Induction of bone formation by recombinant human osteogenic protein-1 and sintered porous hydroxyapatite in adult primates

A critical issue in tissue engineering and morphogenesis of bone is the development of novel biomimetic biomaterials that are capable of optimizing the biological activity of recombinant human bone morphogenetic and osteogenic proteins, which are molecules that initiate bone formation in vivo. From a therapeutic perspective, a carrier matrix is required for the local delivery of these proteins to evoke a desired osteogenic effect. In view of the affinity of these proteins for hydroxyapatite, which may reflect the in vivo supramolecular assembly of bone proteins bound to both the extracellular matrix and the mineral component of bone, we investigated the efficacy of single applications of different doses of human osteogenic protein-1 (hOP-1) adsorbed onto sintered porous hydroxyapatites for bone induction in orthotopic calvarial defects in 12 adult male baboons (Papio ursinus) and heterotopically in the rectus abdominis of four additional baboons. In orthotopic specimens, pretreatment of sintered porous hydroxyapatites with 100 microgram of hOP-1 in 500 microliter of 5 mM hydrochloric acid resulted in rapid and diffuse osteoinduction restricted within the porous spaces of the hydroxyapatite, as evaluated by histology and histomorphometry on day 30. Hydroxyapatites treated with 500 microgram of hOP-1 showed a different pattern of bone formation and distribution on day 30 as compared with the lower dose of the recombinant morphogen. Although bone formation was extensive with the higher dose, it was found on the endocranial and pericranial aspects of the specimens, enveloping the implanted hydroxyapatite carrier, and the internal porous spaces were occupied by a rich vascular network without any bone formation. By 90 and 365 days after the implantation of both doses of hOP-1, however, there was remodelling and complete penetration of the newly induced bone within the available porous spaces. The combination of hOP-1 and hydroxyapatite also showed extensive bone formation in heterotopic specimens harvested from the rectus abdominis muscle of the baboon using doses of 5, 25, and 45 microgram of hOP-1 per implant. These findings in the adult primate demonstrate extensive bone formation by hOP-1 adsorbed onto sintered porous hydroxyapatites and suggest that predictable osteogenesis in clinical contexts for treatment of craniofacial bone defects may be engineered using inorganic, nonimmunogenic, and carvable delivery systems that initiate osteogenesis with relatively low doses of recombinant osteogenic proteins, thus mimicking the macrostructure and microstructure of living bone.     

Bone morphogenetic proteins in craniofacial and periodontal tissue engineering: experimental studies in the non-human primate Papio ursinus

The bone morphogenetic and osteogenic proteins (BMPs/OPs), pleiotropic members of the transforming growth factor-beta (TGF-beta) supergene family act as soluble signals for the de novo initiation of bone formation, sculpting the multicellular mineralized structures of the bone-bone marrow organ. The strikingly pleiotropic effects of BMPs/OPs spring from amino acid sequence variations in the carboxy-terminal domain and in the transduction of distinct signalling pathways by individual Smad proteins after transmembrane serine/threonine kinase complexes of type I and II receptors. BMPs/OPs are the common molecular initiators deployed for embryonic development and the induction of bone formation and regeneration in postnatal osteogenesis. Naturally derived BMPs/OPs extracted and purified from baboon and bovine bone matrices induce complete regeneration of non-healing calvarial defects in the non-human primate Papio ursinus as well as the induction of cementogenesis and the morphogenesis of a periodontal ligament system with a faithful insertion of Sharpey's fibers into the newly formed cementum. gamma-Irradiated recombinant human osteogenic protein-1 (hOP-1) delivered by xenogeneic bovine collagenous bone matrices completely regenerated and maintained the architecture of the induced bone after treatment of calvarial defects with single applications of doses of 0.1, 0.5 and 2.5mg hOP-1 per gram of carrier matrix. The long-term implantation of hOP-1 delivered by gamma-irradiated bovine bone matrices induced the regeneration of the three essential components of the periodontium, i.e. cementum, periodontal ligament and alveolar bone. The osteogenic proteins of the TGF-beta superfamily are sculpting tissue constructs that engineer skeletal tissue regeneration in molecular terms. The pleiotropy of the signalling molecules of the TGF-beta superfamily is highlighted by the redundancy of molecular signals initiating bone formation, including the TGF-beta isoforms per se, powerful inducers of endochondral bone formation but in the primate only. The induction of bone develops a mosaic structure in which members of the TGF-beta superfamily singly, synergistically and synchronously initiate and maintain tissue induction and morphogenesis.     

The induction of endochondral bone formation by transforming growth factor-beta(3): experimental studies in the non-human primate Papio ursinus.

Transforming growth factor-beta(3) (TGF-beta(3)), a multi-functional growth modulator of embryonic development, tissue repair and morphogenesis, immunoregulation, fibrosis, angiogenesis and carcinogenesis, is the third mammalian isoform of the TGF-beta subfamily of proteins. The pleiotropism of the signalling proteins of the TGF-beta superfamily, including the TGF-beta proteins per se, are highlighted by the apparent redundancy of soluble molecular signals initiating de novo endochondral bone induction in the primate only. In the heterotopic bioassay for bone induction in the subcutaneous site of rodents, the TGF-beta(3) isoform does not initiate endochondral bone formation. Strikingly and in marked contrast to the rodent bioassay, recombinant human (h)TGF-beta(3), when implanted in the rectus abdominis muscle of adult non-human primates Papio ursinus at doses of 5, 25 and 125 mug per 100 mg of insoluble collagenous matrix as carrier, induces rapid endochondral bone formation resulting in large corticalized ossicles by day 30 and 90. In the same animals, the delivery of identical or higher doses of theTGF-beta(3) protein results in minimal repair of calvarial defects on day 30 with limited bone regeneration across the pericranial aspect of the defects on day 90. Partial restoration of the bone induction cascade by the hTGF-beta(3) protein is obtained by mixing the hTGF-beta(3) device with minced fragments of autogenous rectus abdominis muscle thus adding responding stem cells for further bone induction by the hTGF-beta(3) protein. The observed limited bone induction in hTGF-beta(3)/treated and untreated calvarial defects in Papio ursinus and therefore by extension to Homo sapiens, is due to the influence of Smad-6 and Smad-7 down-stream antagonists of the TGF-beta signalling pathway. RT-PCR, Western and Northern blot analyses of tissue specimens generated by the TGF-beta(3) isoform demonstrate robust expression of Smad-6 and Smad-7 in orthotopic calvarial sites with limited expression in heterotopic rectus abdominis sites. Smad-6 and -7 overexpression in hTGF-beta(3)/treated and untreated calvarial defects may be due to the vascular endothelial tissue of the arachnoids expressing signalling proteins modulating the expression of the inhibitory Smads in pre-osteoblastic and osteoblastic calvarial cell lines controlling the induction of bone in the primate calvarium.     

Soluble, insoluble and geometric signals sculpt the architecture of mineralized tissues

Bone morphogenetic and osteogenic proteins (BMPs/OPs), members of the transforming growth factor-beta (TGF-beta) 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-beta isoforms per se. In the primate and in the primate only, the TGF-beta 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-beta 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-beta 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-beta 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.     

Recombinant human osteogenic protein-1 (hOP-1) induces new bone formation in vivo with a specific activity comparable with natural bovine osteogenic protein and stimulates osteoblast proliferation and differentiation in vitro.

We reported previously that a 32-36-kDa osteogenic protein purified from bovine bone matrix is composed of dimers of two members of the transforming growth factor (TGF)-beta superfamily: the bovine equivalent of human osteogenic protein-1 (OP-1) and bone morphogenetic protein-2a, BMP-2a (BMP-2). In the present study, we produced the recombinant human OP-1 (hOP-1) in mammalian cells as a processed mature disulfide-linked homodimer with an apparent molecular weight of 36,000. Examination of hOP-1 in the rat subcutaneous bone induction model demonstrated that hOP-1 was capable of inducing new bone formation with a specific activity comparable with that exhibited by highly purified bovine osteogenic protein preparations. The half-maximal bone-inducing activity of hOP-1 in combination with a rat collagen matrix preparation was 50-100 ng/25 mg of matrix as determined by the calcium content of day 12 implants. Evaluation of hOP-1 effects on cell growth and collagen synthesis in rat osteoblast-enriched bone cell cultures showed that both cell proliferation and collagen synthesis were stimulated in a dose-dependent manner and increased 3-fold in response to 40 ng of hOP-1/ml. Examination of the expression of markers characteristic of the osteoblast phenotype showed that hOP-1 specifically stimulated the induction of alkaline phosphatase (4-fold increase at 40 ng of hOP-1/ml), parathyroid hormone-mediated intracellular cAMP production (4-fold increase at 40 ng of hOP-1/ml), and osteocalcin synthesis (5-fold increase at 25 ng of hOP-1/ml). In long-term (11-17 day) cultures of osteoblasts in the presence of beta-glycerophosphate and L(+)-ascorbate, hOP-1 markedly increased the rate of mineralization as measured by the number of mineral nodules per well (20-fold increase at 20 ng of hOP-1/ml). Direct comparison of TGF-beta 1 and hOP-1 in these bone cell cultures indicated that, although both hOP-1 and TGF-beta 1 promoted cell proliferation and collagen synthesis, only hOP-1 was effective in specifically stimulating markers of the osteoblast phenotype.     

Osteogenic protein-2. A new member of the transforming growth factor-beta superfamily expressed early in embryogenesis.

Osteogenic protein-2, OP-2, a new member of the transforming growth factor-beta (TGF-beta) superfamily, closely related to the osteogenic/bone morphogenetic proteins, was discovered in mouse embryo and human hippocampus cDNA libraries. The TGF-beta domain of OP-2 shows 74% identity to OP-1, 75% to Vgr-1, and 76% to BMP-5, hence OP-2 may also have bone inductive activity. The genomic locus of OP-2 has seven exons, like OP-1, and spans more than 27 kilobases (kb). In the C-terminal TGF-beta domain, OP-2 has a unique additional cysteine. Mouse embryos express relatively high levels of OP-2 mRNA at 8 days, two species of 3 and 5 kb. A careful study of mRNA expression of the osteogenic proteins in specific organs revealed discrete mRNA species for BMP-3, BMP-4, BMP-5, and BMP-6/Vgr-1 in lung or liver of young and adult mice. OP-1 is expressed in kidney; however, OP-2 and BMP-2 mRNAs were not detected in any organs studied, suggesting an early developmental role.     

Localization of the expression of types I, III, and IV collagen, TGF-beta 1 and c-fos genes in developing human calvarial bones

Total RNA extracted from developing calvarial bones of 15- to 18-week human fetuses was studied by Northern hybridization: in addition to high levels of type I collagen mRNAs, the presence of mRNAs for type III and type IV collagen, TGF-beta and c-fos was observed. In situ hybridization of sections containing calvarial bone, overlying connective tissues, and skin was employed to identify the cells containing these mRNAs. Considerable variation was observed in the distribution of pro alpha 1(I) collagen mRNA in osteoblasts: the amount of the mRNA in cells at or near the upper surface of calvarial bone was distinctly greater than that in cells at the lower surface, indicating the direction of bone growth. High levels of type I collagen mRNAs were also detected in fibroblasts of periosteum, dura mater, and skin. Type III collagen mRNA revealed a considerably different distribution: the highest levels were detected in upper dermis, lower levels were seen in fibroblasts of the periosteum and the fibrous mesenchyme between bone spiculas, and none was seen in osteoblasts. Type IV collagen mRNAs were only observed in the endothelial cells of blood capillaries. Immunohistochemical localization of type III and IV collagens agreed well with these observations. The distribution of TGF-beta mRNA resembled that of type I collagen mRNA. In addition, high levels of TGF-beta mRNA were observed in osteoclasts of the calvarial bone. These cells, responsible for bone resorption, were also found to contain high levels of c-fos mRNA. Production of TGF-beta by osteoclasts and its activation by the acidic environment could form a link between bone resorption and new matrix formation.     

Induction of bone formation by transforming growth factor‐β2 in the non‐human primate Papio ursinus and its modulation by skeletal muscle responding stem cells

Objectives: Four adult non‐human primates Papio ursinus were used to study induction of bone formation by recombinant human transforming growth factor‐β₂ (hTGF‐β₂) together with muscle‐derived stem cells. Materials and methods: The hTGF‐β₂ was implanted in rectus abdominis muscles and in calvarial defects with and without addition of morcellized fragments of striated muscle, harvested from the rectus abdominis or temporalis muscles. Expression of osteogenic markers including osteogenic protein‐1, bone morphogenetic protein‐3 and type IV collagen mRNAs from generated specimens was examined by Northern blot analysis. Results: Heterotopic intramuscular implantation of 5 and 25 μg hTGF‐β₂ combined with 100 mg of insoluble collagenous bone matrix yielded large corticalized mineralized ossicles by day 30 with remodelling and induction of haematopoietic marrow by day 90. Addition of morcellized rectus abdominis muscle to calvarial implants enhanced induction of bone formation significantly by day 90. Conclusions: In Papio ursinus, in marked contrast to rodents and lagomorphs, hTGF‐β₂ induced large corticalized and vascularized ossicles by day 30 after implantation into the rectus abdominis muscle. This striated muscle contains responding stem cells that enhance the bone induction cascade of hTGF‐β₂. Induction of bone formation by hTGF‐β₂ in the non‐human primate Papio ursinus may occur as a result of expression of bone morphogenetic proteins on heterotopic implantation of hTGF‐β₂; the bone induction cascade initiated by mammalian TGF‐β proteins in Papio ursinus needs to be re‐evaluated for novel molecular therapeutics for induction of bone formation in clinical contexts.     

Induction of bone in composites of osteogenin and porous hydroxyapatite in baboons.

A major goal of the combined effort of basic scientists and plastic and reconstructive surgeons is the development of novel bone substitutes based on osteogenic growth and differentiation factors with optimal delivery systems for skeletal repair. Osteogenin is a protein initiator of bone differentiation. The present study examined the osteogenic potential of osteogenin in combination with porous hydroxyapatite replicas obtained after hydrothermal conversion of calcium carbonate exoskeletons of corals. Bovine osteogenin, with an apparent molecular weight of 28 to 42 kDa, purified by hydroxyapatite-Ultrogel adsorption chromatography, heparin-Sepharose affinity chromatography, and HR Sephacryl S-200 molecular sieve chromatography, was delivered into rods of nonresorbable and resorbable hydroxyapatite replicas with an average porosity of 600 microns. A total of 48 rods were bioassayed for osteogenic activity by intramuscular implantation into eight adult baboons (Papio ursinus) as a prerequisite for clinical trials in humans. Bovine osteogenin fractions reconstituted with baboon insoluble collagenous bone matrix were implanted in an additional four adult baboons. Specimens were harvested at 30 and 90 days after implantation and subjected to histomorphometry and alkaline phosphatase activity determination. Differentiation of bone occurred in nonresorbable hydroxyapatite rods, both osteogenin-treated and controls. However, no bone formation was observed in resorbable rods, even in the presence of osteogenin. These results demonstrate that the surface and chemical characteristics of the substratum, independent of the osteogenic stimulus, have a profound influence on the morphogenesis of bone. The demonstration of bone induction in nonhuman primates with porous nonresorbable hydroxyapatite replicas and baboon insoluble collagenous bone matrix reconstituted with bovine osteogenin establishes the therapeutic potential of the principle of bone induction in craniofacial, periodontal, and orthopedic reconstructive surgery.     

Differential effects of osteogenic protein-1 (BMP-7) on gene expression of BMP and GDF family members during differentiation of the mouse MC615 chondrocyte cells

The mRNA expression patterns of several bone morphogenetic proteins (BMPs) and growth differentiation factors (GDFs) in long-term cultures of the clonal mouse chondrocyte cell line MC615 were examined. Distinct spatial and temporal patterns of expression of BMPs and GDFs were observed. The temporal orders of expression were correlated with those of several biochemical markers characteristic of chondrocytic cell differentiation. BMP-1, -2, -5, and -6 mRNA expression increased throughout the chondrogenic process and BMP-4 mRNA expression was not changed. GDF-1 and -3 mRNA expression increased throughout the chondrogenic process, and GDF-5, -6, -8, and -9 mRNA expressions were not changed. Effects of osteogenic protein-1 (OP-1, BMP-7) on the expression patterns of several other members of the BMP family and of the GDF family were also examined. OP-1 downregulated the BMP-1, -4, -5, and -6 mRNA expression by a maximal 3-, 5-, 2.5-, and 3-fold, respectively. The BMP-2 mRNA expression was not changed significantly by a low concentration of OP-1, but was increased at 200 ng/ml at day 7 of treatment. In contrast to the BMPs, OP-1 upregulated significantly the six GDF members examined (GDF-1, -3, -5, -6, -8, and -9) by three- to four-fold. Our findings demonstrate that OP-1 differentially regulates the mRNA expression of several related members of the BMP family and upregulates the mRNA expression of several members of the GDF family. The observations suggest that OP-1 action on cartilage differentiation involves a complex regulation of gene expression of several members of the BMP and the GDF family.     

Endoglin differentially modulates antagonistic transforming growth factor-beta1 and BMP-7 signaling

Transforming growth factor-beta1 (TGF-beta1) and BMP-7 (bone morphogenetic protein-7; OP-1) play central, antagonistic roles in kidney fibrosis, a setting in which the expression of endoglin (CD105), an accessory TGF-beta type III receptor, is increased. So far, endoglin is known as a negative regulator of TGF-beta/ALK-5 signaling. Here we analyzed the effect of BMP-7 on TGF-beta1 signaling and the role of endoglin for both pathways in endoglin-deficient L(6)E(9) cells. In this myoblastic cell line, TGF-beta1 and BMPs are opposing cytokines, interfering with myogenic differentiation. Both induce specific target genes of which Id1 (for BMPs) and collagen I (for TGF-beta1) are two examples. TGF-beta1 activated two distinct type I receptors, ALK-5 and ALK-1, in these cells. Although the ALK-5/Smad3 signaling pathway mediated collagen I expression, ALK-1/Smad1/Smad5 signaling mediated a transient Id1 up-regulation. In contrast, BMP-7 exclusively activated Smad1/Smad5 resulting in a more prolonged Id1 expression. Although BMP-7 had no impact on collagen I abundance, it antagonized TGF-beta1-induced collagen I expression and (CAGA)(12)-MLP-Luc activity, effects that are mediated by the ALK-5/Smad3 pathway. Finally, we found that the transient overexpression of endoglin, previously shown to inhibit TGF-beta1-induced ALK-5/Smad3 signaling, enhanced the BMP-7/Smad1/Smad5 pathway.     

BMP-2 induction and TGF-beta 1 modulation of rat periosteal cell chondrogenesis

Periosteum contains osteochondral progenitor cells that can differentiate into osteoblasts and chondrocytes during normal bone growth and fracture healing. TGF-beta 1 and BMP-2 have been implicated in the regulation of the chondrogenic differentiation of these cells, but their roles are not fully defined. This study was undertaken to investigate the chondrogenic effects of TGF-beta 1 and BMP-2 on rat periosteum-derived cells during in vitro chondrogenesis in a three-dimensional aggregate culture. RT-PCR analyses for gene expression of cartilage-specific matrix proteins revealed that treatment with BMP-2 alone and combined treatment with TGF-beta 1 and BMP-2 induced time-dependent mRNA expression of aggrecan core protein and type II collagen. At later times in culture, the aggregates treated with BMP-2 exhibited expression of type X collagen and osteocalcin mRNA, which are markers of chondrocyte hypertrophy. Aggregates incubated with both TGF-beta 1 and BMP-2 showed no such expression. Treatment with TGF-beta 1 alone did not lead to the expression of type II or X collagen mRNA, indicating that this factor itself did not independently induce chondrogenesis in rat periosteal cells. These data were consistent with histological and immunohistochemical results. After 14 days in culture, BMP-2-treated aggregates consisted of many hypertrophic chondrocytes within a metachromatic matrix, which was immunoreactive with anti-type II and type X collagen antibodies. In contrast, at 14 days, TGF-beta 1 + BMP-2-treated aggregates did not contain any morphologically identifiable hypertrophic chondrocytes and their abundant extracellular matrix was not immunoreactive to the anti-type X collagen antibody. Expression of BMPR-IA, TGF-beta RI, and TGF-beta RII receptors was detected at all times in each culture condition, indicating that the distinct responses of aggregates to BMP-2, TGF-beta 1 and TGF-beta 1 + BMP-2 were not due to overt differences in receptor expression. Collectively, our results suggest that BMP-2 induces neochondrogenesis of rat periosteum-derived cells, and that TGF-beta 1 modulates the terminal differentiation in BMP-2 induced chondrogenesis.     

Osteogenic protein-1 differentially regulates the mRNA expression of bone morphogenetic proteins and their receptors in primary cultures of osteoblasts

The mRNA expression patterns of several bone morphogenetic proteins (BMPs) and their receptors (BMPRs) in long-term primary cultures of fetal rat calvaria (FRC) cells were examined by Northern analysis. Their temporal orders of expression were correlated with those of several biochemical markers characteristic of osteoblastic cell differentiation. Distinct temporal patterns of expression of BMPs and BMPRs during osteoblastic cell differentiation were observed. BMP-2 and BMP-7 mRNA levels did not change significantly. BMP-4 mRNA expression increased and reached a peak prior to matrix formation. BMP-5 mRNA expression increased during the mineralization phase and BMP-6 mRNA expression increased throughout all phases of cell differentiation. Effects of BMP-7 (Osteogenic Protein-1; OP-1) on the expression patterns of several other members of the BMP family and the receptors were also studied. OP-1 downregulated the BMP-4, -5, and -6 mRNA levels by a maximal of 2-fold, 1.5-fold, and 6-fold, respectively. OP-1 did not change significantly the OP-1 and BMP-2 mRNA expression. Of the three type I BMPR examined, OP-1 upregulated ActR-I and BMPR-IA mRNA expression slightly but with statistical significance. OP-1 downregulated BMPR-IB mRNA expression slightly. OP-1 upregulated BMPR-II mRNA expression by a maximum of 2-fold. Our findings demonstrate that OP-1 differentially regulates the mRNA expression of several related members of the BMP family and their receptors in osteoblasts. The observations suggest that OP-1 action on osteoblastic cells involves a complex regulation of gene expression of related members of the BMP family and their receptors in a cell differentiation stage dependent manner.     

Soluble and insoluble signals sculpt osteogenesis in angiogenesis

The basic tissue engineering paradigm is tissue induction and morphogenesis by combinatorial molecular protocols whereby soluble molecular signals are combined with insoluble signals or substrata. The insoluble signal acts as a three-dimensional scaffold for the initiation of de novo tissue induction and morphogenesis. The osteogenic soluble molecular signals of the transforming growth factor-β (TGF-β) supergene family, the bone morphogenetic/osteogenic proteins (BMPs/OPs) and, uniquely in the non-human primate Papio ursinus (P. ursinus), the three mammalian TGF-β isoforms induce bone formation as a recapitulation of embryonic development. In this paper, I discuss the pleiotropic activity of the BMPs/OPs in the non-human primate P. ursinus, the induction of bone by transitional uroepithelium, and the apparent redundancy of molecular signals initiating bone formation by induction including the three mammalian TGF-β isoforms. Amongst all mammals tested so far, the three mammalian TGF-β isoforms induce endochondral bone formation in the non-human primate P. ursinus only. Bone tissue engineering starts by erecting scaffolds of biomimetic biomaterial matrices that mimic the supramolecular assembly of the extracellular matrix of bone. The molecular scaffolding lies at the hearth of all tissue engineering strategies including the induction of bone formation. The novel concept of tissue engineering is the generation of newly formed bone by the implantation of "smart" intelligent biomimetic matrices that per se initiate the ripple-like cascade of bone differentiation by induction without exogenously applied BMPs/OPs of the TGF-β supergene family. A comprehensive digital iconographic material presents the modified tissue engineering paradigm whereby the induction of bone formation is initiated by intelligent smart biomimetic matrices that per se initiate the induction of bone formation without the exogenous application of the soluble osteogenic molecular signals. 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.     

Osteogenic protein-1 (OP-1, BMP-7) induces osteoblastic cell differentiation of the pluripotent mesenchymal cell line C2C12

The effects of Osteogenic Protein-1 (OP-1, BMP-7) on the differentiation of the pluripotent mesenchymal cell line, C2C12, were examined. OP-1 at 50 ng/ml partially inhibited myotube formation in C2C12 cells, while OP-1 at 200 ng/ml completely inhibited myotube formation and induced the formation of cells displaying osteoblastic morphology. High concentrations of OP-1 elevated the alkaline phosphatase (AP) activity dramatically, both as a function of time and OP-1 concentration. Osteocalcin (OC) mRNA expression was detected as early as 8 days in OP-1-treated cultures and subsequently increased considerably. Expression of bone sialoprotein (BSP) mRNA was low in control cultures and stimulated by OP-1. Collagen type I mRNA expression was enhanced by OP-1 during the early days in culture, but gradually decreased thereafter. MyoD mRNA expression, high in control cultures, was suppressed by OP-1 in a dose- and time-dependent manner. OP-1 enhanced ActR-I mRNA expression and significantly elevated the mRNA expressions of BMP-1, BMP-4, BMP-5, GDF-6, and GDF-8. The present results indicate that OP-1 is a potent inducer of C2C12 differentiation into osteoblastic cells.     

PTHrP expression in chick sternal chondrocytes is regulated by TGF-beta through Smad-mediated signaling

PTHrP regulates the rate of chondrocyte differentiation during endochondral bone formation. The expression of PTHrP and its regulation by TGF-beta, BMP-2, and PTHrP was examined in upper sternal chondrocytes following 1, 3, and 5 days of continuous treatment. While TGF-beta stimulated the expression of PTHrP (5-fold), PTHrP caused a slight inhibition, and BMP-2 markedly inhibited PTHrP mRNA expression. The effect of these factors on PTHrP expression was not simply related to the maturational state of the cells, since BMP-2 increased, while both PTHrP and TGF-beta decreased the expression of type X collagen. TGF-beta isoforms 1, 2, and 3 all stimulated PTHrP expression. Signaling events involved in the induction of PTHrP by TGF-beta were further evaluated in a PTHrP-promoter CAT construct. The effect of TGF-beta, BMP-2, and PTHrP on the PTHrP-promoter paralleled their effects on mRNA expression, with TGF-beta significantly increasing CAT activity, BMP-2 decreasing CAT activity, and PTHrP having a minimal effect. Co-transfection of the TGF-beta signaling molecule, Smad 3, mimicked the effect of TGF-beta (induction of PTHrP promoter), while dominant negative Smad 3 inhibited the induction of the PTHrP promoter by TGF-beta. Furthermore, infection with a Smad 3-expressing retrovirus mimicked the effects of exogenously added TGF-beta, and induced PTHrP mRNA expression in the infected chondrocyte culture. In contrast, a dominant negative Smad 3 completely inhibited PTHrP promoter stimulation by TGF-beta, but only partially blocked the effect of TGF-beta on PTHrP mRNA synthesis. These findings demonstrate that PTHrP is expressed in chondrocytes undergoing endochondral ossification, and show regulation, at least in part, by TGF-beta through Smad mediated signaling events.