jap.24 371–392

Simkiss K and Wada K 1980 Cultured pearls – Commercialisedbiomineralisation. Endeavour New Ser. 4 32–37

Scrutton CT 1964 Periodicity in Devonian coral growth.Palaeontology 7 552–558

Srivastava S, Mohabey DM, Sahni A and Pant SC 1986 UpperCretaceous dinosaur egg clutches from Kheda District, Gujarat,India: their distribution, shell ultrastructure and palaeoccology.Palaeontographica A 193 219–233

Srivastava R, Sahni A, Jafar SA and Mishra S 2005Microstructure-dictated resistance properties of some Indiandinosaur eggshells: finite element modeling. Paleobiology31 317–325

Taylor JD and Layman M 1972 The Mechanical Properties ofthe Bivalve (Mollusca) shell structures. Palaeontology 1573–87

Thewissen JGM, Roe LJ, O'Neil JR, Hussain ST, Sahni A andBajpai S 1996 Evolution of Cetacean osmoregulation. Nature301 379–380

Thomas DB, Cushla M, McGoverin M, Fordyce E, Frew RD andGordon KC 2011 Raman spectroscopy of fossil bioapatite — Aproxy for diagenetic alteration of the oxygen isotopecomposition Palaeogeogr. Palaeoclimatol. Palaeoecol. 31062–70

Torney C, Lee MR and Owen AW 2008 Peering into the eyes oftrilobites using EBSD. In 15th International Conference on theTextures of Materials, 1-5 June 2008, Carnegie MellonUniversity Center, Pittsburgh, Pennsylvania USA (http://eprints.gla.ac.uk/4523/)

Ubukata T 2005 Theoretical morphology of bivalve shellsculptures. Paleobiology 31 643–655

Urey HC 1947 The thermodynamics of isotopic substances.J. Chem. Soc. 0 562–581

Xi-Guang Z and Clarkson ENK 1990 The eyes of the LowerCambrian eodiscid trilobites. Palaeontology 33 911–932

934 Ashok Sahni


http://eprints.gla.ac.uk/4523/2009:1%E2%80%938

http://eprints.gla.ac.uk/4523/2009:1%E2%80%938

Young JR, Davis SA, Bown PR and Mann S 1999 Coccolithsultrastructure and biomineralization. J. Struct. Biol. 126195–215

Wada K 1972 Nucleation and growth of aragonite crystals in thenacre of some bivalve mollusks. Biomin. Res. Rep. 6 141–159

Wells JW 1963 Coral growth and geochronometry.Nature 197 948–950

MS received 23 March 2013; accepted 09 October 2013

Corresponding editor: DURGADAS P KASBEKAR



biomineralization: some complex crystallite-oriented skeletal structures

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