Ugo Ripamonti* and Roland Klar
Bone Research Unit, Medical Research Council/University of the Witwatersrand, Johannesburg, South Africa
Almost 10 years ago, Africa’s First On-Line Science Magazine published a paper from our laboratories highlighting tissue engineering of bone by novel macroporous substrata [Ripamonti 2002]. The senior author wanted later to propose to the readership of Science in Africa a manuscript focused on unique discoveries that the Bone Research Laboratory of the South African Medical Research Council and the University of the Witwatersrand, Johannesburg has published against the scientific dogma working in Africa on the African non-human primate Papio ursinus. The Editor of Science in Africa then very sagaciously, and indeed prophetically, suggested preparing a manuscript on the induction of bone formation under the angle of the “great design”, i.e. proposing novel developmental concepts regulating the induction of bone formation in primate species. Almost 10 years later we now put forward novel concepts regulating the induction of bone formation by the mammalian TGF-β proteins and macroporous constructs that when implanted in heterotopic non-bony intramuscular sites of the non-human primate Papio ursinus initiate the cascade of bone differentiation by induction.
Serendipitously, our laboratories have shown that bone morphogenetic/osteogenic proteins (BMPs/OPs), powerful initiators of the induction of bone formation [Wozney et al. 1988; Reddi 1998; Reddi 2000; Ripamonti et al. 2000; Ripamonti 2006; Ripamonti et al. 2006], are not the only soluble molecular signals endowed with the striking prerogative of initiating the induction of bone formation [Ripamonti 2002; Ripamonti 2003; Ripamonti 2006; Ripamonti et al. 2006]. In a series of unique and systematic studies in Papio ursinus, we have shown that the three mammalian transforming growth factor-β (TGF-β) isoforms, the TGF-β1, -β2 and –β3 proteins, are also powerful initiators of endochondral bone formation but in primates only [Ripamonti et al. 1997; Ripamonti et al. 2000; Ripamonti et al. 2008; Ripamonti and Roden 2010a].
Against the scientific dogma, we have also found that the implantation of macroporous calcium phosphate-based coral-derived constructs in intramuscular sites of Papio ursinus results in the morphogenesis of bone within the macroporous spaces. Importantly, the morphogenesis of bone is initiated without the addition of the osteogenic soluble molecular signals (Fig. 1) [Ripamonti 1990; Ripamonti 1991]; a fascinating phenomenon we have defined as the “intrinsic” and/or “spontaneous” osteoinductivity of macroporous substrata [Ripamonti 1990; Ripamonti 1991; Ripamonti et al. 1993]. In a series of systematic studies in the non-human primate Papio ursinus, we have shown the reproducible evidence that heterotopic intramuscular implantation, i.e. in a site where there is no bone, of calcium phosphate-based macroporous constructs in the rectus abdominis muscle of Papio ursinus results in the morphogenesis of bone [Ripamonti 1990; Ripamonti 1991; Ripamonti et al. 1993; Ripamonti 1996; Ripamonti et al. 2001]. Importantly, bone forms without the exogenous application of osteogenic soluble molecular signals of the TGF-β supergene family, which include the BMPs/OPs and the TGF-β proteins, the latter in primates only [Ripamonti 2003; Ripamonti 2006].
Our first observations were recorded in the late eighties after implantation of coral-derived calcium carbonate/hydroxyapatite constructs when implanted in the rectus abdominis muscle of Papio ursinus (Fig. 1) [Ripamonti 1990; Ripamonti 1991]. Our observations were instrumental to define the concept of the “hydroxyapatite-induced osteogenesis model” in heterotopic intramuscular sites of Papio ursinus without the exogenous application of the osteogenic soluble molecular signals of the TGF-β supergene family as well as of raising the concept of the geometric configuration of the implanted constructs [Ripamonti et al. 1993]. Indeed macroporous coral-derived calcium carbonate/hydroxyapatite constructs combined with highly purified naturally-derived bovine BMPs/OPs were instrumental to identify the critical role of the geometry of macroporous hydroxyapatite regulating the induction of bone formation overruling the biological activity of the recombined osteogenic proteins (Fig. 2) [Ripamonti et al. 1992]; importantly, the granular/particulate constructs failed to induce bone even when pre-treated with doses of naturally-derived highly purified bovine osteogenic fractions purified greater than 50.000 fold [Ripamonti et al. 1992].
The critical role of the substratum geometry was additionally shown by implanting intramuscularly in Papio ursinus identical coral-derived calcium carbonate/hydroxyapatite constructs which however differed by the geometric shape of the implanted matrices, i.e. solid blocks of macroporous constructs 20mm in diameter 7 mm in height vs. granular/particulate constructs [van Eeden and Ripamonti 1994]. Implants of granular/particulate coral-derived hydroxyapatite failed to induce bone intramuscularly in Papio ursinus as compared to the spontaneous induction of bone formation as seen within the macroporous spaces of the implanted calcium phosphate blocks (Fig. 3) [van Eeden and Ripamonti 1994].
It was noteworthy that recombinant hBMP-4 binds equally well to macroporous matrices of coral-derived calcium carbonate/hydroxyapatite constructs of both geometric configurations indicating that the binding of hBMP-4 is not affected by the geometry of the substratum, since 125I-radiolabelled hBMP-4 binds equally well to coral-derived hydroxyapatite substrata in granular/particulate or in block configurations [Ripamonti et al. 1992; van Eeden and Ripamonti 1994]. Importantly, the critical role of the geometry has been identified also controlling the induction of bone formation by macroporous calcium phosphate-based biomaterial matrices extending published work on the critical role of the substratum geometry of demineralized collagenous-based extracellular matrices (Reddi and Huggins 1973; Reddi 1974; Sampath and Reddi 1984); screening of potential substrata in primates could thus help tissue engineers to construct carriers and delivery systems with defined geometries and surface characteristics for replacement therapies that are conducive to the initiation and promotion of therapeutic osteogenesis [Ripamonti 1991, van Eeden and Ripamonti 1994].
What it is that the geometry per se has so biologically powerful to control the induction of bone formation? The senior author started to develop the concept of biomimetism and biomimetics, biomimetizing Nature’s phylogenetically long tested repetitive geometries [Ripamonti 2009; Ripamonti and Roden 2010b]; reading, observing and studying several hundred histological sections showing the formation of bone by macroporous coral-derived calcium phosphate-based constructs, the senior author suddenly realized that the specific form and shape of the concavity per se is endowed with the striking prerogative to initiate the ripple-like cascade of bone differentiation by induction within the macroporous constructs even when implanted without the osteogenic soluble molecular signals of the TGF-β supergene family (Fig. 3) [Ripamonti et al. 1999; Ripamonti 2000; Ripamonti 2004; Ripamonti 2006; Ripamonti 2009; Ripamonti and Roden 2010b].
Seeking to unravel the fascinating scenario of the “geometric induction of bone formation” [Ripamonti et al. 1999], the senior author wished to attend a US Gordon’ Research Conference on Biomaterials in the late eighties; one evening discussing the induction of bone formation in non-human primates Papio ursinus with a biomaterial scientist, the simple but critical suggestion was to find out “where a similar if not identical geometry does exist in the mammalian body; if you find it, you shall soon know what it is that this geometry does and control”; whilst listening, the senior author just saw the sequential images of the remodelling cycle of the primate cortico-cancellous bone, the osteoclastic activity cutting resorption lacunae, pits and concavities within the mineralized bone, prompting the remodelling cycle to initiate bone formation within the concavities cut by osteoclastogenesis. The concavities, as prepared within the biomimetic matrices, biomimetize Nature’ archaic but constantly functional designs initiating the induction of bone formation (Fig. 4) [Ripamonti 2009; Ripamonti and Roden 2010b].
In collaboration with the Council for Scientific & Industrial Research (CSIR) Materials Science and Technology Group, we later translated the “spontaneous induction of bone formation” by coral-derived calcium carbonate/hydroxyapatite constructs to sintered highly crystalline biomimetic matrices which were also found to be inductive when implanted in intramuscular sites of Papio ursinus [Ripamonti et al. 1999]. Concavities, prepared on both planar surfaces of highly crystalline hydroxyapatite discs implanted in the rectus abdominis muscle of Papio ursinus classically initiated the induction of bone formation [Ripamonti et al. 1999; Ripamonti 2000; Ripamonti 2004]. Importantly, bone exclusively initiated within the concavities [Ripamonti et al. 1999; Ripamonti 2000; Ripamonti 2004]. Our systematic studies in Papio ursinus have thus 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 biomimetize the remodelling cycle of the primate cortico-cancellous osteonic bone [Ripamonti 2004; Ripamonti 2009; Ripamonti and Roden 2010b].
An important collaborative research work with the Materials Science & Manufacturing Unit of the CSIR resulted in the fabrication and testing of biphasic hydroxyapatite/β-tricalcium phosphate biomimetic matrices [Ripamonti et al. 2008]. Following several papers on the spontaneous induction of bone formation, we have presented a modified tissue engineering paradigm in which the very insoluble signal or substratum resorbs via a downstream of molecular and cellular cascades that sculpt resorption pits and lacunae in the geometric form of concavities within the implanted biphasic matrices [Ripamonti et al. 2008]. The concavities initiate bone differentiation by induction. The operational molecular and cellular resorption and dissolution of the implanted matrices sculpting lacunae and pits in the form of concavities are the biological continuum for the induction of bone formation [Ripamonti et al. 2008].
The morphological observations of the induction of bone formation in a continuum of resorption/dissolution of the implanted biphasic matrix confirmed that resorption lacunae, pits and concavities cut by osteoclastogenesis are regulators of bone formation by induction [Ripamonti et al. 1999; Ripamonti et al. 2008]. The continuum of substratum’ resorption and dissolution with the induction of bone formation was possible by sintering biphasic constructs [Nilen and Richter 2007] which would resorb upon implantation in heterotopic intramuscular and orthotopic bony sites of the non-human primate Papio ursinus and its replacement by newly formed bone [Ripamonti et al. 2008]
Our latest experiments on the intrinsic and/or spontaneous osteoinductivity by coral derived macroporous hydroxyapatite biomatrices to a degree shed important mechanistic insights into the morphogenesis of bone by macroporous calcium-derived constructs [Ripamonti et al. 2010]; more importantly, however, the molecular data strongly support what we have hypothesized since several years by now [Ripamonti et al. 1997], i.e. the hypothesis that TGF-β serves as an essential bone inductive signalling control centre in non-human primates Papio ursinus and thus by extension, in the human primate Homo sapiens [Ripamonti and Roden 2010a; Ripamonti 2010].
To understand this novel concept of cellular and molecular events, we need to back track to the multiple signalling mechanisms controlling the several members of the TGF-β supergene family when orchestrating the ripple-like cascade of bone differentiation by induction [Reddi 2000]. We have learned that molecular and cellular actions are the net results of initiators and inhibitors coupled with several modulators each finely tuning the complex extracellular receptor domains of the responding cells [Dieudonné et al. 1994; Yamaguchi et al. 2008; Eivers et al. 2009; Alarcón et a. 2009]; we have realized after extensive testing in non-human primate models that both naturally-derived highly purified bone BMPs/OPs as well as recombinant hOP-1 are profoundly osteogenic in both intramuscular non-bony and orthotopic bony sites, yielding large ossicles for craniomandibulofacial reconstruction [Ripamonti et al. 1996; Ripamonti et al. 2000; Ripamonti 2003; Ripamonti 2005; Ripamonti et al. 2009; Ripamonti 2010]. We have learned that Noggin protein is a molecular signal that inhibits the cascade of bone differentiation by induction as initiated by BMPs/OPs directly inhibiting BMPs/OPs at the receptor level [Gazzerro et al. 1998; Groppe et al. 2003].
Our recent experimentation has shown unequivocally that the induction of bone formation by coral-derived macroporous constructs when implanted intramuscularly in Papio ursinus is controlled in part by the expression of the OP-1 gene product [Ripamonti et al. 2010]; it is likely that in vivo other BMPs/OPs are expressed after the implantation of macroporous matrices intramuscularly, though in the context of our experiments, the expression of OP-1 mRNA is clearly one of the initiating signals of the spontaneous induction of bone formation in heterotopic intramuscular sites [Ripamonti et al. 2010]. Indeed, pre-treatment of the coral-derived constructs with the biphosphonate zoledronate Zometa® inhibits the spontaneous induction of bone formation by blocking osteoclastic surface modifications on the macroporous surfaces thus halting the development of surface patterned configurations highly suitable for the differentiation of myoblastic/myoendothelial and/or pericytic/endothelial stem cells into osteoblastic-like cells secreting, expressing and embedding soluble osteogenic molecular signals into the biomimetic matrices initiating bone formation as a secondary response (Fig. 5) [Ripamonti et al. 2010].
Osteoclastic resorption results in micro patterned surface topographical modifications and calcium ions release (Ca2+) within the microenvironment of the cut concavities (Ripamonti et al. 2010); this results in angiogenesis [Munaron 2006] and cell differentiation [Dalby et al. 2007] towards the osteoblastic phenotype [Kanatani et al. 1991; Zayzafoon 2006] and the induction o bone formation as a secondary response [Ripamonti et al. 2010].
Osteoclastic activity is thus critical for Ca2+ release, angiogenesis and cell differentiation; specific surface patterning of biomedical devices in pre-clinical and clinical contexts is now mandatory to initiate osteoclastogenesis leading to further micro surface patterning and topographical modifications, Ca2+ release and myoblastic stem cells differentiation. Recent work has shown that topographic landscapes of selected surface patterning nano-topographies are acutely sensed by osteoclasts in vitro and stimulate rings formation on the migrating osteoclasts resulting in enhanced osteoclastogenesis, cutting cones, pits and lacunae [Geblinger et al. 2010]; this may lead to greater osteogenesis after superior cell differentiation, BMPs/OPs expression and secretion within the macroporous microenvironment.
Micro-patterned surface topographies in the range of 1µm are superiorly sensed by osteoclasts leading to activated osteoclastogenesis after enhanced rings’ formation [Geblinger et al. 2010]; we have thus learned that osteoinductive biomedical devices need to be primed, i.e. surface altered and/or modified by invading active osteoclasts migrating along the macroporous surfaces preparing the substratum for selected stem cell differentiation and osteogenesis. Recent experiments have further demonstrated that topography per se influences the dynamic organization of the osteoclast resorption apparatus [Geblinger et al. 2010].
Physical properties of the implanted substratum set into motion the ripple like cascade of cell differentiation and expression of selected gene products [Ingber 2006; Nelson et al. 2005; Gjorevski and Nelson 2009]. Mechanical forces generated in the cytoskeleton by topographical cues are critical during development and morphogenesis [Ingber 2006]. Indeed, mechanical forces that originate from the contraction of cells result in patterns of growth [Nelson et al. 2005]. Topographical geometric patterning is essential throughout the assembly of biomimetic matrices for bone replacement therapies to sculpt the invading cellular microenvironment and the induction of bone formation. The above studies thus report a mechanical form of morphogenetic control at cellular and sub-cellular levels driving the expression of specific morphogenetic gene products to induce morphogenesis [Ingber 2006; Nelson et al. 2005; Gjorevski and Nelson 2009].
What next in bone tissue engineering using macroporous “smart” substrata? Since the isolation and purification of the entirely new family of proteins, the BMPs/OPs, powerful initiators of bone differentiation, we have learned, and learned the hard way, that a complementary substratum is needed to evoke the osteogenic activity of the soluble osteogenic molecular signals [Reddi 2000]. BMPs/OPs induce local bone formation when reconstituted with the insoluble collagenous matrix, the inactive residue obtained after dissociative extraction of the bone matrix with cahotropic agents such as 6M urea or 4M guanidinium hydrochloride [Sampath and Reddi 1981]. As a proteinaceous substratum, the use of insoluble collagenous matrices and other collagenous-based matrices as delivery systems for the now available recombinant hBMPs/OPs is associated with a number of drawbacks. These include weak mechanical performance, immunogenic response and potential transmission of viral antigens; the latter seriously limit the widespread use of the recombinant human proteins in clinical contexts.
We have learned that adsorption of naturally-derived BMPs/OPs onto hydroxyapatite gels is a critical chromatographic step for its purification [Reddi 2000]. The senior author has thus had the idea to test macroporous coral-derived calcium carbonate/hydroxyapatite substrata as carrier for naturally-derived highly purified BMPs/OPs purified greater than 50.000 fold with respect to the crude guanidinium extract after heparin-Sepharose and hydroxyapatite-Ultrogel affinity and adsorption chromatography with final purification on Sephacryl S-200 [Ripamonti et al. 1992; Ripamonti 2006]. Because of the therapeutic advantage of an inorganic, non-immunogenic substratum in controlling the osteogenic activity of naturally-derived highly purified BMPs/OPs, we have exploited the adsorption of BMPs/OPs for hydroxyapatite to construct an osteogenic delivery system after chromatographic adsorption of highly purified osteogenic fraction onto coral-derived macroporous constructs [Ripamonti et al. 1993].
The chromatographic procedure and the results of the various bioassays in both rodents and primates are summarized in Fig. 6 [Ripamonti et al. 1993]. Proteins fractions were purified by sequential chromatography on heparin-Sepharose, hydroxyapatite-Ultrogel and Sephacryl S-200 (Ripamonti et al. 1993). Highly purified osteogenic fractions were pooled, concentrated and exchanged to 5mM HCl; proteins were tested for biological activity in the rodent subcutaneous space [Ripamonti et al. 1993]. Osteogenic fractions were loaded onto a chromatography column with a recipient bed of several discs of coral-derived macroporous hydroxyapatite-based constructs that would fit the 25 mm diameter of the Pharmacia Inc. chromatography column, equilibrated in 5 mM HCl for adsorption chromatography (Fig. 6) [Ripamonti et al. 1993]. Protein fractions before and after adsorption chromatography were analyzed on a 15% acrylamide SDS gel and silver stained (Fig. 6D).
Protein fractions obtained after the adsorption and purification procedures were bioassayed in the subcutaneous space of the rat after reconstitution with 25 mg of rat insoluble collagenous bone matrix [Sampath and Reddi 1981; Ripamonti et al. 1993]. Aliquots of pooled BMPs/OPs fractions before adsorption chromatography, and increasing volumes of the hydroxyapatite-unbound eluate after adsorption chromatography were bioassayed in the rat [Ripamonti et al. 1993]. Loaded highly purified BMPs/OPs osteogenic fractions induced prominent osteogenesis in the rodent heterotopic subcutaneous space (Fig. 6E). As expected, increasing volumes of eluates did not induce bone formation in the rodent bioassay (Fig. 6F) indicating that all the loaded osteogenic fractions were adsorbed onto the macroporous hydroxyapatite-based bed of the chromatography column [Ripamonti et al. 1993]. This was confirmed by the apparent lack of protein in the hydroxyapatite eluate as evaluated on 15% acrylamide SDS silver stained gel under non-reducing conditions (Fig. 6D). To test the hypothesis that all the loaded osteogenic fractions were adsorbed onto the macroporous coral-derived constructs inserted into the chromatography column, loaded discs were implanted in the rectus abdominis muscle of 6 non-human primates Papio ursinus. Unloaded discs were implanted as control [Ripamonti et al. 1993]. Histological examination on day 30 of macroporous loaded discs after chromatographic adsorption of naturally-derived highly purified osteogenic fractions showed extensive bone differentiation and prominent vascular invasion within the macroporous spaces (Fig. 6G-M). Macroporous constructs implanted without osteogenic fractions showed lack of bone differentiation (Fig. 6 N). The data were found to be important since the quantities and rate of morphogenetic proteins could be tightly controlled by the chromatographic adsorption of controlled amounts of morphogenetic proteins [Ripamonti et al. 1993].
Apparent redundancy of osteogenic molecular signals initiating the induction of bone formation in the rectus abdominis muscle of the non-human primate Papio ursinus. A-F: Tissue induction and morphogenesis by 125 µg recombinant human transforming growth factor-β3 (TGF-β3) combined with insoluble collagenous matrix as carrier; large corticalized newly formed ossicles harvested on day 30 from the rectus abdominis muscle of adult baboons. Newly formed bone in blue corticalized the newly formed ossicles with large osteoid seams populated by contiguous osteoblasts (B). The TGF-β3 protein implanted in the rectus abdominis muscle of Papio ursinus has been found to be the most powerful inducer of endochondral bone formation in the record of experimentation at the Bone Research Laboratory in non-human primates Papio ursinus.
Our laboratories have shown that the mammalian TGF-β isoforms are determinant of the induction of bone formation in the non-human primates Papio ursinus (Fig. 7) (Ripamonti et al. 1997; Ripamonti et al. 2000; Ripamonti et al. 2008; Ripamonti and Roden 2010a) and Macaca mulatta (Bone Research Laboratory, unpublished observations). The lack of endochondral bone differentiation by the three mammalian TGF-β isoforms in murine, rodent and canine models underscores the finely tuned molecular, cellular and morphological cascades of bone: formation by autoinduction [Urist 1965]. Only “morphogens unbound”, following the emergence and definition of morphogens as “forms generating substances” [Turing 1953], organize pattern formation and development ultimately leading to tissue induction and morphogenesis [Lander 2007].
Our latest experimental data have indicated that when OP-1 transcription increases, TGF-β3 increases exponentially and always remains at a higher level than OP-1 expression even in extracted macroporous constructs pre-treated with hOP-1 [Ripamonti et al. 2010]. The clinical scenario of “bone: formation by autoinduction” [Urist 1965] now requires the treatment of human skeletal defects by the mammalian TGF-β proteins, with particular relevance to the TGF-β3 isoform, the most powerful osteoinductive morphogen so far tested in non-human primates of the species Papio ursinus [Ripamonti et al. 2008].
Ultimately, the induction of bone formation by the mammalian TGF-β proteins in non-human primate models cannot and should not be ignored any longer. Newly designed therapeutic strategies in clinical contexts should be now implemented after implantation of hTGF-β3 –based osteogenic devices to treat human skeletal disorders.
Acknowledgments The reported research work against the dogma on the “intrinsic” osteoinductivity by macroporous biomimetic calcium phosphate-based constructs and on the endochondral osteoinductivity of the non-canonical transforming growth factor-β isoforms has been constantly supported by the Medical Research Council of South Africa, the University of the Witwatersrand, Johannesburg, and the National Research Foundation of South Africa since the first histological sections cut by Barbara van den Heever in the late eighties demonstrating the induction of bone formation in coral-derived macroporous constructs; ad hoc grants to the Bone Research Laboratory have most strongly supported the never ending publication effort against the current scientific dogma. This manuscript summarizing more than 23 years of unique research in Africa in the African non-human primate Papio ursinus is dedicated to Barbara van den Heever for her impeccable decalcified and undecalcified histological sections now published around the world and who has cut the first sections with evidence of the morphogenesis of bone alerting the senior author on the formation of new bone within the macroporous coral-derived constructs; the authors acknowledge the constructive and unique work of a series of technologists and scientists who have worked at the Bone Research Laboratory, in particular Jean Crooks, June Teare, Ruqayya Parak, Louise Renton, Laura Yates, Manolis Heliotis, Carlo Ferretti; special thanks to the Materials Science and Manufacturing Unit of the Council for Scientific and Industrial Research Paul W Richter, Roger Nilen and the late Michael Thomas for the long standing collaboration on self-inducing sintered macroporous constructs for implantation in Papio ursinus. Special thanks to Edwin Clayton Shors and Mike Ponticiello of Interpore International, Irvine, California, for the continuous supply of coral-derived calcium carbonate/calcium phosphate constructs for cranial and intramuscular implantation in primate species. We thank Professors Urist and Reddi for inspirational scientific insights into “bone: formation by autoinduction” and Daniella Bella for the drive to create, write and make it happen.
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