Soluble, insoluble and geometric signals sculpt the architecture of mineralized tissues
U. Ripamonti *
Bone Research Unit, Medical Research Council, University of the Witwatersrand, Johannesburg, South Africa
* Correspondence to: Professor Ugo RIPAMONTI Bone Research Unit, MRC/University of the Witwatersrand, Johannesburg, 7 York Road, 2193 Parktown, South Africa. Tel./Fax: + 12 11 717 2300 E-mail: firstname.lastname@example.org
Bone morphogenetic and osteogenic proteins (BMPs/OPs), members of the transforming growth factor-β (TGF-β) superfamily, are soluble mediators of tissue morphogenesis and induce de novo endochondral bone formation in heterotopic extraskeletal sites as a recapitulation of embryonic development. In the primate Papio ursinus, the induction of bone formation has been extended to the TGF-β isoforms per se. In the primate and in the primate only, the TGF-β isoforms are initiators of endochondral bone formation by induction and act in a species-, site- and tissue-specific mode with robust endochondral bone induction in heterotopic sites but with limited new bone formation in orthotopic bone defects. The limited inductive capacity orthotopically of TGF-β isoforms is associated with expression of the inhibitory Smads, Smad6 and Smad7. In primates, bone formation can also be induced using biomimetic crystalline hydroxyapatite matrices with a specific surface geometry and without the exogenous application of osteogenic proteins of the TGF-β superfamily, even when the biomimetic matrices are implanted heterotopically in the rectus abdominis muscle. The sequence of events that directs new bone formation upon the implantation of highly crystalline biomimetic matrices initiates with vascular invasion, mesenchymal cell migration, attachment and differentiation of osteoblast-like cells attached to the substratum, expression and synthesis of osteogenic proteins of the TGF-β superfamily resulting in the induction of bone as a secondary response. The above findings in the primate indicate enormous potential for the bioengineering industry. Of particular interest is that biomimetic matrices with intrinsic osteoinductivity would be an affordable option in the local context.
Keywords: bone induction • primates • TGF-β • BMPs/OPs • hydroxyapatite biomimetic matrices • geometry • bioengineering
J. Cell. Mol. Med. Vol 8, No 2, 2004 pp. 169-180
The initiation of bone formation during embryonic development and postnatal osteogenesis involves a complex cascade of molecular and morphogenetic processes that ultimately lead to the architectural sculpture of precisely organized multicellular structures. These include mineralized bone composed of osteoid which is the novel bone matrix as yet to be mineralized, and cellular elements including osteoblasts or bone forming cells, osteoclasts or remodelling and resorbing cells, and osteocytes, living bone cells within newly formed mineralized bone matrix . Elucidating the nature and interaction of the signalling molecules that direct the generation of tissue-specific patterns during the initiation of bone formation is a major challenge of contemporary molecular, cellular, developmental and tissue engineering biology .
Nature relies on common but limited molecular mechanisms tailored to direct the morphogenesis of specialized tissues and organs. The bone morphogenetic and osteogenic proteins family (BMPs/OPs), members of the transforming growth factor-β (TGF-β) supergene family, is indeed an elegant example of Nature’s parsimony in controlling multiple specialized functions or pleiotropy, deploying molecular isoforms with minor variations in amino acid motifs within highly conserved carboxy-terminal regions [2-6].
A striking and discriminatory prerogative of these morphogens, either naturally-derived or produced by DNA recombinant technologies, is their ability to induce de novo endochondral bone formation, i.e. via a cartilage phase, in heterotopic extraskeletal sites [2, 5, 7, 11–14] (Fig. 1A). The remarkable process of tissue morphogenesis and bone induction in postnatal life is a recapitulation of embryonic development whereby both embryonic development and tissue regeneration are regulated by a select few, highly conserved families of morphogens namely the BMPs/OPs family of the TGF-β superfamily [2, 5, 6, 12, 15, 16] (Fig. 1A).
BMPs/OPs are morphogens defined by Turing  as form generating substances, and when interacting with responding cells are capable of initiating the sequential cascade of events leading to pattern formation and the attainment of tissue form and function, i.e. morphogenesis [2, 6]. Thus these pleiotropic morphogens have potent and diverse effects on cell proliferation, differentiation, motility and matrix synthesis leading ultimately to cartilage and bone differentiation and regeneration [1, 2, 6, 12, 14].
The fact that a single recombinant human BMP/OP initiates bone formation by induction does not preclude interactions with other morphogens deployed synchronously, sequentially and synergistically during the cascade of bone formation by induction. Thus osteogenesis can occur via the combined action of several BMPs/OPs resident within the natural milieu of the extracellular matrix of bone [7, 10–12] (Fig. 1B).
The osteoinductive prerogative, originally solely ascribed to the BMPs/OPs, has now been extended to other TGF-β superfamily members, including decapentaplegic (DPP) and 60A gene products expressed early in Drosophila development , and growth and differentiation factor-5 . Of particular interest is that in the species studied to date, the TGF-β isoforms in contrast to BMPs/OPs, initiate endochondral bone formation only in the primate [20–22] (Fig. 1C). In this animal model, the induction of bone by TGF-β isoforms is characterised by a marked site and tissue specificity [2, 10, 12, 20–23].
The presence of several molecular forms with osteogenic activity poses important questions about the biological significance of this apparent redundancy , indicating multiple interactions, or cross-talk between components of the signalling pathways initiating de novo bone formation and tissue morphogenesis in primate species.
In the assay for heterotopic bone induction in rodents [1–6], neither naturally-derived nor recombinant TGF-β isoforms initiate endochondral bone formation [24–27]. In marked contrast to the rodent bioassay, the TGF-β isoforms so far tested in our laboratories have shown a marked site and tissue-specific endochondral osteoinductivity in the pri
Our studies in the primate Papio ursinus have shown differential osteogenic responses following heterotopic and orthotopic implantation of TGF-β isoforms. Whereas TGF-β1 and TGF-β2 induced substantial endochondral bone formation in the rectus abdominis muscle (Fig. 1C) resulting in large corticalized ossicles by day 90 post implantation [9, 10, 21, 22], repair of calvarial defects was poor  (Fig. 2A), restricted to limited bone regeneration at the pericranial perimeter only 90 days post-implan-tation [2, 9, 10, 21] (Fig. 2B).
Given the ubiquitous nature of TGF-β family members, receptors are expressed on all cell types examined to date  and the pleiotropic nature of their activity within the responding cell, it is unsurprising that the TGF-β signalling pathway is strictly regulated. We now know many of the complex mechanisms controlling TGF-β signal transduction to ensure specificity of response by the target cell, and regulation is at multiple levels, from the cell surface to within the nucleus .
The Smad pathway is the major intracellular signalling system activated by TGF-β superfamily members. Over expression of Smad6 and Smad7, inhibitors of the vertebrate Smad-based TGF-β signalling pathway [31–34], would mechanistically explain the observed site and tissue specificity of endochondral osteoinductivity of the TGF-β isoforms in the primates . Suppression of TGF-β-initiated signal transduction by two inhibitory Smad proteins Smad6 and Smad7 represents a negative autocrine feedback loop since their expression is rapidly induced by TGF-β itself .
Using Northern blot analysis and RT (reverse transcription)-PCR, we have shown mRNA of Smad6 and Smad7 to be expressed where TGF-β-induced bone formation was limited, but almost absent heterotopically (Fig. 3A), in sites of exuberant new bone formation by induction. [2, 10, 23]. These exciting findings in the adult non-human primate highlight the importance of the inhibitory Smads in regulating de novo bone formation in clinical contexts.
Limited bone formation in orthotopic bony sites by the TGF-β isoforms so far tested in our laboratories is potentially due to decreased osteoblastic proliferation and differentiation. Expression of the inhibitory Smad6 and Smad7, as later shown by RTPCR, is clearly suggested by the morphologic analysis of calvarial specimens treated with 100µg hTGF-β2 and harvested on day 90 post-implanta-tion. The examined specimens showed bone formation at both interfacial regions that seemed, at least morphologically, inhibited to proceed or grow centripetally with the generation of a rather substantial fibrogenic response between the inactive particles of the collagenous matrix [21, 23] (Fig. 2B).
The present data indicate that the observed morphological effects of Smad6 and Smad7 on osteoblasts synthesis and bone matrix formation by induction are due to its specific inhibition of the TGF-β and BMPs/OPs signalling pathway in orthotopic sites.
It is worth noting that tissues generated via induction by TGF-β1 and β2 isoforms expressed OP-1 and BMP-3 mRNAs synthesis (Fig. 3B), indicating that bone formation induced by TGF-βs in the rectus abdominis muscle requires, at least in part, synthesis of members of the BMPs-OPs family [10, 21, 22].mate Papio ursinus. Remarkably this occurs in primates only [2, 10, 12, 20–23] (Fig. 1C).
To trigger the cascade of tissue morphogenesis and endochondral bone differentiation, the soluble signals require reconstitution with an insoluble signal, or substratum [2-6]. Whilst molecular biology has elucidated many of the cellular and sub-cellular events activated by the molecular soluble signals, significantly less is known of optimal delivery systems to act as insoluble substrata [2, 9, 10, 12].
The requisite properties of the substratum are that it should be nonimmunogenic, inorganic, carvable and amenable to contouring for optimal adaptation to the various shapes of bone defects. Optimal osteogenic activity should be induced with relatively low doses of recombinant human (rh) BMPs/OPs [2, 10–12, 35]. In addition, an ideal substratum for bone tissue engineering should promote angiogenesis and mesenchymal tissue invasion to be brought in contact with rhBMPs/OPs previously adsorbed on the substratum. The processes of bone remodelling finally occur once the regenerative processes are well under way.
Significantly, the surface characteristics and geometric configurations of the delivery system are critical for bone induction to occur with and without the exogenous application of BMPs/OPs [2, 9, 10, 12, 36–45].
A major objective of our research has been to develop biomaterials that are specifically designed to optimise upregulation of specific BMPs/OPs genes upon implantation of geometrically and biologically correct matrices [10, 11, 40, 41]. After many studies in the primate Papio ursinus we have shown in collaboration with the Council for Scientific and Industrial Research (CSIR) Pretoria [40, 46], that a specific geometry of the substratum of sintered and highly crystalline hydroxyapatite drives bone formation by induction within the porous hydroxyapatite, a phenomenon which we have labelled as geometric induction of bone formation [40, 46]. We have found that the optimal morphogenetic geometry has the shape of a concavity of a specific dimension, and that the concavity is smart in the sense that it anchors specific endogenous BMPs/OPs at the interface of the hydroxyapatite with the fibrovascular tissue invading the concavities with induction of bone as a secondary response [2, 9–12, 40, 47] (Figs. 4 and 5).
To investigate intrinsic osteoinductivity imparted by surface geometry, monolithic discs of hydroxyapatite with concavities on both planar surfaces were implanted in the rectus abdominis muscle of the primate Papio ursinus. Histological analysis of the specimens harvested 30 and 90 days after implantation revealed that bone differentiation was initiated exclusively in the concavities of the substratum (Figs. 4 and 5). On day 30, BMP-3 and OP-1 (also known as BMP-7) were detected by immunolocalization at the mesenchymal tissue-hydroxyapatite interfaces within the concavities of the substratum . Thus the sintered hydroxyapatite acts as a solid state matrix for adsorption and anchorage of endogenously produced BMP-3 and OP-1, and new bone formation occurs as a secondary response [2, 10, 11, 40, 47]. Importantly, porous discs of sintered hydroxypatite implanted in non-healing calvarial defects in non-human primates showed substantial bone formation, culminating in complete deposition of bone spanning the defects .
The programme for sculpting the architecture of the cortico-cancellous structures of bone, or the regulation of bone regeneration and bone tissue engineering requires three key components: an osteoinductive molecular signal, an insoluble substratum which delivers the signal and acts as a scaffold for the induction of new bone formation, and host recipient cells capable of differentiation into bone cells in response to the osteoinductive soluble signal [2, 4, 6, 9, 10, 12]. All three components are subject to manipulation including the signalling molecules to be delivered and the nature of the carrier biomimetic matrices, which additionally can be loaded with responding cells and tissues [2, 6, 48].
Based on studies performed to determine whether BMPs/OPs present in the concavities are derived from the circulation or are produced after local expression by cells present in the concavity, Northern blot analyses have shown the expression of the mRNA of osteogenic markers of the TGF-β superfamily to be present within the concavities of the biomimetic matrices. We now can propose the following sequence of events culminating in de novo bone formation:
1) Vascular invasion and capillary sprouting within the invading tissue with capillary elongation in intimate contact with the implanted hydroxyapatite biomatrix (Fig. 4A). Vascular invasion is a prerequisite for bone formation. Indeed, in the concavities of the hydroxyapatite substratum bone forms only when a strong vascular invasion is histologically present in proximity to the newly formed bone [2, 12, 40, 47] (Figs. 4A and C).
2) Following the attachment and differentiation of mesenchymal cells at the hydroxyapatite/soft tissue interface of the concavities, expression of BMPs/OPs gene products by differentiating osteoblast-like cells. This has been shown by Northern blot analyses of RNA extracted from tissue harvested from the concavities of the substratum as well as immunolocalization [2, 12].
3) Synthesis of specific TGF-β superfamily gene products as markers of bone formation by induction, as shown by immunolocalisation of OP-1 and BMP-3 within the cell cytoplasm and at the interface of the hydroxyapatite biomatrix with the mesenchymal tissue [2, 12, 40, 47].
4) Intrinsic osteoinduction with further differentiation of osteoblastic cells. This is dependant upon a critical threshold of endogenously produced BMPs/OPs initiating bone formation by induction as a secondary response [2, 9, 10, 12].
In primates and in primates only, heterotopic bone induction is initiated by BMPs/OPs and TGF-βs as well as by sintered highly crystalline hydroxyapatites with a specific geometric configuration [2, 9, 10, 12, 40]. This indicates that in the primate, heterotopic ossicles develop as a mosaic structure, and that members of the TGF-β superfamily singly, synergistically and synchronously initiate and maintain the developing morphological structures, playing different roles at different time points of the morphogenetic cascade [2, 9, 10, 12].
Importantly, predictable osteogenesis in clinical contexts may be engineered for treatment of bone defects using delivery systems that initiate osteogenesis with relatively low doses of recombinant morphogens [2, 10–12, 49]. The intrinsic osteoinductivity regulated by the geometry of the substratum in the absence of exogenously applied osteogenic proteins of the TGF-β superfamily is helping to engineer morphogenetic responses for therapeutic osteogenesis [2, 9–12, 40, 46, 47]. The versatile nature of these biomimetic biomaterials makes them more practical and more cost effective in their clinical application than devices requiring the combination of both biomaterial matrices and DNA recombinantly produced BMPs/OPs [10, 11].
To induce the cascade of bone differentiation, the soluble osteogenic signals of the TGF-β superfamily must be reconstituted with an insoluble signal or substratum that triggers the bone differentiation cascade. Insoluble biomimetic matrices will have optimal surface characteristics and geometric configurations that are of critical importance for the induction of bone formation [2, 10–12].
Self-induced bone tissue induction and regeneration has been achieved via the deployment of molecular signals expressed and embedded by site specific modifications within bioactive biomimetic matrices endowed with the striking prerogative of inducing de novo bone formation even in the absence of exogenously applied osteogenic proteins of the TFG-β superfamily [2, 10–12, 40, 47].
In collaboration with the Council for Scientific and Industrial Research (CSIR, Pretoria) Materials Science and Technology, we have constructed sintered biomimetic biomatrices of highly crystalline hydroxypatite that mimics the super-smart functionality of living tissue, thereby highly crystalline porous hydroxypatites when implanted in extraskeletal heterotopic sites of the primate Papio ursinus induce the de novo initiation of bone within the smart concavities assembled within the porous spaces of the biomimetic matrices (Figs. 4 and 5) [10, 11, 40, 46, 47].
Our research in the past several years has been focused to add functionality to smart biomimetic matrices for bone repair and regeneration i.e. geometric cues initiating de novo bone formation [40, 46, 47]. The specific geometry of the substratum i.e. concavities of specific dimensions, is conducive and inducive to the generation of a micro-environ-ment that initiate the cascade of bone differentiation deploying site specific surface modifications imprinted during the synthesis of the smart biomimetic matrices (Figs. 4 and 5).
The idea that synthetic biomimetic matrices do mimic the super-smart functionality of living tissue has a futuristic appeal for the construction of smart self-inducing biomimetic matrices for tissue engineering of bone. The assembly of a series of repetitive concavities of specific dimensions within the porous biomimetic matrices adds selected functionalities or super-smart functionalities to the sintered biomimetic matrices when implanted in extraskeletal heterotopic and orthotopic sites of the primate Papio ursinus [40, 47].
By carving and sculpting a series of repetitive concavities into solid and porous biomimetic matrices of highly crystalline hydroxyapatite, we did perform 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 [40, 46, 47].
There are several fundamental issues related to the reported biomimetic geometry or topography that displays specificity of expression of and binding affinity for selected osteogenic proteins of the TGF-β superfamily. The specific geometric configuration initiates spontaneous bone formation as a secondary response even when the biomimetic matrices are implanted in heterotopic sites and without the addition of exogenously applied osteogenic proteins of the TGF-β superfamily [40, 47].
A most important parameter for the spontaneous induction of bone formation is the geometric configuration of the biomimetic matrices in the form of specific concavities on the surface of the substratum that drive the morphogenetic cascade. The substratum may be either porous i.e. with a sequence of repetitive concavities embedded within the porous spaces or solid i.e. with concavities prepared on the outer surface of both planar surfaces, to enhance bone induction and hasten osteointegration when implanted in orthotopic sites.
Our research has shown that the concavities of our biomimetic biomaterial matrices are geometric regulators of growth endowed with shape memory, recapitulating events that occur in the normal course of embryonic development and appearing to act as gates, giving or withholding permission to growth and differentiation [40, 41, 47].
Our molecular, biochemical, histological and morphological data further show that the specific geometric configuration in the form of concavities is the driving micro-environment conducive and inducive to the sequence of events leading to bone formation by induction preceeded by angiogenesis and vascular invasion, mesenchymal cellular
attachment, orientation and aggregation to the biomimetic matrix of the smart concavity. Attached to the matrix and resident within the smart concavities, differentiating osteoblast-like cells express BMPs/OPs gene products as shown by Northern blot analyses and immunolocalization on tissue harvested from the concavities [2, 9–12, 40, 47].
The concavities are thus regulators of bone initiation and deposition during remodeling processes of the skeleton. Remodeling of the cortico-cancellous bones of the skeleton both of endochondral and membranous origins entails, at any given time along the trabeculae of bone, three fundamental biochemical and morphological processes that characterize the remodeling cycle of cancellous and cortical bone: 1) Resting, i.e. surfaces lined by resting lining cells; 2) Resorption, i.e. areas of trabeculae actively resorbed by osteoclasts, multinucleated bone cells that actively and specifically resorb bone. The bone resorption lacunae as formed by the osteoclastic activity are in the form of concavities; 3) Formation, i.e. the concavities formed by osteoclastic resorption reach dimensions similar if not equal to the concavities created in the above described biomimetic matrices. When these dimensions are reached, bone formation occurs as osteoblasts or bone forming cells are then recruited to the concavities, leading to bone formation until the resorption lacunae or concavities are filled with newly formed bone and the specific area reaches a resting status again.
There is a direct spatial and temporal relationship of morphological and molecular events that emphasize the similarity between the remodeling cycles of cancellous bone vs the geometric induction of bone formation, i.e. the induction of bone in smart concavities assembled in biomimetic matrices of highly crystalline hydroxyapatites. In the adult skeleton, the demand for osteoblasts is created by bone resorption [50, 51], i.e. by the concavities induced by osteoclastogenesis, whereas the demand for osteoclasts is governed by the purposes of bone remodeling .
The basic multicellular unit (BMU) of corticocancellous bone excavates a trench across the surface rather than a tunnel leaving in its wake (with some geometrical latitude) a hemiosteon rather than an osteon [50-52], i.e. a trench with cross-sectional geometric cues of concavities at different stages of osteoclastogenesis eventually leading to osteogenesis.
The morphogenetic and molecular mechanisms initiating the spontaneous induction of bone formation within concavities of the smart biomimetic matrices originates and progress with blood vessels arborizing within the mesenchymal tissue invading the concavities, i.e. capillary sprouting [40, 47].
Progression is sustained by the continued recruitment of mesenchymal cells eventually resting on the surface of the smart concavities and later targeted to differentiate into osteoblasts-like cells. Osteogenic proteins of the TGF-β superfamily are then expressed and secretion of the gene products is followed by the binding of the expressed gene products to the smart concavities of the biomimetic matrices. The necessary molecular signals initiating the cascade of bone formation by induction must originate from within the concavity itself and only deployed when osteoclastogenesis has created concavities of specific dimension. Reversal, i.e. osteoblastogenesis, is initiated after synthesis and expression of osteogenic proteins of the TGF-β superfamily are bound to the smart concavities of both the corticocancellous microarchitecture of the mammalian skeleton and intrinsically osteoinductive biomimetic matrices.
The concavities per se are regulators of growth, inducing specific tissue formation and bone induction as in the remodeling processes of bone and act as powerful geometric attractant for bone forming cells i.e. osteoblasts initiating bone formation. Soluble signals induce morphogenesis, physical forces imparted by the geometric topography of the insoluble signal or substratum dictate biological patterns, constructing the induction of bone and regulating the expression of selective messanger RNA of gene products as a function of the structure [2, 10, 12]. The specific geometric concavity being a geometrical and physical regulator of bone remodeling is of paramount importance in skeletal disorders such as systemic bone loss as in osteoporosis.
Biomimetic biomaterial matrices can now be designed to obtain specific biological responses such that the use of biomaterials capable of initiating bone formation via osteoinduction, even in the absence of exogenously applied BMPs/OPs, is fast altering the horizons of therapeutic bone regeneration. Molecular signals of the TGF-β superfamily induce morphogenesis, and physical forces imparted by the geometric topography of the substratum dictate biological patterns and regulate the expression of osteogenic gene transcripts and their translation products initiating bone formation as a function of the structure.
The concavities of the substratum are geometric regulators of growth endowed with shape memory, recapitulating events that occur in the normal course of embryonic development and appearing to act as gates that give or withhold permission to grow and differentiate [10, 41].
Since regenerative phenomena recapitulate events that occur in the normal course of embryonic development, the multiple patterns of expression of BMPs/OPs and TGF-βs in developing tissues and organs should now be exploited to devise novel therapeutic approaches based on recapitulation of embryonic development.
Bone tissue engineering starts by erecting scaffolds of smart biomimetic matrices affecting the release of soluble molecular signals. The molecular scaffolding lies at the heart of all new tissue engineering strategies. This molecular evidence is based on a surprisingly simple and fascinating concept: morphogens exploited in embryonic development can be re-exploited for the initiation of postnatal morphogenesis and regeneration.
The work presented is derived from all at the Bone Research Unit and at the Manufacturing and Materials Technology Group, CSIR Pretoria, who contributed significantly to the understanding of the fascinating phenomenon of the geometric induction of bone formation: Jean Crooks, Thato Matsaba, Nathaniel L Ramoshebi, Louise Renton, William Ricther, June Teare, Michae Thomas, Barbara van den Heever. This work is supported by grants from the South African Medical Research Council, the University of the Witwatersrand, Johannesburg, the National Research Foundation and by ad hoc grants of the Bone Research Unit. K. Miyozono kindly donated the cDNAs for Smad-6 and Smad-7. We thank Janet Patton, Bone Research Unit, for the RT-PCR analyses and Michael Thomas and William Richter, CSIR Pretoria, for the preparation of the sintered hydroxyapatite biomatrices.
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Fig. 1 Tissue induction and morphogenesis by bone morphogenetic/osteogenic proteins (BMPs/OPs). (A) Islands of chondrogenesis with vascular invasion and osteoblastic differentiation and matrix synthesis as a recapitulation of embryonic development, 8 days after heterotopic implantation in a Long-Evans rat of 2.5µg of recombinant hOP-1 in conjunction with 25 mg of bovine insoluble collagenous bone matrix as carrier (original magnification x45). (B) Bone induction by naturally-derived BMPs/OPs purified from baboon bone matrix and implanted orthotopically in a calvarial defect of an adult baboon Papio ursinus: 30 days after implantation of 280µg of baboon osteogenic fractions after gel filtration chromatography there is induction of large osteoid seams in orange-red surfacing newly developed mineralized bone matrix in blue (undecalcified section, original magnification x25). (C) Low-power view of a corticalized ossicle induced after the implantation of 125µg of recombinant hTGF-β in the rectus abdominis muscle of an adult primate Papio ursinus showing vigorous osteogenesis with osteoid synthesis 30 days after heterotopic implantation (undecalcified section, original magnification x4.5).
Fig. 2 Morphology of calvarial regeneration by recombinant hTGF-β2 in conjunction with collagenous matrix as carrier. Low-power photomicrographs of calvarial defects treated by 100µg hTGF-β2 delivered by insoluble collagenous bone matrix and harvested on day 30 (A) and 90 (B) after implantation in non-healing calvarial defects of the primate Papio ursinus. Minimal bone formation at the edges of the defect on day 30 (A) and osteogenesis albeit limited is found in a specimen harvested 90 days after implantation with bone formation only pericranially. Note the trabeculae of newly formed bone facing scattered remnants of collagenous matrix particles, embedded in a loose but highly vascular connective tissue matrix (undecalcified sections, original magnification x3).
Fig. 3 Northern analyses of type IV collagen and Smad6 (A) and OP-1, BMP-3, TGF-β1 and type II and IV collagens mRNA expression (B) in tissues generated by rhTGF-β alone (A) and in ossicles generated by doses of porcine platelet-derived TGF-β1 alone and in combination with 25ìg hOP-1 implanted heterotopically with insoluble collagenous bone matrix as carrier in the rectus abdominis muscle of adult primates Papio ursinus and harvested on day 30. Minimal mRNA expression of Smad6 in ossicles generated by rhTGF-β when implanted heterotopically (A). (B) Upon implantation of 5 µg doses of recombinant hTGF-β1 there is expression of osteogenic markers of the TGF-β superfamily, namely OP-1 and BMP-3 gene products. Note the two to three fold increase of expression of collagen type IV mRNA, a marker of angiogenesis, in ossicles generated by TGF-β1 alone and in synergistic binary application with hOP-1 (B).
Fig. 4 Effect of the geometry of the substratum of biomimetic matrices on tissue morphogenesis and bone induction on day 30. (A) Capillary sprouting, invasion and elongation of the capillaries within a concavity of a biomimetic matrix of highly crystalline hidroxyapatite implanted heterotopically in the rectus abdominis muscle of Papio ursinus and harvested 30 days after implantation. Top right note the newly formed bone in direct contact with the hydroxyapatite substratum (decalcified section, original magnification x45). (B) Spontaneous initiation of bone formation within a concavity of the biomimetic matrix without the addition of exogenously applied BMPs/OPs. The newly formed bone in light blue in direct contact with the hydroxyapatite substratum is surfaced by contiguous osteoblasts (decalcified section, original magnification x60). (C) Detail of another specimen showing the spontaneous initiation of bone formation in direct contact to the crystalline hydroxyapatite together with capillary invasion within the fibrovascular tissue invading the concavity (decalcified section, original magnification x60).
Fig. 5 Tissue morphogenesis in concavities of the substratum 90 days after heterotopic implantation in the rectus abdominis muscle of the primate Papio ursinus. Low power view of a histological section of a monolithic disc of highly crystalline hydroxyapatite: bone has formed only within the concavities prepared on both planar outer surfaces (decalcified section, original magnification x12).
Source: J. Cell. Mol. Med. Vol 8, No 2, 2004 pp. 169-180