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.

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.

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.

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.

Developmental effects of ectopic expression of the glucocorticoid receptor DNA binding domain are alleviated by an amino acid substitution that interferes with homeodomain binding.

Steroid hormone receptors are distinguished from other members of the nuclear hormone receptor family through their association with heat shock proteins and immunophilins in the absence of ligands. Heat shock protein association represses steroid receptor DNA binding and protein-protein interactions with other transcription factors and facilitates hormone binding. In this study, we investigated the hormone-dependent interaction between the DNA binding domain (DBD) of the glucocorticoid receptor (GR) and the POU domains of octamer transcription factors 1 and 2 (Oct-1 and Oct-2, respectively). Our results indicate that the GR DBD binds directly, not only to the homeodomains of Oct-1 and Oct-2 but also to the homeodomains of several other homeodomain proteins. As these results suggest that the determinants for binding to the GR DBD are conserved within the homeodomain, we examined whether the ectopic expression of GR DBD peptides affected early embryonic development. The expression of GR DBD peptides in one-cell-stage zebra fish embryos severely affected their development, beginning with a delay in the epibolic movement during the blastula stage and followed by defects in convergence-extension movements during gastrulation, as revealed by the abnormal patterns of expression of several dorsal gene markers. In contrast, embryos injected with mRNA encoding a GR peptide with a point mutation that disrupted homeodomain binding or with mRNA encoding the DBD of the closely related mineralocorticoid receptor, which does not bind octamer factors, developed normally. Moreover, coinjection of mRNA encoding the homeodomain of Oct-2 completely rescued embryos from the effects of the GR DBD. These results highlight the potential of DNA-independent effects of GR in a whole-animal model and suggest that at least some of these effects may result from direct interactions with homeodomain proteins.

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.

Bone morphogenetic proteins: an unconventional approach to isolation of first mammalian morphogens.

It is conventional to identify morphogens from fly and frog embryos during morphogenesis using gene-screens, subtractive hybridizations, differential displays and expression cloning. This information is then extended to mice and men. The bone morphogenetic proteins (BMPs) are a family of pleiotropic morphogens/cytokines isolated and cloned from the demineralized extracellular matrix of adult bone. Thus, BMPs were isolated from mammalian bone by an unconventional approach. BMPs initiate the sequential developmental cascade of bone morphogenesis in ectopic sites. The pleiotropic effects of BMPs on chemotaxis, mitosis and differentiation are based on concentration-dependent thresholds. Recent work has demonstrated the critical role of BMPs in pattern formation in amphibian and chick limb development. Targeted disruption of gene function by homologous recombination has demonstrated the actions of BMPs beyond bone in such disparate tissues as kidney, eye, testis, teeth, skin and heart. The successful isolation of first mammalian morphogens has laid the foundation for the elucidation of molecular signalling during morphogenesis in bones and beyond.

Role of morphogenetic proteins in skeletal tissue engineering and regeneration.

Morphogenesis is the developmental cascade of pattern formation and body plan establishment, culminating in the adult form. It has formed the basis for the emerging discipline of tissue engineering, which uses principles of molecular developmental biology and morphogenesis gleaned through studies on inductive signals, responding stem cells, and the extracellular matrix to design and construct spare parts that restore function to the human body. Among the many organs in the body, bone has considerable powers for regeneration and is a prototype model for tissue engineering. Implantation of demineralized bone matrix into subcutaneous sites results in local bone induction. This model mimics sequential limb morphogenesis and has permitted the isolation of bone morphogens, such as bone morphogenetic proteins (BMPs), from demineralized adult bone matrix. BMPs initiate, promote, and maintain chondrogenesis and osteogenesis, but are also involved in the morphogenesis of organs other than bone. The symbiosis of the mechanisms underlying bone induction and differentiation is critical for tissue engineering and is governed by both biomechanics (physical forces) and context (microenvironment/extracellular matrix), which can be duplicated by biomimetic biomaterials such as collagens, hydroxyapatite, proteoglycans, and cell adhesion glycoproteins, including fibronectins and laminin. Rules of tissue architecture elucidated in bone morphogenesis may provide insights into tissue engineering and be universally applicable for all organs/tissues, including bones and joints.

[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.

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.

[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.

Enamel matrix derivative gel stimulates signal transduction of BMP and TGF-{beta}.

It has been shown that Emdogain Gel (Emd-Gel) containing enamel matrix proteins promotes biomineralization, such as osteogenesis and cementogenesis, during the regeneration of periodontal tissues. However, the growth factors involved in these activities of Emd-Gel remain unclear. In this study, Emd-Gel was fractionated into 22 sub-fractions by size exclusion chromatography. The osteoinductive factors, TGF-beta and BMP, were examined by a specific luciferase reporter gene assay. In the unfractionated Emd-Gel, TGF-beta-like activity was detected, while BMP activity was not. In contrast, in the fractionated Emd-Gel samples, TGF-beta-like activity was detected from fractions 8 to 13, and BMP-like activity was detected from fractions 4 to 6. Also, it was confirmed that the BMP-like activity in Emd-Gel was inhibited by authentic TGF-beta1 and TGF-beta-like activity. These results indicate that Emd-Gel contains both TGF-beta- and BMP-like growth factors that contribute to the induction of biomineralization during periodontal regeneration.

Crystallization and preliminary X-ray diffraction analysis of human growth and differentiation factor 5 (GDF-5).

Growth and differentiation factor 5 (GDF-5) belongs to the large TGF-beta superfamily of secreted signalling proteins and plays a pivotal role in skeletal development during embryogenesis. The gene for human GDF-5 was cloned, expressed in Escherichia coli and purified to homogeneity. Crystals were obtained that diffracted to 2.2 A resolution. A native data set was acquired, showing that the crystals belong to a trigonal space group, i.e. P3(1)21 or P3(2)21, with unit-cell parameters a = b = 97.1, c = 48.3 A. Initial analysis suggest the presence of only one monomer in the asymmetric unit, resulting in a high solvent content of 72% in the crystal.

The isolation, characterization, and expression of a novel GDF11 gene and a second myostatin form in zebrafish, Danio rerio.

In the current study, the first non-mammalian growth/differentiation factor (GDF) 11-like homolog was cloned from zebrafish. At the nucleotide level, zebrafish GDF11 is most similar to human GDF11 (79%), while the peptide is most similar to mouse GDF11 (78%). Phylogenetic analysis showed that the zebrafish GDF11 clusters with mammalian GDF11s. This study also cloned a second MSTN form in zebrafish most similar to Salmonid MSTN2 forms. Based on real time PCR, GDF11 is expressed in multiple adult tissues, with levels highest in whole heads and gonads, and expression is less ubiquitous when compared to MSTN expression. During embryonic development, real time PCR demonstrated increasing GDF11 mRNA levels 10 h post-fertilization (hpf), while MSTN mRNA levels remain low until 48 hpf. This is the first report of a transforming growth factor (TGF)-beta superfamily member in a non-mammalian species that is more closely related to GDF11 than MSTN, and also a second form of MSTN in zebrafish; suggesting that a more complex TGF-beta superfamily array exists in primitive vertebrates than previously thought.