Biomimetism, biomimetic matrices and the induction of bone formation

Bone formation by induction initiates by invocation of osteogenic soluble molecular signals of the transforming growth factor-β (TGF-β) superfamily; when combined with insoluble signals or substrata, the osteogenic soluble signals trigger the ripple-like cascade of cell differentiation into osteoblastic cell lines secreting bone matrix at site of surgical implantation. A most exciting and novel strategy to initiate bone formation by induction is to carve smart self-inducing geometric concavities assembled within biomimetic constructs. The assembly of a series of repetitive concavities within the biomimetic constructs is endowed with the striking prerogative of differentiating osteoblast-like cells attached to the biomimetic matrices initiating the induction of bone formation as a secondary response. Importantly, the induction of bone formation is initiated without the exogenous application of the osteogenic soluble molecular signals of the TGF-β superfamily. This manuscript reviews the available data on this fascinating phenomenon, i.e. biomimetic matrices that arouse and set into motion the mammalian natural ability to heal thus constructing biomimetic matrices that in their own right set into motion inductive regenerative phenomena initiating the cascade of bone differentiation by induction biomimetizing the remodelling cycle of the primate cortico-cancellous bone.


Introduction: the induction of bone formation, the emergence of the skeleton, of the vertebrates and of Homo species
In a number of reviews on the induction of bone formation, we have often asked how bone differentiation by induction is initiated? Or how the soluble molecular signals of the transforming growth factor-␤ (TGF-␤) supergene family are deployed so as to initiate de novo bone formation by induction [1][2][3]? Somehow more simply but very confusing in a scenario of redundancy of multiple soluble molecular signals initiating bone differentiation by induction in the primate, which are the molecular signals that initiate de novo bone formation by induction [1][2][3][4][5][6][7][8][9][10]?
To initiate the induction of bone formation and thus to ultimately erect the skeleton, Nature has had a powerful lesson to teach. A lesson that is continuously taught to biomaterial scientists, molecular biologists and tissue engineers alike, who wish to design, manufacture and sculpt new tissue constructs for replacement of lost parts in human patients [2,5,[11][12][13][14][15][16][17]. The induction of bone formation requires three key components [17]: an osteoinductive soluble signal, an insoluble signal or substratum and responding host's cells. The insoluble signal delivers the osteogenic soluble molecular signal and acts as a scaffold for bone formation to occur after transmembrane serine-threonine kinase receptors' phosphorilation of responding host's cells [1,12,14,16,17].
The osteogenic soluble molecular signals of the TGF-␤ supergene family need thus to be reconstituted, more figuratively perhaps, recombined, with an insoluble signal or substratum to initiate the cascade of bone differentiation by induction [9][10][11][12][17][18][19]. This fundamental rule in molecular and cellular biology has now become the cardinal rule to initiate tissue morphogenesis after the molecular dissection of the fascinating phenomenon of 'bone: formation by autoinduction' [20], though regretfully not always completely understood. The classic experiments of the dissociative extraction and reconstitution of the bone matrix components showed that partially purified [1,[21][22][23][24] or highly purified recombinant human bone morphogenetic proteins (BMPs) [25,26] need to be reconstituted with an insoluble signal or substratum to trigger the cascade of bone differentiation by induction [21]. The experiments were also critical to learn that putative BMPs within the bone matrix could be solubilized by the dissociative extraction of the bone matrix [17,21,22]. This has set into motion a ripple-like cascade of biochemical, chromatographic and molecular events that ultimately has resulted in the isolation, characterization and molecular cloning of an entirely new family of protein initiators collectively called the BMPs/OPs [17][18][19][25][26][27].
When writing about osteoinduction, it is important to properly define the terminology related to 'bone: formation by autoinduction' [20]. The acid test of the induction of bone formation is the de novo generation of endochondral bone after heterotopic extraskeletal implantation of the osteogenic soluble molecular signals of the TGF-␤ supergene family [5,17,18]. The heterotopic implantation site avoids the ambiguities of the orthotopic site where some degree of bone formation by conduction may occur from the viable bone interfaces [20].
The dissociative extraction and reconstitution of the bone matrix components [21] and of the homology of BMPs/OPs among mammals [22] have provided the key to implement the phenomenon of the induction of bone formation in pre-clinical and clinical contexts [17][18][19]. Before implantation, recombinant human BMPs/OPs, either hBMP-2 and/or hOP-1, [39,40] need to be reconstituted with a variety of biomaterial matrices to form the osteogenic device for orthotopic orthopedic and craniofacial applications in clinical contexts [2-5, 17, 39-41]. Alternative approaches are continuously under laboratory and pre-clinical evaluation, i.e. directing the differentiation of stem cells into the osteogenic lineage firstly in vitro and then in vivo when implanted in skeletal defects of animal models [42,43]. Additional approaches are based on the manipulation of a deliberately created space within the body, such that it serves as an in vivo bioreactor [44]. Osteogenesis forms within the surgically manipulated space of the bone bioreactor, which deploys the essential ingredients to induce bone formation, i.e. osteogenic soluble molecular signals, responding cells and the extracellular matrix scaffolds of the operated patients. The induced bone is later transplanted in osseous defects of the same patient [44]. The in vivo bone bioreactor has available a mesenchymal layer rich in pluripotent cells endowed with the potential to rapidly differentiate into osteoblastic-like cells secreting bone matrix remodelled into newly formed bone for autogenous transplantation [44].
Custom-made bone grafts grown in extraskeletal sites in human beings by hOP-1 and marrow aspirated from the iliac crest have been implanted in the latissimus dorsi muscle [45]. Heliotis et al. [46] went further by demonstrating that with a single recombinant OP combined with a porous hydroxyapatite (HA) carrier, a prefabricated heterotopic bone graft could be constructed in a human outside the skeleton without the addition of cortical bone, bone marrow aspirates or any other bone precursors to engineer a custom-made prefabricated bone flap for human mandibular reconstruction (Fig. 1) [46]. characterized for naturally derived and several human recombinant BMPs/OPs in different animal models [3,5,17,19,25,26]. Importantly and conclusively, in the bioassay for bone induction in rodents, the TGF-␤ isoforms do not initiate the induction of bone formation [47]. In marked contrast, however, the mammalian TGF-␤ isoforms do induce rapid and substantial endochondral bone formation when implanted in heterotopic extraskeletal sites of Papio ursinus [3,5,18,48]. In the primate, the TGF-␤ isoforms may act upstream to the BMPs/OPs and may induce the induction of heterotopic bone by expressing BMPs/OPs-related gene products resulting in the cascade of bone differentiation by induction [49][50][51]. Heterotopic implantation of the three mammalian TGF-␤ isoforms results in expression of BMP-3 and OP-1 as evaluated by Northern blot [49,50] and RT-PCR analyses [51]. The presence of several molecularly related but different proteins with osteogenic activity in the primate [3,5,18] poses important questions about the biological significance of this apparent redundancy, additionally indicating multiple interactions during both embryonic development and the induction of bone formation in postnatal life [5,18].
We have shown a potent synergistic induction of endochondral bone formation after binary applications of hOP-1 and TGF-␤1 in both heterotopic and orthotopic sites of the non-human primate Papio ursinus [48,49]. The level of tissue induction induced by hOP-1 was raised several fold by the binary application of comparatively low doses of the TGF-␤1 isoform implanted in the rectus abdominis muscle of adult baboons [48]. The rapid induction of large corticalized ossicles in the rectus abdominis muscle of nonhuman primate species using binary applications of osteogenic molecular signals has important clinical implications. In the human, the primate Homo sapiens, regenerative phenomena are slower than in several other animals including non-human primate species. In human patients, regenerative medicine is often ineffective with delayed healing, contamination and scarring. The rapidity of tissue morphogenesis complete with mineralization of the outer cortex and bone marrow formation by binary application of recombinant hOP-1 and relatively low doses of hTGF-␤1 indicates that osteogenesis and bone induction in Homo sapiens should rather be engineered by the synergistic induction of bone formation [3,52,53]. The latter overcomes the temporally delayed repair phenomena in human patients where healing progresses slower than in experimental animals including non-human primate species [3,48,52,53].

Biomimetism and biomimetic matrices self-assembling the induction of bone formation
A most fascinating and novel strategy to initiate the induction of bone formation is to construct biomimetic bioactive biomaterial matrices that per se initiate the morphogenesis of bone. This is initiated within the porous spaces of smart self-inducing biomaterial matrices even when implanted in heterotopic extraskeletal sites and without the addition of osteogenic soluble molecular signals of the TGF-␤ superfamily (Fig. 2) [2,3,5,7,27,54,55].  After millions of years of evolution, Nature has had the capacity to nucleate and evolve highly sophisticated tissues and organs. A classical example is the evolution of the skeleton, thus providing for the emergence of the vertebrates, deambulation and body erection freeing the upper limbs for superior foraging and more industrious Homo like activities including the use of tools for hunting, foraging above all, however, for maternal care contributing thus to the speciation of the genus Homo ultimately directing the emergence of Homo sapiens. The emergence of osteogenesis and later in evolution of the vertebrates, of the skeleton and Homo sapiens after a billion years from Drosophila melanogaster has permitted the precision self-assembly of the bone unit or osteosome [56] and the remodelling processes of the skeleton [57,58]. The concavities as sculpted in the bio-inspired biomimetic bioceramics biomimetize the remodelling cycle of the osteonic corticocancellous bone (Fig. 3) [5,8,9,59].
Nature has relied on common yet limited molecular mechanisms to direct morphogenesis and the emergence of specialized tissues and organs [3,5,[17][18][19]. The BMPs/OPs reflect Nature's parsimony in controlling multiple specialized functions or pleiotropy, deploying several osteogenic molecular signals with minor variation in amino acid motifs within highly conserved carboxy terminal regions [2,5]. The pleiotropic activity of the BMPs/OPs gene products is vast and spans from neurothropism to nephrogenesis, from cementogenesis to chondrogenesis, from air follicle induction to tooth morphogenesis, from angiogenesis to neurogenesis and from osteogenesis to cardiogenesis. The pleiotropic activity of the secreted proteins indicates that they are critical in development and are involved in several unrelated events that control pattern formation in embryonic development, morphogenesis and regeneration in postnatal life [3,5,[17][18][19].
The evolutionary conservation of the OPs of the TGF-␤ superfamily is superbly demonstrated by the remarkable observation that recombinant decapentaplegic and 60A proteins, gene products of the fruit fly Drosophila melanogaster, induce bone formation in mammals [60].
Decapentaplegic and 60A in D. melanogaster are highly homologous to BMP-2, BMP-4 and BMP-5 and BMP-7, respectively, indicating the primordial role of BMPs/OPs sequences for the emergence of the vertebrates [5,60]. Nature has thus usurped phylogenetically ancient amino acid sequences deployed for dorsal-ventral patterning in D. melanogaster to set the unique vertebrate trait of the induction of bone and of the skeleton rather than evolving novel gene products initiating the induction of bone formation [5,60]. The skeleton with its supramolecular assembly of structural proteins, collagens and vascular structures permeating the osteonic walls bathed by the bone marrow organ is a superior example of design architecture and engineering [56]. The skeleton has evolved through millions of years of evolution and the extant skeleton has appeared as a result of expression and secretion of complex soluble molecular signals. Molecular signals interacting with insoluble signals or substrata of the extracellular matrix of bone populated by several different responding cells within the bone bone/marrow organ. The remodelling of the skeleton, the formation of bone by osteoblasts and the resorption of bone by osteoclasts, is a closely integrated homeostatic system [56]. When thinking about the molecular cell biology of bone remodelling and maintenance, it is important to visualize the geometric pattern of the remodelling cycle as initiated in any given time on each trabecula of the cortico-cancellous bone during the remodelling cycle (Fig. 3). The sequential phases of bone remodelling in the primate cortico-cancellous bone are quiescence, remodelling activation, resorption, reversal, formation/induction and quiescence again [56][57][58].
Remodelling of the cortico-cancellous bones entails, at any given time along the trabeculae of bone, three fundamental molecular, cellular and morphological processes that characterize the remodelling cycle of the primate cortico-cancellous bone: (i) resting, i.e. surfaces lined by resting lining cells as yet to be differentiated; (ii) resorption, i.e. areas of trabeculae actively resorbed by activated osteoclasts. Of critical importance for tissue engineering of bone, the bone resorption lacunae as formed by osteoclastogenesis are in the form of concavities (Fig. 3) [8,9,[57][58][59]. The concavities are thus regulators of bone initiation and deposition during the remodelling processes of the skeleton.
Independently, other research groups using different bioceramics and animal models reported the intrinsic induction of bone formation [62][63][64][65][66][67][68][69]. de Groot [70] hypothesized that after heterotopic implantation of calcium phosphate ceramics in vivo, there is 'de-and re-mineralization processes that may concentrate and immobilize the experimental animal's own naturally derived BMP/OP complex' [70]. Whether BMPs/OPs attached to biomimetic matrices implanted heterotopically in animal models are originating from systemic circulation and/or extracellular matrices invading the porous spaces and concavities or de novo expressed in cellular elements resting on the biomimetic matrices, has been elucidated by Northern blot analyses showing the local expression of mRNA of osteogenic soluble molecular signals in tissues formed within the concavities of the implanted substrata [61]. Concavities are endowed with the striking prerogative of de novo initiating bone differentiation by induction in heterotopic extraskeletal sites of adult non-human primates Papio ursinus (Figs. 2, 3 and 7) [5,27,55,61]. Our systematic studies have shown that the driving force of the intrinsic induction of bone formation by bioactive biomimetic matrices is the shape of the implanted substratum; the language of shape is the language of geometry; the language of geometry is the language of a sequence of repetitive concavities which biomimetizes the remodelling cycle of the primate osteonic bone (Figs. 2, 3 and 7) [5,8,9,55,61,71].
The term biomimetism has been recently introduced in the lexicon of regenerative medicine and tissue engineering with the intended meaning of the creative imitation of various specific biological systems [72][73][74]. Biomimetism is particularly appealing in the creation and assembly of biomaterials matrices which are endowed with specific smart functionalities as imparted by the

(C, D and E) Cortico-cancellous bone remodelling and osteoclastogenesis (blue arrows) in extant non-human primate Papio ursinus. Concavities induced by osteoclastogenesis along trabeculae of bone biomimetize the concavities prepared in biomimetic bioceramics setting formation after resorption (magenta arrows in D and E), (F) self-inducing the spontaneous induction of bone formation (blue arrows) in biomimetic matrices when implanted in heterotopic extraskeletal sites of an adult baboon Papio ursinus. (F) Arrows indicate the rich vascular network and capillary sprouting that always correlate with the induction of bone formation (magenta arrows) within the porous biomimetic matrices. Original magnification: (
biomimetic organization coupled to the bioactive matrices. Biomaterial biomimetic matrices biomimetize specific biological systems with multifunctional shape memories with self-assembly capacities initiating and promoting angiogenesis and osteogenesis. The induction of angio-osteogenesis exploits and recapitulates events that initiate the induction of bone formation. Bone forms in the concavities as initiated by osteoclastogenesis during the remodelling cycle of the osteonic cortico-cancellous bone of nonhuman and human primates (Figs. 2 and 3) [5,9,17,61,71]. (Fig. 2) [7-10, 55, 61, 71].

In collaboration with the Council for Scientific and Industrial Research division of Materials Science and Technology we have thus encapsulated into solid matrices repetitive sequences of concavities as biologically programmed morphogenetic cues that result in the differentiation of resident mesenchymal cells into osteoblastic-like cells expressing, secreting and embedding soluble osteogenic molecular signals into the concavities initiating bone formation by induction as a secondary response
We have proposed that there is a direct spatial and temporal relationship of molecular and morphological events that emphasize the pronounced biomimetism of the geometric induction of bone formation, i.e. the induction of bone in smart concavities assembled in biomimetic matrices with the remodelling cycles of cancellous bone (Fig. 3) [8-10, 59, 61, 71]. The basic multicellular unit of the cortico-cancellous bone excavates a trench across the surface rather than a tunnel leaving in its wake (with some degree of geometrical latitude) a hemi-osteon rather than an osteon [58], i.e. a trench with a cross-sectional geometric cue of a concavity at different stages of osteoclastogenesis, i.e. concavities with different radii of curvatures and depths as induced by osteoclastic activity.
Predating formation during the remodelling cycle of primate cortico-cancellous bone, osteoblasts eventually appear at the resorption site, i.e. lacunae with a geometric configuration of concavities (Fig. 3). Which are the molecular signals and/or physical forces imparted by the geometric topography of the substratum that terminate osteoclastogenesis and recruit osteoblastic-like cells initiating bone formation by induction within the concavities (Fig. 3)?
The available molecular and morphological data on the subject from several research groups have permitted to formulate the following conceptualization of the spontaneous induction of bone formation within porous biomimetic matrices: the net result of the induction of bone formation in concavities of the substratum is nothing but the language of geometry set by the concavities assembled within the biomimetic biomaterials.
In the adult skeleton, the demand of osteoblasts is created by bone resorption, i.e. the concavities induced by osteoclastogen-esis, whereas the demand of osteoclasts is governed by the purpose of bone remodelling [58]. The concavities assembled in the biomimetic matrices are endowed with multifunctional pleiotropic self-assembly capacities initiating and promoting angiogenesis and differentiating resident mesenchymal cells into osteoblastic cell lines expressing, secreting and embedding osteogenic molecular signals within the concavities of the biomimetic matrices [27,61,71]. The molecular and morphogenetic mechanisms initiating the spontaneous induction of bone formation within concavities of the smart biomimetic matrices originate and progress with blood vessels and capillary sprouting developing within the mesenchymal tissue invading the concavities [7][8][9][10]. The extracellular matrix components of type IV collagen and laminin around the invading capillaries bind morphogenetic proteins involved both in angiogenesis and osteogenesis [59,71,[75][76][77][78][79]. Bound morphogenetic proteins are then presented locally in an immobilized form to responding mesenchymal cells and osteoprogenitors alike to initiate osteogenesis in angiogenesis [5,9,59,71].

Morphogenetic progression is sustained by continuous recruitment of mesenchymal cells and capillary invasion within the concavities. Resting mesenchymal cells attach to the matrix and differentiate into osteoblastic-like cells within the concavities of the biomimetic matrices. Differentiating osteoblastic-like cells express
OPs of the TGF-␤ superfamily; mRNA expression, as evaluated by Northern blot analyses, is then followed by secretion and embedding of the expressed gene products within the smart concavities of the biomimetic matrices ( Fig. 2D and E) [61,71]. The induction of bone formation then follows as a secondary response [2,5,27,55,71].
Synthetic biomimetic matrices mimic the super-smart functionality of living tissue and allow the engineering of smart selfinducing biomimetic matrices for tissue engineering of bone [9]. The assembly of a series of repetitive concavities within porous biomimetic matrices adds selected functionalities or super-smart functionalities to the biomimetic matrices that intrinsically per se induce the spontaneous induction of bone formation without the exogenous application of osteogenic soluble molecular signals of the TGF-␤ superfamily (Figs 2 and 7) [9]. A critical step is the embedding of smart biological functions within intelligent scaffolds for tissue engineering of bone, i.e. embedding biological signals into biomaterials designed with super-smart biomimetic functionalities, ultimately resulting in the intrinsic induction of bone formation [61,71].
Secreted BMPs/OPs within the concavities of the biomimetic matrices may be the result of osteoblastic-like cell differentiation or macrophages/osteoclasts expressing selected BMPs/OPs during the remodelling cycle of the primate cortico-cancellous bone. Macrophages/osteoclastic cells are known to express BMPs/OPs during the remodelling cycle of the cortico-cancellous bone [80]. Macrophages and osteoclastic cells are continuously involved during the osteointegration processes of implanted biomaterials [81] and are essential for effective tissue regeneration as they regulate the recruitment, proliferation and attachment of fibroblasts, osteoblasts and endothelial cells, significantly contributing to construct osteogenesis in angiogenesis [81][82][83][84][85]. Whether BMPs/OPs are secreted and embedded into the smart concavities by differentiating osteoblast-like cells or macrophages/osteoclastic cells still deserves experimental investigation. The embedded soluble molecular signals will then differentiate osteoblastic-like cells, further secreting bone matrix and the induction of bone formation as a secondary response. Besides BMPs/OPs [82,83], macrophages are also known to secrete TGF-␤1 [86]. While the evidence is still lacking, it is tempting to suggest that the induction of bone within concavities of smart biomimetic matrices recapitulates embryonic development by expressing and secreting BMPs/OPs synchronously and synergistically with TGF-␤ proteins, initiating the synergistic cascade of bone differentiation [18,48].

Influence of geometry on the expression of the osteogenic phenotype
Experiments by Reddi and Huggins [87] have shown that the temporal sequence of fibroblast-chondroblast-osteoblast transformation is profoundly influenced by the geometry of the transformant, i.e. demineralized rodent incisors [87]. The specific geometric configuration of the inductor ultimately results in the induction of endochondral bone either with an anlage of cartilage or bone with bone marrow without the induction of chondrogenesis [87]. Subcutaneous implantation of coarse demineralized bone matrix powders (particle size 420-850 m) resulted in the local differentiation of endochondral bone. On the other hand, implantation of fine matrix with particle size of 44-74 m did not induce the bone [87].
Since implantation of fine demineralized bone matrix (particle size 44-74 m) did not induce endochondral bone differentiation [87], it was interesting to determine whether extracts of fine bone matrix contain endochondral bone differentiation activity or, if the geometry of the implanted matrix particles solely drives the induction of bone [88]. When protein extracts of fine matrix were combined with coarse inactive collagenous matrix residues, there was restoration of the induction of endochondral bone formation [88]. Fine particles thus contain inductive proteins and the geometry of the matrix carrier is critical for the initiation of the bone induction cascade [88].
In previous experiments, we have shown that the biological activity of BMPs/OPs could be expressed and delivered by a substratum other than the insoluble collagenous bone matrix as carrier [90]. The dramatic differences observed between substrata of granular and disc configuration underscored the importance of geometry in bone formation by induction [90]. Substrata of coralderived HAs in disc configuration reconstituted with bovine osteogenic fractions purified greater than 50,000-fold with respect to crude guanidinium extract induced heterotopic endochondral bone formation when implanted subcutaneously in rodents [90]. Identical coral-derived HAs but in granular configuration did not induce bone differentiation even if pre-treated with identical doses of highly purified naturally derived BMPs/OPs [90]. Remarkably thus, the geometric configuration of the substratum can inhibit and overrule the osteogenic activity of BMPs/OPs both in rodents and non-human primates Papio ursinus [90,94].
Identical coral-derived porous HAs with distinct geometric configurations were implanted in the rectus abdominis muscle of adult baboons. Bone differentiation occurred only in blocks of HA in rod configuration [94]. As in the rodent bioassay, though without the addition of highly purified osteogenic fractions, implants of particulate granular HA implanted in the rectus abdominis muscle of adult baboons, failed to induce bone differentiation within the porous spaces [94]. Instrumental for our understanding of the critical role of the concavity in driving the cascade of bone differentiation, minimal yet some bone formation was only found in a concavity of a particulate granular coral-derived HA specimen harvested on day 90 from the rectus abdominis muscle (Fig. 4H) [94]. The lack of bone differentiation in implants of granular HA implies a critical role of implant geometry on bone differentiation by induction [90,94]. This has important implications for the construction of appropriate porous bone substitutes for reconstructive bone tissue engineering in clinical contexts [90,94].
Predating the induction of bone formation, and in close proximity to developing bone, there is always a rich capillary network invading the porous spaces of the biomimetic matrix, particularly in concavities of the substratum [27,54,55]. Angiogenesis and vascular invasion are prerequisites for osteogenesis [34,95]. Trueta has stressed the importance of the blood vessels in osteogenesis, and defined the vascular invasion during bone formation as osteogenetic vessels, suggesting that the endothelium may be capable of osteoblastic differentiation [34]. While circumstantial evidence is lacking, it is tempting to suggest that osteogenetic vessels, penetrating the porous spaces of the substratum, might have provided a temporally regulated flow of cell populations capable of expression of the osteogenic phenotype (Figs 2H and  4C, D) [34,54,95]. (Fig. 4) [54,96,97]. The development of mesenchymal condensation at the HA interface is a critical developmental event predating the initiation of spontaneous bone differentiation in porous bioceramics when implanted in the rectus abdominis muscle of Papio ursinus [54,87,88]. Indeed, mesenchymal condensations are critical for the initiation of skeletal development [98].

The implantation of calcium carbonate coral-derived HAs in the rectus abdominis of adult baboons showed that osteocyte-like cells are embedded within a tissue that had intermediate features between fibrous tissue and bone
By including the analysis of early periods of observation (i.e. days 30 and 60), morphological and histochemical data have shown that the differentiation of large, hyper-chromatic and intensely alkaline phosphatase positive cells at the HA interface is a critical morphogenetic event preceding the differentiation of bone ( Fig. 4B and C) [54]. A critical step during the developmental cascade of the spontaneous and/or intrinsic bone induction in porous bioceramics without the addition of exogenously applied BMPs/OPs is the differentiation of resident mesenchymal cells in contact with the biomimetic substrata into osteoblastic cell lines resting upon the surface of the implanted matrices ( Fig. 4B and C) [54,61]. We now propose the following cascade of molecular, cellular and morphological events culminating in the induction of bone formation in heterotopic sites of non-human primates Papio ursinus initiating within concavities of 'smart' biomimetic matrices: [99]. In the late 1980s, systematic studies were started in nonhuman primates Papio ursinus after the remarkable finding of intrinsic osteoinductivity in the porous spaces of coral-derived HAs (Figs. 4-6) [5,54,96,97,100,101]. Coral-derived HAs induced substantial amounts of membranous bone when implanted heterotopically in the rectus abdominis muscle of adult Papio ursinus (Figs. 5 and 6) [54,96,97,100,101]. In the same animals, coral-derived (Figs 8 and 9) and sintered porous HAs (Fig. 10) implanted in calvarial defects also induced extensive bone deposition culminating in complete calvarial regeneration [5,7,55,101].

Importantly for application in clinical contexts, the extent of the spontaneous induction of bone formation in calcium phosphate ceramics varies significantly in different animal models.
Using coral-derived HA substrata, minimal, if any, bone formation was shown in dogs and rabbits as compared to adult baboons [100]. In contrast to other studies, calcium phosphate ceramics showed bone formation within the porous spaces of the implanted matrices in canine models [70,102]. The bone also formed in direct contact of calcium phosphate ceramic particles implanted heterotopically in sheep muscles [66,103,104] and in goats [62]. Further studies on the cross-species comparison of heterotopic bone induction in biphasic calcium phosphate HA scaffolds showed that the extent of heterotopic bone formation is controlled by the animal species as well as the nature of the implanted scaffolds [67]. Implantation of calcium phosphate bioceramics in the rat heterotopic bioassay results in lack of bone differentiation although specimens harvested from heterotopic sites of murine models showed the differentiation of bone to a varying degree [67].
Several papers have stressed the importance of biomimetic matrices capable of concentrating several proteins including BMPs/OPs from the body fluids and/or the extracellular matrix [70]. It has been proposed that ceramics sintered at a lower temperature would have the ability to concentrate more circulating and/or locally produced BMPs/OPs [62,67,70]. The capacity to concentrate BMPs/OPs as found in the circulation or in the microenvironment of the extracellular matrix suggests that the incidence of the spontaneous induction of bone formation varies with animal species as well as the implanted biomimetic matrix. Additional critical parameters for the spontaneous induction of bone formation in a variety of porous bioceramics are the porosity, pore size and distribution, interconnectivity, fenestration, distribution as well as orientation of pores [62,67]. Constructs of porous ␤-TCP also showed the induction of heterotopic bone in dogs [85]. Kondo et al. have suggested that the microporous surfaces of the ␤-TCP constructs enhanced protein adsorption and cell attachment contributing to the osteoinductivity of the tested ␤-TCP biomatrices also showing in vitro that microstructure is a key factor for osteoblastic differentiation and proliferation [85].
Data from several investigations in vivo in canine and ovine models have suggested that the microporosity of the implanted bioceramics play important roles as the storage microenvironment for several extracellular matrix components, including BMPs, and are ultimately responsible for the induction of bone formation. The reason why, however, the microstructure and interconnected pores are so critical for osteoblastic-like cells differentiation, expression of OPs, and thus the induction of bone formation as a secondary response still remain unclear. Similarly, the reason for the differences in biomaterials-induced bone formation in the tested animal species is as yet to be defined [66,67,69,100]. More importantly perhaps, morphological and molecular experiments are now mandatory to assign to specific cellular elements the expression and secretion of the OPs of the TGF-␤ superfamily.
Porous biomaterials with a series of repetitive concavities with optimal surface topography and microstructure provide porous spaces that are architecturally conducive to differentiate resident mesenchymal cells into osteoblastic cell lines. Expression, secretion and embedding of osteogenic molecular signals are followed by rapid vessel ingrowths, capillary sprouting and the induction of bone formation as a secondary response [71]. The surface topography and the geometry of the concavity affect cellular morphology, and cellular shape will influence function [105][106][107] during the differentiation of osteoblastic cell lines expressing and secreting osteogenic molecular signals. The differentiation of osteoblastic cell lines expressing and secreting osteogenic soluble molecular signals of the TGF-␤ superfamily is at the crux of the intrinsic osteoinductivity of a variety of porous calcium phosphate and biphasic TCP HA bioceramics in a variety of animal models including man.
Morphological analyses of coral-derived HAs implanted in the rectus abdominis muscle of Papio ursinus showed the differentiation of large hyper-chromatic cells arranged in one or two layers directly opposed to the HA substratum [54]. Such cells, interpreted as differentiating osteoblasts, were close to a rich capillary network lined by large and hyper-chromatic endothelial cells [54].   [54,55].

The differentiation of large, hyper-chromatic and intensely alkaline phosphatase positive osteoblastic cells in an intimate relationship with endothelial cells and the implanted biomimetic matrix is a critical morphogenetic event preceding the differentiation of bone
The induction of bone is constructed by regulating the expression of selected mRNA of gene products as a function of the structure [2,9,10,61]. Biomimetic matrices of highly crystalline HAs [9,55] or biphasic HA/TCP bioceramics [61] constructed with a series of repetitive concavities offer a geometric configuration which vividly reproduces and biomimetizes the remodelling processes of the primate osteonic bone [5,8,9,57,58].
Ultimately, which are the resident mesenchymal cells capable of transformation/ differentiation into secreting osteoblastic-like cells at the HA interface? It is obvious that the rectus abdominis muscle of adult baboons is endowed with a stem cell niche [108,109] that provides a large number of differentiating cells including osteogenic progenitors which attach and differentiate onto the biomimetic matrices. The presence of a stem cell niche in rectus abdominis muscles where stem cells reside and undergo selfrenewal continuously producing large number of progenitor cells [108,109] is additionally supported by the recent identification of myendothelial cells in human skeletal muscle [110]. Myogenic and endothelial cells may derive from a common somatic precursor, and cells co-expressing myogenic and endothelial cell mark-ers residing in the interstitial spaces of skeletal muscle, i.e. myoendothelial cells, may contribute to postnatal tissue morphogenesis [110]. Importantly, in the context of the spontaneous induction of bone formation in porous biomimetic matrices implanted in the rectus abdominis muscle, clonally expanded myoendothelial cells differentiate into myogenic, chondrogenic and osteogenic cells under appropriate culture conditions [110]. Multipotent myoendothelial cells residing in stem cell niches within the rectus abdominis muscle do respond to endothelial cell mitogens including angiogenic factors [110]; myoendothelial cells may also respond to BMPs previously bound to collagen type IV and other extracellular matrix components further inducing the ripple-like cascade of the induction of bone formation within the porous spaces of the biomimetic matrices biomimetizing the cortico-cancellous remodelling cycle of the primate bone [8,9,57,58].
The final leap into the 'intrinsic' induction of bone formation will rest upon the mechanistic understanding of how microstructured surface areas of biphasic or not calcium phosphate biomaterial matrices differentiate inducible stem cells into osteoblastic-like cells initiating the induction of bone formation as a secondary response. The effect of calcium phosphate microstructure on the 'intrinsic' osteoinductivity of biphasic calcium phosphate biomatrices has been elegantly investigated by Li et al. [69]. The   biomaterial matrices are endowed with osteoinductivity; microstructured surfaces with increased surface areas might promote superior protein adsorption [69]; selected adsorbed proteins would directly influence cell attachment and proliferation to further promote the induction of the osteogenic phenotype in attached and resident stem cells into osteoblastic-like cells synthesizing, expressing, secreting and embedding osteogenic soluble molecular signals into the biomimetic matrices inducing the ripple-like cascade of bone differentiation by induction [61,71].

Conclusions and therapeutic perspectives of porous biomimetic matrices with intrinsic osteoinductivity
Tissue engineers, molecular biologists, reconstructive surgeons and developmental biologists alike have learned that tissue engineering in postnatal life recapitulates events that occur in the normal course of embryonic development [2,5,17]. More figuratively perhaps, tissue engineering of postnatal tissues recapitulates and exploits the very mechanisms of embryonic development though with different ratios and quantities of synchronously expressed soluble molecular signals and insoluble extracellular matrix substrata that nature has so parsimoniously developed through million years of evolution. The summary of several hundred years of research into the mechanisms of regenerative medicine is surprisingly simple: morphogens exploited in embryonic development are re-exploited and re-deployed for postnatal tissue induction and morphogenesis.
Perhaps indeed the different ratios and quantities of synchronously expressed soluble molecular signals interacting with insoluble signals or substrata pinpoints the very different capabilities to regenerate between embryos and adult animals, particularly the regenerative potential of Homo sapiens. Embryonic development and postnatal tissue regeneration are equally regulated by selected few and highly conserved families of secreted proteins, members of the TGF-␤ superfamily [2,17,18]. We have learned that in primates and in primates only, there is an apparent redundancy of soluble molecular signals initiating the induction of bone formation in heterotopic extraskeletal sites. We have learned though that we still have to grapple with the reality of our discoveries, that when implanted in heterotopic sites of the rectus abdominis muscle of non-human primates Papio ursinus, the mammalian TGF-␤ iso-forms do induce substantial endochondral bone formation by induction [5,[48][49][50][51].
We have further learned, and again biomimetizing nature that combines signals to induce tissue morphogenesis, that combining osteogenic soluble molecular signals with relatively low doses of the TGF-␤1 isoform, the rapidly generated tissue constructs are the result of a synergistic interaction which we have labelled 'synergistic induction of bone formation' [2,5,18,48]. The 'synergistic induction of bone formation' has remarkably showed to us that bone tissue develops as a mosaic structure in which members of the TGF-␤ superfamily singly, synergistically and synchronously initiate and maintain tissue induction and morphogenesis [2,5].
The morphogenesis of structurally organized chondrogenic zones, highly reminiscent of rudimentary embryonic growth plates [48], is a finding that vividly illustrates the concept that regeneration of cartilage and bone in postnatal life shares common cellular and molecular mechanisms with embryonic bone development, and that the 'memory' of developmental events in embryo can be redeployed postnatally by the application of morphogen combinations [48]. The 'synergistic induction of bone formation' reflects nature's parsimony in deploying low doses of molecularly different molecular signals yet resulting in rapid and complete tissue induction and regeneration [48,49]. Nature's key is to synchronously and synergistically deploy low doses of molecularly different single gene products during both embryogenesis and tissue  Fig. 8 (A, B and C regeneration in postnatal life, parsimoniously achieving complete tissue regeneration [18,111]. Supra-physiological doses of single recombinant human BMPs/OPs [111,112] contravene nature's elegant deployment of synchronously active molecular signals; this often generates incomplete and partial tissue induction in clinical contexts [112]. Biominetic matrices of HA/␤-TCP bioceramics have shown remarkable intrinsic oteoinduction in a variety of animal models [113][114][115][116]. Long-term experiments in the non-human primate Papio ursinus were set to investigate the induction of bone formation by biphasic biomimetic matrices (HA/␤-TCP) 40 to 60 and 20 to 80, respectively [117,118]. One year after implantation in orthotopic calvarial sites there was prominent osteogenesis coupled with resorption/dissolution of the implanted biomimetic matrices (Fig. 11) [118]. Solid discs of biphasic HA/␤-TCP, with concavities prepared on one planar surface only, were implanted heterotopically in the rectus abdominis muscle of adult baboons [117,118]. Histological analyses of heterotopic specimens harvested on days 90 and 365 showed the induction of bone formation also on the planar surfaces of the implanted biphasic HA/␤-TCP discs. Osteoclasts/macrophages excavate resorption lacunae and pits upon which osteoblastic cell lines secrete bone matrix within the microconcavities cut by osteoclastogenesis [117,118]. Morphological analyses 1 year after heterotopic implantation showed the induction of substantial bone with marrow on both planar surfaces with further dissolution of the implanted HA/␤-TCP biomimetic matrix with bone formation within the excavated resorption lacunae [117,118]. Orthotopic calvarial specimens showed complete induction of bone with resorption and dissolution of the implanted biphasic biomimetic matrices (Fig. 11) [117,118].
Resorption thus initiates the induction of bone formation in a 'continuum' of molecular and morphological processes that ultimately results in significant amounts of bone formation by induction Fig. 9 (A, B, C and D 'in toto' replacing the implanted 'smart' biomimetic matrices [117,118]. Above all, the overall geometric configuration of novel biomimetic matrices will provide biomimetic constructs to optimally deliver low doses of OPs of the TGF-␤ superfamily. The incorpora-tion of specific biological activities into biomimetic biomaterial matrices by manipulating the geometry of the substratum, defined as 'geometric induction of bone formation' [5,8,9,55] is now helping to engineer therapeutic osteogenesis in clinical contexts.