Evaluation of composition and mineral structure of callus tissue in rat femoral fracture.

Callus formation is a critical step for successful fracture healing. Little is known about the molecular composition and mineral structure of the newly formed tissue in the callus. The aim was to evaluate the feasibility of small angle x-ray scattering (SAXS) to assess mineral structure of callus and cortical bone and if it could provide complementary information with the compositional analyses from Fourier transform infrared (FTIR) microspectroscopy. Femurs of 12 male Sprague-Dawley rats at 9 weeks of age were fractured and fixed with an intramedullary 1.1 mm K-wire. Fractures were treated with the combinations of bone morphogenetic protein-7 and/or zoledronate. Rats were sacrificed after 6 weeks and both femurs were prepared for FTIR and SAXS analysis. Significant differences were found in the molecular composition and mineral structure between the fracture callus, fracture cortex, and control cortex. The degree of mineralization, collagen maturity, and degree of orientation of the mineral plates were lower in the callus tissue than in the cortices. The results indicate the feasibility of SAXS in the investigation of mineral structure of bone fracture callus and provide complementary information with the composition analyzed with FTIR. Moreover, this study contributes to the limited FTIR and SAXS data in the field.

Complete subchondral bone defect regeneration with a tricalcium phosphate collagen implant and osteoinductive growth factors: a randomized controlled study in Göttingen minipigs.

The restoration and reconstruction of osseous defects close to the joint, constitutes a challenging field for reconstructive surgery. A dual-layer implant of ß-tricalcium phosphate (TCP) and a collagens I/III scaffold was evaluated in a prospective, randomized comparison in a larger animal model. For this purpose, a standardized osteochondral defect was created in the medial facet of the patellar groove in both stifle joints of Göttingen minipigs. Critical-size osseous defects were either left empty (spontaneous healing; group 1; n = 12) or treated with the two-layer TCP collagen implant (group 2; n = 12). In group 3 (n = 12), additional growth factor mixture (GFM) was supplemented (bone morphogenetic proteins 2, 3, 4, 6, 7, and TGF-ß1, 2, 3). Osseous defect regeneration was assessed at 6, 12, and 52 weeks postoperatively (n = 4). Qualitative and quantitative histomorphometric assessment of defect regeneration and bone substitute resorption was conducted by means of light microscopy, fluorescence microscopy, and microradiography. Critical-size defects did not heal spontaneously throughout follow-up (group 1: max. 21.84 ± 2.81% defect area at 52 weeks). The TCP layer of the implant significantly increased the amount of new bone formation with 29.8 ± 9.68% at 6 weeks and 40.09 ± 4.76% at 12 weeks when compared with controls. After 52 weeks, the TCP was almost fully degraded (4.35 ± 3.70%) and the defect was restored with lamellar trabecular bone (31.28 ± 5.02%). Growth factor supplementation resulted in earlier resorption of the TCP implant and faster defect regeneration. The dual-layer TCP collagen implant is suitable to restore subchondral osseous defects. Additional use of GFM increased the resorption of the TCP layer, but did not foster new bone formation.

Bio-composites composed of a solid free-form fabricated polycaprolactone and alginate-releasing bone morphogenic protein and bone formation peptide for bone tissue regeneration.

Biomedical scaffolds should be designed with highly porous three-dimensional (3D) structures that have mechanical properties similar to the replaced tissue, biocompatible properties, and biodegradability. Here, we propose a new composite composed of solid free-form fabricated polycaprolactone (PCL), bone morphogenic protein (BMP-2) or bone formation peptide (BFP-1), and alginate for bone tissue regeneration. In this study, PCL was used as a mechanical supporting component to enhance the mechanical properties of the final biocomposite and alginate was used as the deterring material to control the release of BMP-2 and BFP-1. A release test revealed that alginate can act as a good release control material. The in vitro biocompatibilities of the composites were examined using osteoblast-like cells (MG63) and the alkaline phosphatase (ALP) activity and calcium deposition were assessed. The in vitro test results revealed that PCL/BFP-1/Alginate had significantly higher ALP activity and calcium deposition than the PCL/BMP-2/Alginate composite. Based on these findings, release-controlled BFP-1 could be a good growth factor for enhancement of bone tissue growth and the simple-alginate coating method will be a useful tool for fabrication of highly functional biomaterials through release-control supplementation.

Apatite containing aspartic acid for selective protein loading.

Physico-chemical modifications of hydroxyapatite (HAp) materials are considered as pre-requisites for the development of new bioactive carrier materials for drug delivery and tissue engineering applications. Since acidic amino acids have well-documented affinities to both HAp and basic proteins, HAp modified by aspartic acid (Asp, acidic amino acid) might be one of the candidate substrates for a basic protein carrier. Here, we synthesized HAp in the presence of various concentrations of Asp and observed that HAp crystallinity and other physico-chemical properties were effectively modulated. Detailed studies indicated that Asp was not incorporated in the HAp crystal lattice, but rather was trapped in HAp crystals. Protein adsorption studies indicated that the HAp particles modified by Asp had a selective loading capacity for basic protein. Therefore, HAp particles containing Asp might have potential in drug delivery applications, especially as the carrier of basic proteins including bFGF and BMP.

Non-invasive screening method for simultaneous evaluation of in vivo growth factor release profiles from multiple ectopic bone tissue engineering implants.

The purpose of this study was to develop and validate a screening method based on scintillation probes for the simultaneous evaluation of in vivo growth factor release profiles of multiple implants in the same animal. First, we characterized the scintillation probes in a series of in vitro experiments to optimize the accuracy of the measurement setup. The scintillation probes were found to have a strong geometric dependence and experience saturation effects at high activities. In vitro simulation of 4 subcutaneous limb implants in a rat showed minimal interference of surrounding implants on local measurements at close to parallel positioning of the probes. These characteristics were taken into consideration for the design of the probe setup and in vivo experiment. The measurement setup was then validated in a rat subcutaneous implantation model using 4 different sustained release carriers loaded with (125)I-BMP-2 per animal. The implants were removed after 42 or 84 days of implantation, for comparison of the non-invasive method to ex vivo radioisotope counting. The non-invasive method demonstrated a good correlation with the ex vivo counting method at both time-points of all 4 carriers. Overall, this study showed that scintillation probes could be successfully used for paired measurement of 4 release profiles with minimal interference of the surrounding implants, and may find use as non-invasive screening tools for various drug delivery applications.

Gene cloning and biochemical characterization of the BMP-2 of Pinctada fucata.

The bone morphogenetic proteins (BMPs) constitute a subfamily of the transforming growth factor type beta (TGF-beta) supergene family. BMP-2 plays an important role not only in osteoblast differentiation but also in pattern formation during development. To determine the function of BMP-2 in Pinctada fucata development and hard tissue formation, we isolated a BMP-2 genomic DNA clone and the BMP-2 cDNA. The deduced BMP-2 sequence consisted of 447 amino acids. The BMP-2 gene was composed of three exons. The C-terminal portion (149 amino acids) had 86% and 66% identity to the Crassostrea gigas and the human BMP-2 sequence respectively. The 5' flanking promoter region contained putative glioma transcription factor (Gli) and retinoic acid receptor (RAR) responding elements. The BMP-2 gene was expressed strongly in the inner part of the mantle tissue, corresponding to the nacreous aragonite shell layer. This finding suggests that BMP-2 has a key role in nacreous layer formation.

The protease domain of procollagen C-proteinase (BMP1) lacks substrate selectivity, which is conferred by non-proteolytic domains.

Procollagen C-proteinase (PCP) removes the C-terminal pro-peptides of procollagens and also processes other matrix proteins. The major splice form of the PCP is termed BMP1 (bone morphogenetic protein 1). Active BMP1 is composed of an astacin-like protease domain, three CUB (complement, sea urchin Uegf, BMP1) domains and one EGF-like domain. Here we compare the recombinant human full-length BMP1 with its isolated proteolytic domain to further unravel the functional influence of the CUB and EGF domains. We show that the protease domain alone cleaves truncated procollagen VII within the short telopeptide region into fragments of similar size as the full-length enzyme does. However, unlike full-length BMP1, the protease domain does not stop at this point, but degrades its substrate completely. Moreover, the protease domain cleaves other matrix proteins such as fibronectin, collagen I and collagen IV, which are left intact by the full-length enzyme. In addition, we show for the first time that thrombospondin-1 is differently cleaved by both BMP1 and its catalytic domain. In summary, our data support the concept that the C-terminal domains of BMP1 are important for substrate recognition and for controlling and restricting its proteolytic activity via exosite binding.

Identification of Sparus aurata bone morphogenetic protein 2: molecular cloning, gene expression and in silico analysis of protein conserved features in vertebrates.

Bone morphogenetic protein 2 (BMP-2) is a secreted signaling molecule that acts as an inducer of bone formation and a regulator of embryonic development. The objectives of this work were as follows: (1) to clone the full-length cDNA of BMP-2 in a marine fish model, (2) analyze its gene expression during development, in adult tissues and in cell lines, and (3) identify protein conserved features of vertebrate BMP-2. Using a combination of RT- and 5'-RACE-PCR, a 1653-bp fragment corresponding to Sparus aurata BMP-2 cDNA (SaBMP-2) was amplified. Levels of SaBMP-2 gene expression were estimated using quantitative real-time PCR and shown to be strongly increased (150-fold induction) at gastrulation, thus suggesting a key role for BMP-2 in fish development. Tissue distribution of SaBMP-2 mRNA revealed highest levels in the calcified tissues bone, caudal fin and scales and in liver. BMP-2 was also found to be highly expressed in S. aurata bone-derived cell lines VSa13 and VSa16 and to be up-regulated (more than 10-fold induction) in mineralized VSa13 chondrocyte-like cells. Using bioinformatic tools and all vertebrate protein sequences available, conserved features of BMP-2 were characterized. The mature protein was shown to be highly conserved across 20 species indicating that BMP-2 function has been conserved throughout evolution, a finding that is in agreement with the widely accepted view of the important role played by BMPs in vertebrate development.

Identification of receptors and signaling pathways for orphan bone morphogenetic protein/growth differentiation factor ligands based on genomic analyses.

There are more than 30 human transforming growth factor beta/bone morphogenetic protein/growth differentiation factor (TGFbeta/BMP/GDF)-related ligands known to be important during embryonic development, organogenesis, bone formation, reproduction, and other physiological processes. Although select TGFbeta/BMP/GDF proteins were found to interact with type II and type I serine/threonine receptors to activate downstream Smad and other proteins, the receptors and signaling pathways for one-third of these TGFbeta/BMP/GDF paralogs are still unclear. Based on a genomic analysis of the entire repertoire of TGFbeta/BMP/GDF ligands and serine/threonine kinase receptors, we tested the ability of three orphan BMP/GDF ligands to activate a limited number of phylogenetically related receptors. We characterized the dimeric nature of recombinant GDF6 (also known as BMP13), GDF7 (also known as BMP12), and BMP10. We demonstrated their bioactivities based on the activation of Smad1/5/8-, but not Smad2/3-, responsive promoter constructs in the MC3T3 cell line. Furthermore, we showed their ability to induce the phosphorylation of Smad1, but not Smad2, in these cells. In COS7 cells transfected with the seven known type I receptors, overexpression of ALK3 or ALK6 conferred ligand signaling by GDF6, GDF7, and BMP10. In contrast, transfection of MC3T3 cells with ALK3 small hairpin RNA suppressed Smad signaling induced by all three ligands. Based on the coevolution of ligands and receptors, we also tested the role of BMPRII and ActRIIA as the type II receptor candidates for the three orphan ligands. We found that transfection of small hairpin RNA for BMPRII and ActRIIA in MC3T3 cells suppressed the signaling of GDF6, GDF7, and BMP10. Thus, the present approach provides a genomic paradigm for matching paralogous polypeptide ligands with a limited number of evolutionarily related receptors capable of activating specific downstream Smad proteins.

Identification of the minimal domain structure of bone morphogenetic protein-1 (BMP-1) for chordinase activity: chordinase activity is not enhanced by procollagen C-proteinase enhancer-1 (PCPE-1).

Bone morphogenetic protein 1 (BMP-1), which is a tolloid member of the astacin-like family of zinc metalloproteinases, is a highly effective procollagen C-proteinase (PCP) and chordinase. On the other hand, mammalian tolloid like-2 (mTLL-2) does not cleave chordin or procollagen; procollagen is cleaved by mTLL-2 in the presence of high levels of procollagen C-proteinase enhancer-1 (PCPE-1), for reasons that are unknown. We used these differences in activity between BMP-1 and mTLL-2 to narrow in on the domains in BMP-1 that specify PCP and chordinase activity. Using a domain swap approach, we showed that: 1) the metalloproteinase and CUB2 domains of BMP-1 are absolutely required for PCP activity; swaps with either of the corresponding domains in BMP-1 and mTLL-2 did not result in procollagen cleavage and 2) the proteinase domain of mTLL-2 can cleave chordin if coupled to the CUB1 domain of BMP-1. Therefore, the minimal structure for chordinase activity comprises a metalloproteinase domain (either from BMP-1 or from mTLL-2) and the CUB1 domain of BMP-1 (the CUB1 domain of mTLL-2 cannot substitute for the CUB1 domain of BMP-1). We showed that the minimal procollagen C-proteinase (BMP-1 lacking the EGF and CUB3 domain) was enhanced by PCPE-1 but not as well as BMP-1 retaining the CUB3 domain. Further studies showed that PCPE-1 had no effect on the ability of BMP-1 to cleave chordin. The data support a previously suggested mechanism of PCPE-1 whereby PCPE-1 interacts with procollagen, but in addition, the CUB3 domain of BMP-1 appears to augment the interaction.

Novel PCL-based honeycomb scaffolds as drug delivery systems for rhBMP-2.

This study investigated a novel drug delivery system (DDS), consisting of polycaprolactone (PCL) or polycaprolactone 20% tricalcium phosphate (PCL-TCP) biodegradable scaffolds, fibrin Tisseel sealant and recombinant bone morphogenetic protein-2 (rhBMP-2) for bone regeneration. PCL and PCL-TCP-fibrin composites displayed a loading efficiency of 70% and 43%, respectively. Fluorescence and scanning electron microscopy revealed sparse clumps of rhBMP-2 particles, non-uniformly distributed on the rods' surface of PCL-fibrin composites. In contrast, individual rhBMP-2 particles were evident and uniformly distributed on the rods' surface of the PCL-TCP-fibrin composites. PCL-fibrin composites loaded with 10 and 20 microg/ml rhBMP-2 demonstrated a triphasic release profile as quantified by an enzyme-linked immunosorbent assay (ELISA). This consisted of burst releases at 2 h, and days 7 and 16. A biphasic release profile was observed for PCL-TCP-fibrin composites loaded with 10 microg/ml rhBMP-2, consisting of burst releases at 2 h and day 14. PCL-TCP-fibrin composites loaded with 20 microg/ml rhBMP-2 showed a tri-phasic release profile, consisting of burst releases at 2 h, and days 10 and 21. We conclude that the addition of TCP caused a delay in rhBMP-2 release. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and alkaline phosphatase assay verified the stability and bioactivity of eluted rhBMP-2 at all time points.

Bone morphogenetic protein-2 decorated silk fibroin films induce osteogenic differentiation of human bone marrow stromal cells.

Bone morphogenetic protein (BMP)-2 has a critical role in bone formation and regeneration. Therefore, the ability to immobilize this molecule in certain matrices may be crucial in bone tissue engineering. Using carbodiimide chemistry, BMP-2 was directly immobilized on silk fibroin films. Whereas human bone marrow stromal cells cultured on unmodified silk fibroin films in the presence of osteogenic stimulants exhibited little if any osteogenesis, the same cells cultured on BMP-2 decorated films in the presence of osteogenic stimulants differentiated into an osteoblastic lineage as assessed by their significantly elevated alkaline phosphatase activity, calcium deposition, and higher transcript levels of collagen type I, bone sialoprotein, osteopontin, osteocalcin, BMP-2, and cbfa1. Using cell culture inserts, it was demonstrated that differentiation was induced by the immobilized protein and not by protein released into the culture medium. Comparison with a similar amount of medium-supplemented BMP-2, where no additional protein was added with medium changes, showed that delivery of BMP-2 immobilized on the biomaterial surface was more efficient than soluble delivery. The results illustrate that BMP-2 covalently coupled on silk biomaterial matrices retains biological function in vitro based on the induction of osteogenic markers in seeded bone marrow stromal cells.

Processing and transport of matrix gamma-carboxyglutamic acid protein and bone morphogenetic protein-2 in cultured human vascular smooth muscle cells: evidence for an uptake mechanism for serum fetuin.

Matrix gamma-carboxyglutamic acid protein (MGP) is a member of the vitamin K-dependent protein family with unique structural and physical properties. MGP has been shown to be an inhibitor of arterial wall and cartilage calcification. One inhibitory mechanism is thought to be binding of bone morphogenetic protein-2. Binding has been shown to be dependent upon the vitamin K-dependent gamma-carboxylation modification of MGP. Since MGP is an insoluble matrix protein, this work has focused on intracellular processing and transport of MGP to become an extracellular binding protein for bone morphogenetic protein-2. Human vascular smooth muscle cells (VSMCs) were infected with an adenovirus carrying the MGP construct, which produced non-gamma-carboxylated MGP and fully gamma-carboxylated MGP. Both forms of MGP were found in the cytosolic and microsomal fractions obtained from the cells by differential centrifugation. The crude microsomal fraction was shown to contain an additional, more acidic Ser-phosphorylated form of MGP believed to be the product of Golgi casein kinase. The data suggest that phosphorylation of MGP dictates different transport routes for MGP in VSMCs. A proteomic approach failed to identify a larger soluble precursor of MGP or an intracellular carrier protein for MGP. Evidence is presented for a receptor-mediated uptake mechanism for fetuin by cultured human VSMCs. Fetuin, shown by mass spectrometry not to contain MGP, was found to be recognized by anti-MGP antibodies. Fetuin uptake and secretion by proliferating and differentiating cells at sites of calcification in the arterial wall may represent an additional protective mechanism against arterial calcification.

Bone morphogenetic protein-1 (BMP-1). Identification of the minimal domain structure for procollagen C-proteinase activity.

Bone morphogenetic protein-1 (BMP-1) is a shorter spliced variant of mammalian tolloid (mTld), both of which cleave the C-propeptides of type I procollagen during the synthesis of extracellular matrix collagen fibrils. The fact that BMP-1 and mTld both exhibit procollagen C-proteinase (PCP) activity and that BMP-1 is the smaller variant might indicate that BMP-1 comprises the minimal required sequences for PCP activity. BMP-1 comprises a metalloproteinase domain, three CUB domains, and an epidermal growth factor (EGF)-like domain, which is located between the second and third CUB (complement components C1r/C1s, the sea urchin protein Uegf, and BMP-1) domains. In this study we showed the following. 1) The CUB1 domain is required for secretion of the molecule. Domain swapping experiments, in which CUB1 and other CUB domains were interchanged, resulted in retention of the proteins by cells. Therefore, CUB1 and its location immediately adjacent to the metalloproteinase domain are essential for secretion of the protein. 2) Mutants lacking the EGF-like and CUB3 domains exhibited full C-proteinase activity. In contrast, mutants lacking the CUB2 domain were poor C-proteinases. 3) Further studies showed that Glu-483 on the beta4-beta5 loop of CUB2 is essential for C-proteinase activity of BMP-1. In conclusion, the study showed that the minimal domain structure for PCP activity is considerably shorter than expected and comprises the metalloproteinase domain and the CUB1 and CUB2 domains of BMP-1.

Paired basic/Furin-like proprotein convertase cleavage of Pro-BMP-1 in the trans-Golgi network.

Bone morphogenetic protein (BMP)-1 is a zinc-dependent metalloproteinase that cleaves a variety of extracellular matrix substrates, including type I procollagen. Little is known about the site of action of BMP-1, although the extracellular matrix seems likely to be it. BMP-1 is synthesized with an N-terminal prodomain. The removal of the prodomain presumably activates the proteinase. In this study we show that the prodomain is cleaved in the trans-Golgi network (TGN) and by furin-like/paired basic proprotein convertases. Inhibitors of furin resulted in the secretion of pro-BMP-1, which could not cleave procollagen. Recombinant furin cleaved the prodomain from pro-BMP-1. Site-directed mutagenesis of the prodomain cleavage site (RSRR) to RSAA resulted in efficient secretion of pro-BMP-1. Therefore, prodomain cleavage was not required for secretion. Using peptide N-glycosidase and neuraminidase digestion to determine the post-translational status of pro-BMP-1 during its conversion to BMP-1, we showed that BMP-1 first appears in the TGN during sialylation of the molecule. Furthermore, immunofluorescence studies using an antibody to the nascent N terminus of BMP-1 showed localization to the TGN and plasma membrane. The observation that BMP-1 occurs inside the cell raises the possibility that BMP-1 might begin to cleave its substrates prior to secretion to the extracellular matrix.

Fabrication of homogeneously cross-linked, functional alginate microcapsules validated by NMR-, CLSM- and AFM-imaging.

Cross-linked alginate microcapsules of sufficient mechanical strength can immunoisolate cells for the long-term treatment of hormone and other deficiency diseases in human beings. However, gelation of alginate by external Ba(2+) (or other divalent cations) produces non-homogeneous cross-linking of the polymeric mannuronic (M) and guluronic (G) acid chains. The stability of such microcapsules is rather limited. Here, we show that homogeneous cross-linking can be achieved by injecting BaCl(2) crystals into alginate droplets before they come into contact with external BaCl(2). The high effectiveness of this crystal gun method is demonstrated by confocal laser scanning microscopy and by advanced nuclear magnetic resonance imaging. Both techniques gave clear-cut evidence that homogeneous cross-linkage throughout the microcapsule is only obtained with simultaneous internal and external gelation. Atomic force microscopy showed a very smooth surface topography for microcapsules made by the crystal gun method, provided that excess Ba(2+) ions were removed immediately after gelation. In vitro experiments showed greatly suppressed swelling for crystal gun microcapsules. Even alginate extracted from Lessonia nigrescens (highly biocompatible) yielded microcapsules with long-term mechanical stability not hitherto possible. Encapsulation of rat islets, human monoclonal antibodies secreting hybridoma cells and murine mesenchymal stem cells transfected with cDNA encoding for bone morphogenetic protein (BMP-4) revealed that injection of BaCl(2) crystals has no adverse side effects on cell viability and function. However, the release of low-molecular weight factors (such as insulin) may be delayed when using alginate concentrations in the usual range.