Nature relies on common but limited conserved molecular mechanisms tailored to provide the emergence of specialized tissues and organs. The bone morphogenetic/osteogenic proteins (BMPs/OPs), members of the transforming growth factor-β (TGF-β) supergene family (Wozney et al., 1988), are an elegant example of Nature’s parsimony in programming multiple specialized functions deploying several molecular isoforms with minor variation in amino acid motifs within highly conserved carboxy-terminal regions (Reddi, 1997, 2000; Miyazono et al., 2001; Ripamonti, 2006a; Lander, 2007).

BMPs/OPs singly induce de novo endochondral bone formation in extraskeletal heterotopic sites of rodents as a recapitulation of embryonic development (Reddi, 1994, 1998, 2000; Ripamonti, 2006a). The molecular cloning of the BMPs/OPs (Wozney et al., 1988; Özkaynak et al., 1990) and the results obtained in numerous pre-clinical studies in mammalian species including non-human primates (Reddi, 2000; Ripamonti, 2004, 2006a) have prematurely convinced molecular biologists, tissue engineers, and skeletal reconstructionists alike to believe that a single recombinant human bone morphogenetic protein would result in clinically acceptable tissue induction and morphogenesis (Friedlaender et al., 2001; Govender et al., 2002).

This theoretical potential has not been translated to acceptable results in clinical contexts. Clinical trials of craniofacial and orthopedic applications such as mandibular reconstruction and sinus-lift operations have indicated that supra physiological doses of a single recombinant human protein are needed to induce often unacceptable tissue induction whilst incurring significant costs without equivalence to autogenous bone grafts (Ripamonti et al., 2006, 2007a, 2009a; Garrison et al., 2007; Mussano et al., 2007).

The need for alternatives to the recombinant human BMPs/OPs is now felt more acutely following the reported complications and performance failure associated with the clinical use of recombinant hBMP-2 (Carragee et al., 2011a,b). Additional concerns are the apparent association of high doses of hBMP-2 with malignancies (Fauber, 2011a). Because of the often substandard regeneration of clinical defects implanted with hBMPs/OPs, similar if not inferior to autogenous bone grafts (Garrison et al., 2007; Mussano et al., 2007; Ripamonti et al., 2007a), we now need to finally ask the provocative question: are the bone morphogenetic proteins the only initiators of the induction of bone formation in clinical contexts?

The osteoinductive prerogative, originally solely assigned to naturally derived and recombinantly produced BMPs/OPs, has been extended to the three mammalian TGF-β isoforms themselves. The three mammalian TGF-β isoforms are determinant of a prominent endochondral osteoinductivity in heterotopic intramuscular sites of the non-human primate Papio ursinus (Ripamonti et al., 1997, 2000a, 2008; Ripamonti and Roden, 2010). Using the insoluble collagenous bone matrix as carrier, the TGF-β₃ isoform induces rapid and substantial endochondral bone formation in the rectus abdominis muscle of P. ursinus (Ripamonti et al., 2008).

We now describe the induction of bone formation by a variety of macroporous calcium phosphate-based constructs implanted in heterotopic intramuscular rectus abdominis and orthotopic calvarial sites of P. ursinus to engineer a super activated bone bioreactor by the hTGF-β₃ isoform for the rapid induction of bone formation in pre-clinical and clinical contexts. We further report that the ratio by weight of the binary application of hTGF-β₃ and hOP-1 is a critical parameter that controls the synergistic induction of bone formation. The reported unprecedented tissue induction and morphogenesis by single doses of 250 μg hTGF-β₃ indicate the novel molecular and morphological frontier for the induction of bone formation in clinical contexts.

Regenerative medicine is the grand multidisciplinary challenge of molecular, cellular, and evolutionary biology requiring the integration of tissue biology, tissue engineering, developmental and experimental surgery to trigger de novo and ex novo induction of tissues and organs of the mammalian body (Sampath and Reddi, 1981, 1983; Khouri et al., 1991; Reddi, 1998, 2000; Viola et al., 2003).

The need for alternatives to recombinant hBMPs/OPs is now felt more acutely after the reported complications and performance failure in clinical contexts (Carragee et al., 2011a,b; Fauber, 2011b). Contrary to study in rodents and lagomorphs, heterotopic implantation of recombinant hTGF-β₃ reconstituted with insoluble and inactive collagenous bone matrix, induces rapid and substantial bone formation in the non-human primate P. ursinus (Ripamonti et al., 2008, 2009a).

We have shown that sintered HA/β-TCP and coral-derived macroporous constructs combined with doses of the mammalian hTGF-β₃ form a super activated bioreactor inducing rapid and substantial bone formation. Of note, bone had predominantly formed at the periphery of the implanted super activated biomimetic constructs. The peripheral induction of tissue morphogenesis prominently characterized the induction of bone formation by high doses of the recombinant protein. Peripheral tissue induction also characterized coral-derived bioreactors pre-loaded with binary applications of the recombinant morphogens. Provocatively, binary applications of hOP-1 and hTGF-β₁ or platelet-derived TGF-β₁ and hTGF-β₃ synergize to induce massive ossicles in orthotopic calvarial and heterotopic rectus abdominis intramuscular sites (Ripamonti et al., 1997, 2010; Duneas et al., 1998). Of note, the temporal/spatial distribution of bone formation is rapid with the induction of large expanded and corticalized ossicles outgrowing the rectus abdominis intramuscular space and orthotopically, the pericranial calvarial space (Ripamonti et al., 1997, 2010; Duneas et al., 1998).

Responding mesenchymal stem cells, either myoblastic/pericytic of striated muscle or osteoprogenitor pericranial stem cells surrounding the implanted scaffolds, rapidly transform to the synergistic induction of bone formation. TGF-β₁ and TGF-β₃ pre-loaded collagenous bone matrix as carrier singly or in binary application with hOP-1 induce rapid bone formation, with limited if any tissue formation in the central void-like areas of the newly formed ossicles (Ripamonti et al., 1997, 2010; Duneas et al., 1998).

In previous studies using three different doses of the hTGF-β₁ isoform combined with the 25 μg selected dose of hOP-1, synergy was found to be optimal at a ratio of 1:20 by weight of hTGF-β₁:hOP-1, respectively (Ripamonti et al., 1997). We have previously reported 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 and recapitulated postnatally by the application of morphogens’ combination (Ripamonti et al., 1997). Changing the morphogen ratio, as reported in the present experiment, substantially alters the impetus of the morphogenetic cascade; the inversion of hTGF-β₃ vs. hOP-1 ratios significantly reduces the induction of bone formation. Synergistic interactions amongst secreted morphogens are a general principle adopted in embryonic development, later invocated in postnatal life. Synergistic synchronous expression of related gene products tightly controlled by threshold levels of finely tuned secreted morphogens, engineers morphogenesis of specialized tissues and organs.

By day 20, in specimens pre-loaded with 250 μg hTGF-β₃, there was extensive spatial/temporal tissue induction and morphogenesis. Large mineralized ossicles formed well outside the profile of the implanted matrix with multiple trabeculation of newly formed mineralized bone. Newly formed trabeculae were covered by contiguous rows of plump osteoblasts. The rapid induction and corticalization of the newly formed heterotopic ossicles is due to the rapid transformation of all available responding stem cells at the periphery of the implanted carriers, either insoluble collagenous bone matrix (Ripamonti et al., 1997; Duneas et al., 1998) or macroporous calcium phosphate-based constructs (Ripamonti et al., 2010), including the present experiments. The recruitment and transformation of all the available responding stem cells at the periphery of the implanted bioactive matrices result in the rapid induction of bone formation outside the profile of the implanted macroporous constructs. The rapid peripheral induction further delays differentiating stem cells migration within the central areas of the macroporous spaces. This results in limited if any bone formation within the center of the implanted macroporous constructs when evaluated on day 30 and 90. Different doses of the hTGF-β₃ isoform are responsible for variation in the temporo/spatial induction of bone formation; 250 and 125 μg hTGF-β₃ resulted in greater peripheral induction of bone formation when compared to 25 μg hTGF-β₃ which resulted in greater bone induction within the macroporous spaces owing to the less chemotactic inductive signal of less amounts of the recombinant protein.

High doses of the recombinant morphogen showed the pronounced induction of bone formation well outside the profile of the implanted macroporous scaffolds. The morphological results imply that the ligand attached to the most peripheral regions of the implanted macroporous constructs rapidly sets into motion the ripple-like cascade of bone differentiation. This is potentiated and expanded as a wave throughout the surrounding spaces by the invading rich capillary network with paravascular myoendothelial/pericytic stem cells available for rapid differentiation and transformation toward osteoblastic cell lines under the powerful osteogenic soluble signal of the hTGF-β₃ protein. Indeed the invading rich vascular and paravascular “niches” with accompanying progenitor stem cells (Kovacic and Boehm, 2009) provide stem cells to “feel their way into organogenesis,” paraphrasing the concept that cells feel and respond to the stiffness of their substrata (Disher et al., 2005).

The extensive osteoinduction could also be explained by desorption of the morphogen from the implanted macroporous constructs. Desorption as suggested in previous experiments in the primate after the generation of large islands of heterotopic ossification away from the implanted collagenous matrix as carrier (Ripamonti et al., 1996, 2000b). Similarly, desorption of the hTGF-β₃ with subsequent diffusion of the recombinant protein within the newly formed highly vascularized matrix including type IV collagen and laminin might have provided an ideal microenvironment for cell differentiation and induction of tissue formation (Trueta, 1963; Reddi, 1984; Duneas et al., 1998). Whether such an extended range of action is due to a diffusion gradient or to the initiation of a sequential chain of cellular induction (Slack, 1987; Lander, 2007) cannot be deduced from the available data.

Of interest, the morphological induction of bone formation by 125 μg hTGF-β₃ is somehow equal when delivered by either insoluble collagenous bone matrix (Figure 10A) or macroporous calcium phosphate-based constructs (Figure 10B), i.e., well enlarged ossicles forming outside the profile of the implanted matrix prominently growing between the ventral fascia and the muscular tissue of the rectus abdominis muscle. This suggests that the biological activity of 125 μg hTGF-β₃ is not best delivered by either collagenous matrix or coral-derived macroporous constructs; Figures 12 and 13 clearly indicate that the delivery system for the hTGF-β₃ isoform needs to contend with the most powerful chemotactic and inductive activities inherent to the third mammalian TGF-β isoform, which rapidly induces large amount of bone spatially away from the site of the implanted morphogen.

Hyper cellular osteoblastic activity, osteoid synthesis, angiogenesis, and capillary sprouting characterize the newly formed heterotopic ossicles by the hTGF-β₃ isoform (Ripamonti et al., 2008). Together with the induction of prominent osteogenesis with mineralization in mandibular defects of P. ursinus (Ripamonti, 2006b), the induction of large corticalized mineralized ossicles in the rectus abdominis muscle with large void-like spaces within the newly generated tissues (Ripamonti et al., 2008, 2009a), has suggested that the hTGF-β₃ isoform is the most powerful osteogenic gene product so far tested in non-human primates. It is likely that the associated expression of osteogenic gene products upon the implantation of the mammalian TGF-β isoforms particularly type IV collagen mRNA (Duneas et al., 1998) further enhances the bone induction cascade (Ripamonti et al., 1997, 2000a, 2008; Ripamonti and Roden, 2010). The temporal window during which TGF-β₁ mRNA is expressed upon implantation of doses of hOP-1 both heterotopically and orthotopically in P. ursinus is mandatory for the induction of optimal osteogenesis (Ripamonti, 2005). This has been unambiguously demonstrated by the endochondral osteoinductivity of the mammalian TGF-β isoforms in non-human primates (Ripamonti et al., 1997, 2000b, 2008; Ripamonti and Roden, 2010). We have previously indicated that the hTGF-β₃ isoform may regulate the expression of the homologous but molecularly different BMPs/OPs, acting upstream of the bone morphogenetic proteins which may initiate the induction of bone formation (Ripamonti et al., 2008; Ripamonti and Roden, 2010).

Regenerative medicine in clinical contexts is on a different scale altogether when compared to animal models that may not adequately translate and reproduce morphogen-related therapeutic responses in Homo sapiens. Tissue engineering is a field of tremendous promises particularly after successful pre-clinical studies including non-human primate species (Ripamonti et al., 2001, 2006, 2007a). Importantly however novel methods and procedures dramatically shown in animal models have not yet been translated in clinical contexts (Ripamonti et al., 2006, 2007a; Garrison et al., 2007; Mussano et al., 2007). In spite predictions to the contrary (O’Keefe and Mao, 2011), ultimately it is still doubtful whether tissue engineering will emerge as a winning medical technology or abruptly fail (Williams, 2006).

Presently, the use of recombinant hBMP-2 and hOP-1 in clinical contexts is in the balance (US Food and Drug Administration, 2008; Department of Justice, 2009; Williams et al., 2010; Centre for Devices and Radiological Health, 2011; Fauber, 2011a,b), and the US suppliers for both proteins are currently under US federal and FDA investigations. The recombinant proteins are now no longer available for use in clinical contexts (US Food and Drug Administration, 2008; Department of Justice, 2009; Williams et al., 2010; Centre for Devices and Radiological Health, 2011; Fauber, 2011a). The possible association of high doses of hBMP-2 with malignancies is now additionally under scrutiny (Fauber, 2011a). There is an acute need for morphogen-related bone tissue engineering particularly after the current FDA investigations into hOP-1 and hBMP-2 (US Food and Drug Administration, 2008; Department of Justice, 2009; Williams et al., 2010; Centre for Devices and Radiological Health, 2011; Fauber, 2011a). To establish a robust method to engineer a calcium phosphate-based bone bioreactor for heterotopic induction of bone formation, macroporous calcium phosphate-based constructs were implanted with relatively high doses of the hTGF-β₃ isoform in heterotopic sites of the rectus abdominis muscle in P. ursinus. The addition of the hTGF-β₃ isoform constructed a bone bioreactor that enabled the rapid formation of heterotopically induced ossicles with significant induction of bone formation outside the profile of the implanted biomimetic matrices.

Orthotopic applications of hTGF-β₃ induced statistically significant larger volumes of bone at 90 days when loaded onto macroporous single-phase hydroxyapatites as compared to biphasic HA/TCP constructs. Binary applications of hTGF-β₃ and hOP-1 on coral-derived HA/CC macroporous carriers in heterotopic sites produced larger amounts of bone at a ratio of 1:5 than the inverse ration in both 5 and 13% conversions’ specimens. The prominent induction of bone formation by 250 μg hTGF-β₃ has been translated in clinical contexts to treat a large mandibular defect in a pediatric patient (Ripamonti and Ferretti, 2012). Long term follow up will confirm whether these early results will translate into a complete reconstruction of the avulsed mandibular segment, and to confirm that the implanted 250 μg hTGF-β₃ per gram of demineralized human bone matrix is the novel dose required for bone tissue regeneration in clinical contexts.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.