Growth Factor Bone Morphogenetic Protein Research Articles

Nanotemplated polyelectrolyte films as porous biomolecular delivery systems. Application to the growth factor BMP-2.

Biomaterials capable of delivering controlled quantities of bioactive agents, while maintaining mechanical integrity, are needed for a variety of cell contacting applications. We describe here a nanotemplating strategy toward porous, polyelectrolyte-based thin films capable of controlled biomolecular loading and release. Films are formed via the layer-by-layer assembly of charged polymers and nanoparticles (NP), then chemically cross-linked to increase mechanical rigidity and stability, and finally exposed to tetrahydrofuran to dissolve the NP and create an intra-film porous network. We report here on the loading and release of the growth factor bone morphogenetic protein 2 (BMP-2), and the influence of BMP-2 loaded films on contacting murine C2C12 myoblasts. We observe nanotemplating to enable stable BMP-2 loading throughout the thickness of the film, and find the nanotemplated film to exhibit comparable cell adhesion, and enhanced cell differentiation, compared with a non-porous cross-linked film (where BMP-2 loading is mainly confined to the film surface).

BMP growth factor signaling in a biomechanical context

Bone Morphogenetic Proteins (BMPs) are members of the transforming growth factor-β superfamily of secreted polypeptide growth factors and are important regulators in a multitude of cellular processes. To ensure the precise and balanced propagation of their pleiotropic signaling responses, BMPs and their corresponding signaling pathways are subject to tight control. A large variety of regulatory mechanisms throughout different biological levels combines into a complex network and provides the basis for physiological BMP function. This regulatory network not only includes biochemical factors but also mechanical cues. Both BMP signaling and mechanotransduction pathways are tightly interconnected and represent an elaborate signaling network active during development but also during organ homeostasis. Moreover, its dysregulation is associated with a number of human pathologies. A more detailed understanding of this crosstalk in respect to molecular interactions will be indispensable in the future, in particular to understand BMP-related diseases as well as with regard to an efficient clinical application of BMP ligands.

Synergistic effect of Indian hedgehog and bone morphogenetic protein-2 gene transfer to increase the osteogenic potential of human mesenchymal stem cells

IntroductionTo stimulate healing of large bone defects research has concentrated on the application of mesenchymal stem cells (MSCs).MethodsIn the present study, we induced the overexpression of the growth factors bone morphogenetic protein 2 (BMP-2) and/or Indian hedgehog (IHH) in human MSCs by adenoviral transduction to increase their osteogenic potential. GFP and nontransduced MSCs served as controls. The influence of the respective genetic modification on cell metabolic activity, proliferation, alkaline phosphatase (ALP) activity, mineralization in cell culture, and osteogenic marker gene expression was investigated.ResultsTransduction had no negative influence on cell metabolic activity or proliferation. ALP activity showed a typical rise-and-fall pattern with a maximal activity at day 14 and 21 after osteogenic induction. Enzyme activity was significantly higher in groups cultured with osteogenic media. The overexpression of BMP-2 and especially IHH + BMP-2 resulted in a significantly higher mineralization after 28 days. This was in line with obtained quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analyses, which showed a significant increase in osteopontin and osteocalcin expression for osteogenically induced BMP-2 and IHH + BMP-2 transduced cells when compared with the other groups. Moreover, an increase in runx2 expression was observed in all osteogenic groups toward day 21. It was again more pronounced for BMP-2 and IHH + BMP-2 transduced cells cultured in osteogenic media.ConclusionsIn summary, viral transduction did not negatively influence cell metabolic activity and proliferation. The overexpression of BMP-2 in combination with or without IHH resulted in an increased deposition of mineralized extracellular matrix, and expression of osteogenic marker genes. Viral transduction therefore represents a promising means to increase the osteogenic potential of MSCs and the combination of different transgenes may result in synergistic effects.

The Skeletal Phenotype of Chondroadherin Deficient Mice

Chondroadherin, a leucine rich repeat extracellular matrix protein with functions in cell to matrix interactions, binds cells via their α2β1 integrin as well as via cell surface proteoglycans, providing for different sets of signals to the cell. Additionally, the protein acts as an anchor to the matrix by binding tightly to collagens type I and II as well as type VI. We generated mice with inactivated chondroadherin gene to provide integrated studies of the role of the protein. The null mice presented distinct phenotypes with affected cartilage as well as bone. At 3–6 weeks of age the epiphyseal growth plate was widened most pronounced in the proliferative zone. The proteome of the femoral head articular cartilage at 4 months of age showed some distinct differences, with increased deposition of cartilage intermediate layer protein 1 and fibronectin in the chondroadherin deficient mice, more pronounced in the female. Other proteins show decreased levels in the deficient mice, particularly pronounced for matrilin-1, thrombospondin-1 and notably the members of the α1-antitrypsin family of proteinase inhibitors as well as for a member of the bone morphogenetic protein growth factor family. Thus, cartilage homeostasis is distinctly altered. The bone phenotype was expressed in several ways. The number of bone sialoprotein mRNA expressing cells in the proximal tibial metaphysic was decreased and the osteoid surface was increased possibly indicating a change in mineral metabolism. Micro-CT revealed lower cortical thickness and increased structure model index, i.e. the amount of plates and rods composing the bone trabeculas. The structural changes were paralleled by loss of function, where the null mice showed lower femoral neck failure load and tibial strength during mechanical testing at 4 months of age. The skeletal phenotype points at a role for chondroadherin in both bone and cartilage homeostasis, however, without leading to altered longitudinal growth.

Copolymerization of 2-Methylene-1,3-dioxepane and Glycidyl Methacrylate, a Well-Defined and Efficient Process for Achieving Functionalized Polyesters for Covalent Binding of Bioactive Molecules

The understanding of cell-material interactions is important for creating personalized implants for tissue engineering. This has resulted in an interest in developing polymers with functional groups with the possibility of controlling the macromolecular surface. We have in a one-pot reaction synthesized a series of amorphous and degradable polyester-based copolymers with active functional groups by copolymerization of 2-methylene-1,3-dioxepane and glycidyl methacrylate. The properties of the final polymers were varied by varying the feed ratios of the monomers, and it was seen that it was possible to control the amount of active functional groups. The resulting epoxy-functionalized polyester was further modified by covalent immobilization of heparin. The heparinization was done in order, in a future aspect, to enhance the osteogenic differentiation of mesenchymal stem cells. Heparin binds directly with the growth factor bone morphogenetic protein-2 and helps to retain its activity. The molecular structure of the copolymers was characterized by nuclear magnetic resonance, size exclusion chromatography, and Fourier transform infrared spectroscopy. Differential scanning calorimetry and tensile testing showed that the monomer feed ratio had a great influence on the properties of the final polymer and that it thus was possible to control the mechanical properties to suit an intended application. The presence of heparin was verified by toluidine blue staining, and all of the films tested showed positive signals for heparin.

Effects of titanium surface anodization with CaP incorporation on human osteoblastic response

In this study we investigated whether anodization with calcium phosphate (CaP) incorporation (Vulcano®) enhances growth factors' secretion, osteoblast-specific gene expression, and cell viability, when compared to acid etched surfaces (Porous®) and machined surfaces (Screw®) after 3 and 7days. Results showed significant cell viability for Porous and Vulcano at day 7, when compared with Screw (p=0.005). At the same time point, significant differences regarding runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and bone sialoprotein (BSP) expression were found for all surfaces (p<0.05), but with greater fold induction for Porous and Vulcano. The secretion of transforming growth factor β1 (TGF-β1) and bone morphogenetic protein 2 (BMP-2) was not significantly affected by surface treatment in any experimental time (p>0.05). Although no significant correlation was found for growth factors' secretion and Runx2 expression, a significant positive correlation between this gene and ALP/BSP expression showed that their strong association is independent on the type of surface. The incorporation of CaP affected the biological parameters evaluated similar to surfaces just acid etched. The results presented here support the observations that roughness also may play an important role in determining cell response.

Immobilization of chondroitin sulfate to lipid membranes and its interactions with ECM proteins

Glycosaminoglycans (GAGs) in the extracellular matrix (ECM) have multiple functions in tissues including providing support, mediating cell division and differentiation, and taking part in important interactions with proteins, e.g. growth factors. Studying GAG related interactions is inherently difficult and requires suitable interaction platforms. We show two strategies to covalently couple the GAG chondroitin sulfate (CS) to supported lipid bilayers (SLBs), either by (a) activating carboxy-functionalized phospholipids in the lipid bilayer, followed by the addition of hydrazide-functionalized CS, or by (b) activating naturally occurring carboxyl groups on CS prior to addition to an amino-functionalized SLB. Bilayer formation and subsequent immobilization was followed in real-time using the Quartz Crystal Microbalance with Dissipation monitoring, a technique that provides unique information when studying highly hydrated molecular films. The two strategies yielded thin CS films (in the nanometer range) with similar viscoelastic properties. Fluidity of the lipid bilayer was retained when CS was coupled. The application of the CS interaction platform was exemplified for type I collagen and the bone inducing growth factor bone morphogenetic protein-2 (BMP-2). The addition of collagen to immoblized CS resulted in soft layers whereas layers formed by addition of BMP-2 were denser, independent on the immobilization strategy used.

Cholesteryl group- and acryloyl group-bearing pullulan nanogel to deliver BMP2 and FGF18 for bone tissue engineering

To create a drug delivery system that allows the controlled release of proteins, such as growth factors, over a long-term period, cholesteryl group- and acryloyl group-bearing pullulan (CHPOA) nanogels were aggregated to form fast-degradable hydrogels (CHPOA/hydrogels) by cross-linking with thiol-bearing polyethylene glycol. The gold standard of clinical bone reconstruction therapy with a physiologically active material is treatment with recombinant human bone morphogenetic protein 2 (BMP2); however, this approach has limitations, such as inflammation, poor cost-efficiency, and varying interindividual susceptibility. In this study, two distinct growth factors, BMP2 and recombinant human fibroblast growth factor 18 (FGF18), were applied to a critical-size skull bone defect for bone repair by the CHPOA/hydrogel system. The CHPOA-FGF18/hydrogel displayed identical results to the control CHPOA-PBS/hydrogel, and the CHPOA-BMP2/hydrogel treatment imperfectly induced bone repair. By contrast, the CHPOA-FGF18 + BMP2/hydrogel treatment strongly enhanced and stabilized the BMP2-dependent bone repair, inducing osteoprogenitor cell infiltration inside and around the hydrogel. This report indicates that the CHPOA/hydrogel system can successfully deliver two different proteins to the bone defect to induce effective bone repair. The combination of the CHPOA/hydrogel system with the growth factors FGF18 and BMP2 might be a step towards efficient bone tissue engineering.

Isolation and Characterization of Human Amniotic Mesenchymal Stem Cells and Their Chondrogenic Differentiation

Freshly isolated human amniotic mesenchymal (fHAM) cells contain somatic stem cells possessing proliferative ability and pluripotency, including a chondrogenic lineage. However, little is known about the biology of amnion-derived mesenchymal stem cells (MSCs) because fHAM cells can barely survive to expand under culture conditions in vitro for a long time. In this study, we separated fHAM cells and seeded them to isolate MSCs and analyze its character. In addition, suitable chondrogenic growth factor was determined by pellet culture, and their viability under xenogenic environment was examined by transplantation into rabbit knee joints. We succeeded in purifying proliferative subpopulations of fHAM cells, which could continue to proliferate more than 50 cumulative population doubling levels, and designated them as HAMα cells. Flow cytometry analysis revealed that they were positive for MSC markers (CD44, CD73, CD90, and CD105) and negative for hematopoietic cell markers (CD34, CD14, and CD45) and major histocompatibility complex class II antigen (human leukocyte antigen-DR). The expression of various stem-cell markers such as OCT3/4, C-MYC, SOX2, NANOG, CD44, SSEA-3, and SSEA-4 was also proved by immunocytochemical staining. Pellet culture using chondrogenic medium supplemented with transforming growth factor β3, transforming growth factor β3 plus bone morphogenetic protein (BMP)-2, or BMP-2 implied that supplementation of BMP-2 alone most effectively induced chondrogenesis in vitro. Xenotransplantation of HAMα cells achieved 8-week survival in vivo. These results suggest that HAMα cells correspond to MSCs that are highly proliferative and multipotent. Their chondrogenic potential and low immunogenicity indicate that HAMα cells could be an allotransplantable cell resource for cartilage repair.

Osteoblast proliferation and differentiation on a barrier membrane in combination with BMP2 and TGFβ1

Bioresorbable collagen membranes are routinely utilized in guided bone regeneration to selectively direct the growth and repopulation of bone cells in areas of insufficient volume. However, the exact nature by which alveolar osteoblasts react to barrier membranes as well as the effects following the addition of growth factors to the membranes are still poorly understood. The objective of the present study was therefore to investigate the effect of a bioresorbable collagen membrane soak-loaded in growth factors bone morphogenetic protein 2 (BMP2) or transforming growth factor β1 (TGFβ1) on osteoblast adhesion, proliferation, and differentiation. Prior to experimental seeding, membranes were soaked in either BMP2 or TGFβ1 at a concentration of 10ng/ml for 5min. Human osteoblasts adhered to all soak-loaded membranes as assessed by scanning electron microscopy. Growth factors BMP2 and TGFβ1 increased osteoblast proliferation at 3 or 5days post-seeding when compared to control collagen membranes. Analysis of real-time PCR revealed that administration of BMP2 increased osteoblast differentiation markers such as osterix, collagen I, and osteocalcin. BMP2 also increased mineralization of primary osteoblasts as demonstrated by alizarin red staining when compared to control and TGFβ1 soak-loaded membranes. The combination of a collagen barrier membrane with growth factors TGFβ1 and BMP2 significantly influenced adhesion, proliferation, and differentiation of primary human osteoblasts. The described in vitro effects following the combination of collagen barrier membranes with growth factors TGFβ1 and BMP2 provide further biologic support for the clinical application of this treatment strategy in guided bone regeneration procedures.

Effect of bone morphogenetic protein 2 on tendon‐to‐bone healing in a canine flexor tendon model

Tendon-to-bone healing is typically poor, with a high rate of repair-site rupture. Bone loss after tendon-to-bone repair may contribute to poor outcomes. Therefore, we hypothesized that the local application of the osteogenic growth factor bone morphogenetic protein 2 (BMP-2) would promote bone formation, leading to improved repair-site mechanical properties. Intrasynovial canine flexor tendons were injured in Zone 1 and repaired into bone tunnels in the distal phalanx. BMP-2 was delivered to the repair site using either a calcium phosphate matrix (CPM) or a collagen sponge (COL) carrier. Each animal also received carrier alone in an adjacent repair to serve as an internal control. Repairs were evaluated at 21 days using biomechanical, radiographic, and histologic assays. Although an increase in osteoid formation was noted histologically, no significant increases in bone mineral density occurred. When excluding functional failures (i.e., ruptured and gapped repairs), mechanical properties were not different when comparing BMP-2/CPM groups with carrier controls. A significantly higher percentage of BMP-2 treated specimens had a maximum force <20 N compared to carrier controls. While tendon-to-bone healing can be enhanced by addressing the bone loss that typically occurs after surgical repair, the delivery of BMP-2 using the concentrations and methods of the current study did not improve mechanical properties over carrier alone. The anticipated anabolic effect of BMP-2 was insufficient in the short time frame of this study to counter the post-repair loss of bone.

Degradable Segmented Polyurethane Elastomers for Bone Tissue Engineering: Effect of Polycaprolactone Content

Segmented polyurethanes (PURs), consisting of degradable poly(a-hydroxy ester) soft segments and aminoacid-derived chain extenders, are biocompatible elastomers with tunable mechanical and degradative properties suitable for a variety of tissue-engineering applications. In this study, a family of linear PURs synthesized from poly(ϵ-caprolactone) (PCL) diol, 1,4-diisocyanobutane and tyramine with theoretical PCL contents of 65–80 wt% were processed into porous foam scaffolds and evaluated for their ability to support osteoblastic differentiation in vitro. Differential scanning calorimetry and mechanical testing of the foams indicated increasing polymer crystallinity and compressive modulus with increasing PCL content. Next, bone marrow stromal cells (BMSCs) were seeded into PUR scaffolds, as well as poly(lactic-co-glycolic acid) (PLGA) scaffolds, and maintained under osteogenic conditions for 14 and 21 days. Analysis of cell number indicated a systematic decrease in cell density with increasing PUR stiffness at both 14 and 21 days in culture. However, at these same time points the relative mRNA expression for the bone-specific proteins osteocalcin and the growth factors bone morphogenetic protein-2 and vascular endothelial growth factor gene expression were similar among the PURs. Finally, prostaglandin E2 production, alkaline phosphatase activity and osteopontin mRNA expression were highly elevated on the most-crystalline PUR scaffold as compared to the PLGA and PUR scaffolds. These results suggest that both the modulus and crystallinity of the PUR scaffolds influence cell proliferation and the expression of osteoblastic proteins.

A comparative analysis of the osteogenic effects of BMP-2, FGF-2, and VEGFA in a calvarial defect model.

The utilization of growth factors for bone regeneration is a widely studied field. Since the approval of bone morphogenetic protein-2 (BMP-2) for therapeutic use in humans, the concept of utilizing growth factors for bone regeneration in translational medicine has become even more attractive. Despite many studies published on individual growth factors in various bone models, comparative analysis is largely lacking. The aim of our study was to compare three different proosteogenic factors under identical in vivo conditions. Thus, we tested the bone regeneration capacity of the three different growth factors BMP-2, fibroblast growth factor-2 (FGF-2), and vascular endothelial growth factor A (VEGFA) in a calvarial defect model. We demonstrated that BMP-2 and VEGFA had similar bone healing capacities, resulting in complete calvarial healing as early as week 3. FGF-2 also showed a significantly higher bone regeneration capacity; however, the healing rate was lower than with BMP-2 and VEGFA. Interestingly, these findings were paralleled by an increased angiogenic response upon healing in BMP-2- and VEGFA-treated calvarial defects as compared with FGF-2. Immunohistochemistry for proliferating and osteoprogenitor cells revealed activity at different points after surgery among the groups. In conclusion, we demonstrated an efficient bone regeneration capacity of both BMP-2 and VEGFA, which was superior to FGF-2. Moreover, this study highlights the efficient bone regeneration of VEGFA, which was comparable with BMP-2. These data provide a valuable comparative analysis, which can be used to further optimize growth factor-based strategies in skeletal tissue engineering.

Spatial and Temporal Expression of Molecular Markers and Cell Signals During Normal Development of the Mouse Patellar Tendon

Tendon injuries are common clinical problems and are difficult to treat. In particular, the tendon-to-bone insertion site, once damaged, does not regenerate its complex zonal arrangement. A potential treatment for tendon injuries is to replace injured tendons with bioengineered tendons. However, the bioengineering of tendon will require a detailed understanding of the normal development of tendon, which is currently lacking. Here, we use the mouse patellar tendon as a model to describe the spatial and temporal pattern of expression of molecular markers for tendon differentiation from late fetal life to 2 weeks after birth. We found that collagen I, fibromodulin, and tenomodulin were expressed throughout the tendon, whereas tenascin-C, biglycan, and cartilage oligomeric protein were concentrated in the insertion site during this period. We also identified signaling pathways that are activated both throughout the developing tendon, for example, transforming growth factor beta and bone morphogenetic protein, and specifically in the insertion site, for example, hedgehog pathway. Using a mouse line expressing green fluorescent protein in all tenocytes, we also found that tenocyte cell proliferation occurs at highest levels during late fetal life, and declines to very low levels by 2 weeks after birth. These data will allow both the functional analysis of specific signaling pathways in tenocyte development and their application to tissue-engineering studies in vitro.

In vitro and in vivo evaluations on osteogenesis and biodegradability of a β‐tricalcium phosphate coated magnesium alloy

Magnesium (Mg) alloys have been demonstrated to be potential orthopedic implants due to their biodegradability in vivo. To enhance its corrosion resistance and improve its osteogenesis, β-tricalcium phosphate (β-TCP) was coated on a Mg alloy (Mg-3AI-1Zn) by phosphating process. In vitro, the human osteosarcoma cell line (SaOS-2) showed significantly good adherence and proliferation on the surface of the β-TCP coated Mg alloy after 24-h incubation. The growth factor bone morphogenetic protein 2 (BMP-2) was highly expressed in SaOS-2 cultured with the β-TCP coated Mg alloy by Western blot analysis (p < 0.05). In vivo, the newborn bone at the implant/bone interface was formed at week 1 and matured at week 4 postimplantation. Villous tissue was found at the implant/bone interface at week 12 postimplantation. The contents of phosphorus and calcium on the surface of the β-TCP coated Mg alloy were decreased at week 4 and week 12 postimplantation, respectively. Immunohistochemical analysis of the experiment results demonstrated that the β-TCP coated Mg alloy implants provided a high BMP-2 expression during the first 4 weeks postimplantation. Compared with the naked Mg alloy which was degraded for 33% in vivo, only 17% of the β-TCP coated Mg alloy was degraded at week 12 postimplantation (p < 0.05). The in vitro cell tests showed that the β-TCP coating provided the Mg alloy with a significantly better surface cytocompatibility, and in vivo results also confirmed that the β-TCP coating exhibited greatly improved osteoconductivity and osteogenesis in the early 12 weeks postoperation period. Moreover, in vivo experiment demonstrated that the β-TCP coating layer could slow down the degradation of the naked Mg alloy at the early stage of implantation.

Transcriptional Targeting of Bone Morphogenetic Protein Receptors: Is it Ready for Prime Time?

Bone morphogenetic proteins (BMPs) have been shown to impact a variety of cellular functions, including apoptosis, hemostasis, proliferation, and cellular differentiation [ 1 Arihiro K. Inai K. Expression of CD31, Met/hepatocyte growth factor receptor and bone morphogenetic protein in bone metastasis of osteosarcoma. Pathol Int. 2001; 51: 100 Crossref PubMed Scopus (29) Google Scholar , 2 Raida M. Clement J.H. Ameri K. et al. Expression of bone morphogenetic protein 2 in breast cancer cells inhibits hypoxic cell death. Int J Oncol. 2005; 26: 1465 PubMed Google Scholar ]. Any discussion of the role of this complex set of proteins should begin with a brief overview of their function. BMPs are synthesized as 400–425 amino acid long precursor molecules that become mature after disulfide-linked dimerization with other BMP monomers [ 3 Sakou T. Bone morphogenetic proteins: From basic studies to clinical approaches. Bone. 1998; 22: 591 Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar ]. These active molecules then bind to one of four BMP type I (BMPR1) or two BMP type II (BMPR2) extracellular surface protein receptors [ 4 Kawabata M. Chytil A. Moses H.L. Cloning of a novel type II serine/threonine kinase receptor through interaction with the type I transforming growth factor-β receptor. J Biol Chem. 1995; 270: 5625 Crossref PubMed Scopus (134) Google Scholar ]. BMP binding then facilitates the heteromeric association of BMPR1 and BMPR2 and subsequent phosphorylation of BMPR1 via the kinase activity of BMPR2 [ 5 Rosenzweig B.L. Imamura T. Okadome T. et al. Cloning and characterization of a human type II receptor for bone morphogenetic proteins. Proc Natl Acad Sci U S A. 1995; 92: 7632 Crossref PubMed Scopus (474) Google Scholar ]. SMAD proteins 1, 5, and 8 are then phosphorylated and dimerized with SMAD4 [ 6 Derynck R. Zhang Y. Feng X.H. Smads: Transcriptional activators of TGF-β responses. Cell. 1998; 95: 737 Abstract Full Text Full Text PDF PubMed Scopus (945) Google Scholar , 7 Heldin C.H. Miyazono K. ten Dijke P. TGF-β signalling from cell membrane to nucleus through SMAD proteins. Nature. 1997; 390: 465 Crossref PubMed Scopus (3309) Google Scholar ]. This complex then translocates to the nucleus, where it triggers transcription of various gene targets. Because these molecules can serve as therapeutic targets for myriad diseases, they have been subjected to intense investigation over the past decade [ 8 Blish K.R. Wang W. Willingham M.C. et al. A human bone morphogenetic protein antagonist is down-regulated in renal cancer. Mol Biol Cell. 2008; 19: 457 Crossref PubMed Scopus (55) Google Scholar ]. Given the building interest in this area, one can reasonably wonder if research on molecular control of BMP is ready for primetime. The recent work by Dahdaleh et al. would suggest that BMP is a reasonable molecular target for the treatment of myriad diseases [ 9 Dahdaleh F.S. Carr J.C. Calva D. Howe J.R. Sp1 regulates the transcription of BMPR1A. J Surg Res. 2011; 171: e15 Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar ].

Deciphering Gene Expression Patterns

Large-scale studies in various model systems including Drosophila , fish, and the mouse have documented the exquisite temporal and spatial expression patterns of thousands of genes during development—making sense of it all is a major challenge in the field. The study by Yakoby and colleagues illustrates how very different expression patterns can be generated from a small number of initial patterns. By examining expression patterns for a large number of genes in the Drosophila follicle cell epithelium, the authors proposed that the various expression patterns could be explained by a simple combinatorial code based on six spatial building blocks and the operations of union, difference, intersection, and addition. Importantly, the six spatial building blocks can be linked to the activity of the epidermal growth factor receptor and bone morphogenetic protein signaling pathways that control gene expression along the dorsoventral and anteroposterior axes. A challenge for the future will be to see whether such a powerful approach can be applied to explain the diversity of patterns generated in more complex systems such as embryos. This PaperPick refers to A Combinatorial Code for Pattern Formation in Drosophila Oogenesis by N. Yakoby, C.A. Bristow, D. Gong, X. Schafer, J. Lembong, J.J. Zartman, M.S. Halfon, T. Schupbach, and S.Y. Shvartsman, published in November 2008. Video Abstract Nir Yakoby and Stas Shvartsman discuss the gene expression profiling project that led them to develop this framework for cataloguing and interpreting gene expression patterns and how they are using that framework in current studies of evolutionary diversification.

Dermis isolated adult stem cells for cartilage tissue engineering

Adult stem cells from the dermal layer of skin are an attractive alternative to primary cells for meniscus engineering, as they may be easily obtained and used autologously. Recently, chondroinducible dermis cells from caprine skin have shown promising characteristics for cartilage tissue engineering. In this study, their multilineage differentiation capacity is determined, and methods of expanding and tissue engineering these cells are investigated. It was found that these cells could differentiate along adipogenic, osteogenic, and chondrogenic lineages, allowing them to be termed dermis isolated adult stem cells (DIAS cells). Focusing on cartilage tissue engineering, it was found that passaging these cells in chondrogenic medium and forming them into self-assembled tissue engineered constructs caused upregulation of collagen type II and COMP gene expression. Further investigation showed that applying transforming growth factor β1 (TGF-β1) or bone morphogenetic protein 2 (BMP-2) to DIAS constructs caused increased sulfated glycosaminoglycan content. Additionally, TGF-β1 treatment caused significant increases in compressive properties and construct contraction. In contrast, BMP-2 treatment resulted in the largest constructs, but did not increase compressive properties. These results show that DIAS cells can be easily manipulated for cartilage tissue engineering strategies, and may also be a useful cell source for other mesenchymal tissues.

Identification of a core domain within the proregion of bone morphogenetic proteins that interacts with the dimeric, mature domain

Proregions of bone morphogenetic proteins (BMPs) fulfill important biological functions as intramolecular chaperones and for extracellular targeting of the mature signal ligand. Knowledge of the structures of the proregions would contribute to a more comprehensive picture of the biological activities of the pro-forms of BMP growth factors. In this study, a protease resistant core domain of the proregions of three BMPs was identified. For a more detailed analysis, the core domain of the BMP-2 proregion was recombinantly produced. Unfolding/refolding experiments and spectroscopic analyses proved that the core domain can be obtained as a folded entity. Binding of the core domain to the mature growth factor was demonstrated by SPR. Via peptide microarray analysis, residues within the core fragment could be identified that engage in binding to the dimeric, mature domain. Our study reveals that diverse members of the BMP family share a common, independently folding core domain within the large proregions peculiar to TGF-β superfamily members that may serve as a scaffold for folding and assembly of the dimeric proprotein complexes.

Human adipose derived stem cells reduce callus volume upon BMP-2 administration in bone regeneration

IntroductionThe demand for new therapeutic approaches to treat bone defects and fractures is increasing in trauma surgery and orthopaedics because the number of patients with degenerative diseases is continuously growing. “Tissue Engineering” offers promising new technologies that combine the three components – cells, growth factors and matrix. Efforts are targeted at improving and accelerating recovery, especially for long bone fractures, and reducing the risk of delayed bone healing or pseudoarthrosis. Adult human adipose-derived stem cells (ASC) can differentiate into osteoblasts in an osteogenic surrounding. Bone morphogenetic protein-2 (BMP-2) accelerates and initiates this differentiation. Fibrin, a matrix that promotes wound healing, is a promising carrier for ASCs and BMP-2. Materials and methodsIn this study, a 2mm transcortical drill hole in the femur of male rats served as a small non-critical size defect model for fracture simulation. In vivo bone healing was investigated upon administration of the growth factor BMP-2 embedded with ASCs in a locally applied fibrin matrix. Groups with the components alone were also investigated. After 2 and 4 weeks, μCT and histology were performed to determine the bone and callus volume. Results and discussionAfter only a short period of time (2 and 4 weeks), this animal model discloses comparative information about the osteogenetic potential and bone regeneration with little effort (no osteosynthesis necessary). The most significant result found in this model is that the combination of ASCs and BMP-2 in a fibrin matrix significantly reduces callus formation after 2 weeks compared to BMP-2 alone. BMP-2 alone significantly increased callus formation. ASCs embedded alone in the fibrin matrix did not lead to increased bone regeneration. ConclusionTransplantation of ASC modulated the callus induction by BMP-2 to a normal volume.