Platelet-Rich Plasma, Adipose Tissue, and Scar Modulation.

Level of Evidence: 4.

Identification and expression of a novel transcript of the growth and differentiation factor-11 gene.

The growth and differentiation factor-11 (GDF-11) gene is thought to code for a single protein that plays a crucial role in regulating the development of multiple tissues. In this study, we aimed to investigate if the GDF-11 gene has another transcript and, if so, to characterise this transcript and determine its tissue-specific and developmental expression. We have identified a novel transcript of GDF-11 in mouse muscle, which contains the 3' region of intron 1, exon 2, exon 3 and 3'UTR, and has two transcription initiation sites and a single termination site. We named the novel transcript GDF-11ΔEx1 because it does not contain exon 1 of canonical GDF-11. The GDF-11ΔEx1 transcript was expressed in the skeletal muscles, heart, brain and kidney, but was undetectable in the liver and gut. The concentration of the GDF-11ΔEx1 transcript was increased in gastrocnemius muscles from three to 6 weeks of age, a period of accelerated muscle growth, steadily declined thereafter and was higher in male than female mice (P < 0.001 for age and sex). GDF-11ΔEx1 cDNA was predicted to code for a putative N-terminal-truncated propeptide and the canonical ligand for GDF-11. However, propeptide-specific antibodies could not identify proteins of the expected size in skeletal muscle. Interestingly, in silico analysis of the GDF-11ΔEx1 RNA predicted a secondary structure with the potential to coordinate multiple protein interactions as a molecular scaffold. Therefore, we postulate that GDF-11ΔEx1 may act as a long non-coding RNA to regulate the transcription of canonical GDF-11 and/or other genes in skeletal muscle and other tissues.

Cloning and characterization of a bone morphogenetic protein homologue of Schistosoma japonicum.

Bone morphogenetic proteins (BMPs) are known to play an important role in the regulation of cell proliferation, survival, differentiation and apoptosis in many vertebrates and invertebrates through the TGF-ß signaling pathway. Although the TGF-ß signaling pathway exists in schistosomes, BMP homologue, a ligand of TGF-ß in Schistosoma japonicum, has not yet been identified. In this study, a BMP homologue of S. japonicum was cloned and characterized. The full length SjBMP cDNA is 3,020 bp and encodes 928 amino acids, which include a TGF-ß superfamily conserved domain at the C-terminus. BLAST analysis showed that, SjBMP has 68%, 51% and 43% homology with BMP from Schistosoma mansoni, Schmidtea mediterranea and Dugesia japonica at the amino acid level, respectively. According to data from real-time PCR, SjBMP was expressed in lung-stage schistosomula, 21-day liver-stage schistosomula, 50-day adult worms (the male and female), and eggs. The PCR data also indicated that, there was a ≈ 27- and ≈ 37-fold increase of SjBMP transcripts in the lung-stage schistosomula and eggs, respectively, and that there was relatively more SjBMP transcript in the adult male worm than in the adult female, in which the hepatic schistosomula was set as the calibrator for calculation. In situ hybridization based on FITC-labeled specific antisense oligonucleotide probes showed that SjBMP mRNA localized to the ovary of female worms and the integument and epithelium of female and male worms. After treatment with double-stranded RNA (dsRNA) at a concentration of 8 × 10(-2) µg/ml, which was added to the culture medium every other day for a week, the level of SjBMP mRNA in the cultured adult mixed-sex S. japonicum decreased at a range of ≈ 25-98% within 7 days compared with the level of SjBMP mRNA in the blank control group. On the 2nd day, the number of eggs produced per pair of worms decreased 28.7%, and the percent of normal eggs also decreased (12.7% vs. 4.3%) in the SjBMP dsRNA-treated group when compared with the eggs laid by the blank control group. No difference was detected between the two groups on the 7th day of treatment, because the eggs of the untreated worms were also mostly abnormal, similar to the eggs laid by the treated group. In addition, no significant difference in the morphological structure of the adult worms was observed. Thus, the preliminary in vitro experiment indicated that SjBMP may be involved in the oviposition behavior of S. japonicum, and further studies based on the recombinant virus vector-induced steady knockdown of SjBMP or in vivo experiments are required for more in-depth investigation.

Evaluation of a novel reconstituted bone xenograft using processed bovine cancellous bone in combination with purified bovine bone morphogenetic protein.

BACKGROUND: Xenogeneic grafting represents an alternative to autogenous grafting in osseous reconstruction and exhibits many beneficial properties. However, the usefulness of xenogeneic bone relies on necessary processing procedures for removing antigens and viruses, and preserving biological activities simultaneously. By chemical treatment of bovine cancellous bone to make it an antigen-free scaffold, and extraction of bone morphogenetic protein (BMP) from bovine cortical bone, followed by recombination of the scaffold with the BMP, we developed a new grafting material, reconstituted bone xenograft (RBX). METHODS: In this study, scanning electron microscope and energy dispersive X-ray were first employed to observe the structure and components of RBX. Then the biomechanical property was evaluated by applying compression in a materials testing machine. Subsequently, the immunologic evaluation was performed by measuring galactose-alpha-1,3-galactose (α-gal) epitope in vivo and proinflammatory cytokine (TNF-α) secreted by human monocytic cell line (THP-1) in vitro. Finally, this RBX was implanted into segmental radial defects in a rabbit model, and its ability to treat large bone defects was specifically evaluated. RESULTS: Although the compressive strength of RBX was 10% lower than that of unprocessed bovine cancellous bone (UBCB), the basic porous structure and natural components were still kept in this composite. The α-gal xenoantigen level was significantly lower in RBX (P < 0.05) compared with UBCB. Moreover, the TNF-α level was significantly (P < 0.05) reduced compared with UBCB when THP-1 was exposed to RBX. On the other hand, RBX appeared to induce cartilage formation from immature cell populations and resulted in osteogenesis through endochondral-like ossification from 4 to 12 weeks in repairing segmental bone defects. CONCLUSIONS: These results demonstrate that RBX, with its natural microstructure and components, certain mechanical strength and strong osteoinductivity without evoking immune rejection, has significant potential for the treatment of bone defects.

The role of cell surface markers and enamel matrix derivatives on human periodontal ligament mesenchymal progenitor responses in vitro.

Periodontitis is a chronic-, infectious-disease of the human periodontium that is characterized by the loss of supporting tissues surrounding the tooth such as the periodontal ligament (PDL), cementum and alveolar bone. Regeneration of the periodontium is dependent on the participation of mesenchymal stem/stromal cells (MSC) resident in the PDL. Enamel matrix derivative (EMD), an extract from immature porcine enamel rich in amelogenin protein but that also contain bone morphogenetic protein (BMP), is used to treat periodontal defects. The effects of EMD on MSC cells of the PDL are not well characterized. In this in vitro study, we identify PDL progenitor cells from multiple individuals and demonstrate that EMD stimulates them. We show that the effect of EMD on cell proliferation and migration is mediated through the amelogenin it contains, while the differentiation of these progenitor cells to cell types of mineralized tissue is mainly due to BMP signaling.

Osteoinductivity of partially purified bovine, ostrich and emu bone morphogenetic proteins in vitro.

The aim of this study was to observe the osteogenic activity of native bone morphogenetic proteins (BMPs) obtained from different species including bovine, ostrich and emu sources in order to compare mammalian and avian BMPs. Rat mesenchymal progenitor marrow stromal cells and pre-osteoblastic C2C12 cell cultures, were exposed to the native BMPs and alkaline phosphatase (ALP) and creatine kinase (CK) levels were determined by assay. The results showed that the ALP activity in C2C12 cultures was elevated by bovine BMP by 2- to 10-fold (p < 0.05-0.001) from day 3 during 14 days. There were no significant differences in avian BMP related elevations of ALP activity except with ostrich BMPs at day 14 (p < 0.05). However, exposure of MSCs cultures to BMPs derived from bovine, ostrich or emu sources resulted in elevated ALP from day 3 (p < 0.05). Bovine BMP resulted in more ALP elevation than with either of the avian BMPs. All of BMPs elevated Creatine kinase (CK) activity from day 1 and climbed until peaking at day 7. Compared with control cultures, CK was elevated more with exposure to emu BMP and was more elevated with greater statistical significance than with bovine and ostrich BMP before day 5. These higher levels remained until day 14 (p < 0.05). The results of this study suggest that both bovine and avian BMPs are able to stimulate osteogenesis in mature osteoblasts in vitro. The strongest synergistic effect on osteogenesis was detected in cells stimulated with bovine BMP. Avian BMPs had lower effects on ALP and CK activity, emu BMP being more effective than ostrich BMP.

Expression, purification, and refolding of a recombinant human bone morphogenetic protein 2 in vitro.

In this work, the recombinant human bone morphogenetic protein 2 (rhBMP-2) gene was cloned from MG-63 cells by RT-PCR, and the protein was expressed in Escherichia coli expression system, purified by Ni-NTA column under denaturing conditions and refolded at 4°C by urea gradient dialysis. We found that the protein refolding yield was increased with the increase of pH value from pH 6.0 to pH 9.0. The yield was 42% and 96% at pH 7.4 and pH 9.0, respectively, while that at pH 6.0 was only 3.4%. The cell culture results showed that the rhBMP-2 refolded at pH 7.4 urea gradient dialysis had higher biological activity for MG-63 cell proliferation and differentiation than that refolded at pH 9.0 since pH 7.4 is closer to the conditions in vivo leading to the formation of dimers through the interchain disulfide bond. Moreover, the biological activity for MG-63 was promoted with the increase of rhBMP-2 concentration in the cell culture medium. This work may be important for the in vitro production and biomedical application of rhBMP-2 protein.

Optimized procedure for expression and renaturation of recombinant human bone morphogenetic protein-2 at high protein concentrations.

A prokaryotic expression system has been used to produce recombinant human bone morphogenetic protein-2 (rhBMP-2). However, low rhBMP-2 yields and protein loss during purification and renaturation are the hurdles in the clinical application. Previous studies have indicated that variables such as temperature, host cell, salt concentration, and culture time affect the final rhBMP-2 yield. The optimization of these conditions in an Escherichia coli culture yielded 28.258 mg of rhBMP-2 per liter of culture. To reduce rhBMP-2 loss during purification and renaturation, we performed purification before renaturation in the prokaryotic expression system instead of using the traditional renaturation-before-purification approach. rhBMP-2 was separated on a Sephacryl S-300 HR column and eluted from a DEAE-Sepharose Fast Flow column. The collected protein was refolded by dialysis with urea buffer, which was followed by dialysis with ultrapure water. The purified rhBMP-2 dimer significantly increased alkaline phosphatase (ALP) activity and osteogenic activity in the femoral muscle and showed the same level of bone-forming activity as natural BMP-2. This optimized procedure for expression and renaturation of rhBMP-2 has potential clinical applications.

Receptor oligomerization and beyond: a case study in bone morphogenetic proteins.

BACKGROUND: Transforming growth factor (TGF)beta superfamily members transduce signals by oligomerizing two classes of serine/threonine kinase receptors, termed type I and type II. In contrast to the large number of ligands only seven type I and five type II receptors have been identified in mammals, implicating a prominent promiscuity in ligand-receptor interaction. Since a given ligand can usually interact with more than one receptor of either subtype, differences in binding affinities and specificities are likely important for the generation of distinct ligand-receptor complexes with different signaling properties. RESULTS: In vitro interaction analyses showed two different prototypes of binding kinetics, 'slow on/slow off' and 'fast on/fast off'. Surprisingly, the binding specificity of ligands to the receptors of one subtype is only moderate. As suggested from the dimeric nature of the ligands, binding to immobilized receptors shows avidity due to cooperative binding caused by bivalent ligand-receptor interactions. To compare these in vitro observations to the situation in vivo, binding studies on whole cells employing homodimeric as well as heterodimeric bone morphogenetic protein 2 (BMP2) mutants were performed. Interestingly, low and high affinity binding sites were identified, as defined by the presence of either one or two BMP receptor (BMPR)-IA receptor chains, respectively. Both sites contribute to different cellular responses in that the high affinity sites allow a rapid transient response at low ligand concentrations whereas the low affinity sites facilitate sustained signaling but higher ligand concentrations are required. CONCLUSION: Binding of a ligand to a single high affinity receptor chain functioning as anchoring molecule and providing sufficient complex stability allows the subsequent formation of signaling competent complexes. Another receptor of the same subtype, and up to two receptors of the other subtype, can then be recruited. Thus, the resulting receptor arrangement can principally consist of four different receptors, which is consistent with our interaction analysis showing low ligand-receptor specificity within one subtype class. For BMP2, further complexity is added by the fact that heterooligomeric signaling complexes containing only one type I receptor chain can also be found. This indicates that despite prominent ligand receptor promiscuity a manifold of diverse signals might be generated in this receptor limited system.

Expression, purification and osteogenic bioactivity of recombinant human BMP-4, -9, -10, -11 and -14.

Bone morphogenetic proteins (BMPs) are cytokines from the TGF-beta superfamily, with important roles during embryonic development and in the induction of bone and cartilage tissue differentiation in the adult body. In this contribution, we report the expression of recombinant human BMP-4, BMP-9, BMP-10, BMP-11 (or growth differentiation factor-11, GDF-11) and BMP-14 (GDF-5), using Escherichia coli pET-25b vector. BMPs were overexpressed, purified by affinity his-tag chromatography and shown to induce the expression of early markers of bone differentiation (e.g. smad-1, smad-5, runx2/cbfa1, dlx5, osterix, osteopontin, bone sialoprotein and alkaline phosphatase) in C2C12 cells and in human adipose stem cells. The described approach is a promising method for producing large amounts of different recombinant BMPs that show potential for novel biomedical applications.

Proteomic profiling of CHO cells with enhanced rhBMP-2 productivity following co-expression of PACEsol.

Chinese hamster ovary (CHO) cells are widely used for the production of recombinant protein biopharmaceuticals. The purpose of this study was to investigate differences in the proteome of CHO DUKX cells expressing recombinant human bone morphogenetic protein-2 (rhBMP-2) (G5 cells) compared to cells also expressing soluble exogenous paired basic amino acid cleaving enzyme soluble paired basic amino acid cleaving enzyme (PACEsol) (3C9 cells), which has been previously found to improve the post-translational processing of the mature rhBMP-2 dimer. PACEsol co-expression was also associated with a significant increase (almost four-fold) in cellular productivity of rhBMP-2 protein. Differential proteomic expression profiling using 2-D DIGE and MALDI-TOF MS was performed to compare 3C9 and G5 cells, and revealed a list of 60 proteins that showed differential expression (up/downregulated), with a variety of different cellular functions. A substantial number of these altered proteins were found to have chaperone activity, involved with protein folding, assembly and secretion, as well as a number of proteins involved in protein translation. These results support the use of proteomic profiling as a valuable tool towards understanding the biology of bioprocess cultures.

Soluble expression and purification of synthetic human bone morphogenetic protein-2 in Escherichia coli.

A 345-bp gene that encodes human bone morphogenetic protein-2 (hBMP-2) has been synthesized. The codon usage of the resulting gene was modified to include those triplets that are utilized in highly expressed Escherichia coli genes. The hBMP-2 gene was efficiently expressed in E. coli as a soluble and active protein. Since the recombinant hBMP-2 was readily solublized, no further solublization steps were required throughout purification. No additional tagging residues were introduced into the synthetic hBMP-2 gene product. The developed synthetic gene is a promising approach for scaling-up the soluble expression of hBMP-2.

Brachydactyly type A2 associated with a defect in proGDF5 processing.

We investigated a family with a brachydactyly type A2 and identified a heterozygous arginine to glutamine (R380Q) substitution in the growth/differentiation factor 5 (GDF5) in all affected individuals. The observed mutation is located at the processing site of the protein, at which the GDF5 precursor is thought to be cleaved releasing the mature molecule from the prodomain. In order to test the effect of the mutation, we generated the GDF5-R380Q mutant and a cleavage-resistant proGDF5 mutant (R380A/R381A) in vitro. Both mutants were secreted from chicken micromass cultures, but showed diminished biological activity. Western blot analyses showed that wt GDF5 was processed by the chicken micromass cells, whereas the mutants were not, indicating that the mutations interfere with processing and that this leads to a strong reduction of biological activity. To test the requirements for GDF5 processing in vitro we produced recombinant human (rh) proGDF5 wild-type protein in Escherichia coli. The results show that unprocessed (rh) proGDF5 is virtually inactive but can be proteolytically activated by different enzymes such as trypsin, furin, and MMP3. (rh) proGDF5 could thus be used as a locally administered depot form with retarded release of activity. In contrast to mature rhGDF5, (rh) proGDF5 shows a high solubility at physiological pH, a characteristic that might be useful for therapeutic applications.

Step gradients in 3-zone simulated moving bed chromatography. Application to the purification of antibodies and bone morphogenetic protein-2.

The simulated moving bed (SMB) technology is a proven tool for efficient separation of binary mixtures. However, relying on isocratic conditions limits the applicability of the classical SMB approach when considering the field of bioseparations. Here, the use of gradients opens up new possibilities. A gradient in a SMB process can be established by using different solvent strengths in the incoming feed and desorbent streams, resulting in two internal plateaus of elution strength. Thus, compared to the conventional process, the overall amount of solvent needed can be reduced, productivity can be increased and more concentrated product streams can be obtained. In this contribution, two case studies will be presented. At first, the separation of bovine IgG from lysozyme will be analyzed as a model system. Antibodies are a common target substance in bio-chromatography, as therapeutic monoclonal antibodies are among the most promising biopharmaceuticals. Using adsorption data obtained from single-column experiments, an appropriate SMB process was designed and implemented. The second target component is the active dimeric form of the bone morphogenetic protein-2 (BMP-2). This protein was isolated from a renaturation solution, which also contained its inactive monomeric form as well as other undefined proteins from the bacterial production strain. A 3-zone open-loop gradient-SMB approach was used successfully for both separations.

[Physiological function of osteocytes].

Osteocytes produce DMP1 (dentin matrix protein 1), FGF23 (fibroblast growth factor 23) and sclerostin. FGF23 is a phosphate-regulating hormone that links bone to kidney. DMP1 is a matrix protein that is involved in mineralization. Patients with DMP1 mutations exhibit increased FGF23 and hypophosphatemia, suggesting that DMP1 negatively regulates FGF23 in osteocytes. Sclerostin is secreted by osteocytes and negatively regulates osteoblastic function, and its neutralizing antibody is being developed as a new treatment for osteoporosis. A mouse model that enables targeted ablation of osteocytes tells us about the physiologic and pathologic functions of osteocytes in regulating bone remodeling in response to mechanical environment.

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.

Discontinuous and continuous separation of the monomeric and dimeric forms of human bone morphogenetic protein-2 from renaturation batches.

Bone morphogenetic protein-2 (BMP-2) is one of the most interesting of the approximately 14 BMPs which belong to the transforming-growth-factor-beta (TGF-beta) superfamily. BMP-2 induces bone formation and thus plays an important role as a pharmaceutical protein. Recently, rhBMP-2 has been produced in form of inactive inclusion bodies in Escherichia coli. After solubilization and renaturation the biologically active dimeric form of rhBMP-2 can be generated. However, inactive monomers of BMP-2 are also formed during the renaturation process which must be separated from the active dimeric BMP-2. The purpose of this paper is to present: (a) results of an experimental study of a chromatographic separation of the monomeric and dimeric forms; and (b) a concept for a continuous counter-current simulated moving bed (SMB) process. The capacity of heparin as stationary phase was estimated for different salt concentrations in the mobile phase. A simulation study of a three-zone SMB process was performed applying a two step salt gradient. The results reveal the potential of the process for the purification of the dimeric BMP-2.

Expression of active hBMP2 in transgenic tobacco plants.

Bone morphogenetic protein 2 (BMP2) is important for bone tissue repair. The goal of this research is to construct a high level human BMP2 (hBMP2) expression system using transgenic tobacco plants as a bioreactor. Cauliflower mosaic virus (CaMV) 35S promoter, alfalfa mosaic virus (AMV) enhancer, tobacco mosaic virus (TMV) enhancer, matrix attachment regions (MARs) sequence, and "Kozak" sequence were used to construct recombinant expression vectors and the high-expression vectors were screened out through GUS-fusions assay. The promoter is the most important factor; double-CaMV 35S promoter is more effective than single promoter. The AMV or TMV enhancer is able to promote the foreign protein expression. After four-step purification, the activated hBMP2 (0.02% total soluble protein) was obtained. Our results suggested that the transgenic tobacco has great potential to be used as a bioreactor to produce hBMP2.

The effects of demineralized bone matrix proteins and osteogenic protein-1 on bone cells isolated in culture.

With the growing number of bone-related traumas and the limitations of traditional bone repair, alternative methods of bone management must be investigated. Demineralized bone matrix protein (DBX) has been used to reconstruct bone. DBX, a type of demineralized bone matrix, is a combination of several different proteins including osteogenic protein-1 (OP-1). Osteogenic protein-1 or Bone Morphogenic Protein-7 (BMP-7) was the first BMP approved for clinical use in the United States. Previous studies have shown that proliferation of osteoblasts (bone forming cells) was stimulated by OP-1. However, the effects of DBM and OP-1 at the cellular level have not been clearly defined. MG-63 osteosarcoma cells were utilized as a model and subsequently plated onto 24 well tissue culture plates at a density of 1x 10(5) ml/well. Cells were exposed to different concentrations of DBX demineralized bone matrix and OP-1 for periods of 24, 48, and 72 hours and compared with untreated controls. After each incubation period, cell morphology, cell damage, cell number, and protein concentrations were determined. Results indicate a significant increase in cell number at 72 hours in cells treated with 30% (5.66 x 10(5)) and 100% (6.3 x 10(5)) DBX treated groups when compared with the control (1.4 x 10(5)). OP-1 results do not indicate a significant increase in cell number at the 24 and 48 hour treatment phases when compared with the control (p > 0.05), however, results do show a statistically significant difference (approximately twofold, p < 0.05) between the control cells (1.9 x 10(4)) and those cells treated with low (3.9 x 10(4)) and high (4.1 x 10(4)) concentrations of OP-1 at the 72 hour time phase. The increases in cell number indicate that both DBX and OP-1 are effective in stimulating cell growth. When comparing the results of the DBX treatments with those of the OP-1 treatments, the cells treated with DBX showed a more substantial increase in bone cell proliferation after treatment than those cells treated with OP-1. This does suggest that DBX provides the most effective treatment for bone cell proliferation. Closer evaluation of the morphology especially the changes occurring at the nuclear level need to be addressed in future studies.

[Historical background and recent advance in BMP research].

In 1971, Uurist gave the name bone morphogenetic protein (BMP) to the factor in bone matrix, which retains the activity to induce ectopic bone formation. After that, BMP research has rapidly developed by achieving BMP purification, BMP cloning, and identification of BMP receptors and signal transuding molecules (Smads). In this review, I overview the historical background and recent advance in BMP research.