Olivier Drevelle

Cell responses to bone morphogenetic proteins and peptides derived from them: Biomedical applications and limitations

The bone morphogenetic proteins (BMPs) are cytokines of the transforming growth factor beta family. Some BMPs such as BMP-2 and BMP-7 play a major role in the development of the skeleton and the maintenance of homeostasis during bone remodelling. To date, only BMP-2 and BMP-7 have been approved by the Food and Drug Administration for specific orthopaedic applications. However, due to BMP cost, peptides derived from their knuckle epitope with osteogenic properties have been developed. BMPs are involved in many other biological events, including embryogenesis, angiogenesis and cancer. BMPs therefore have great biomedical potential as osteogenic factors and as anti-cancer agents. This review focuses on the use of BMPs and their derived peptides in biomedical delivery systems and gene therapy.

Bone cells-biomaterials interactions.

With the aging population, the incidence of bone defects due to fractures, tumors and infection will increase. Therefore, bone replacement will become an ever bigger and more costly problem. The current standard for bone replacement is autograft, because these transplants are osteoconductive and osteoinductive. However, harvesting an autograft requires additional surgery at the donor site that is related to high level of morbidity. In addition, the quantity of bone tissue that can be harvested is limited. These limitations have necessitated the pursuit of alternatives using biomaterials. The control of bone tissue cell adhesion to biomaterials is an important requirement for the successful incorporation of implants or the colonization of scaffolds for tissue repair. Controlling cells-biomaterials interactions appears of prime importance to influence subsequent biological processes such as cell proliferation and differentiation. Therefore, interactions of cells with biomaterials have been widely studied especially on two-dimensional systems. This review focuses on these interactions.

Effect of functionalized polycaprolactone on the behaviour of murine preosteoblasts

The efficiency of biomaterials used in bone repair depends greatly on their ability to interact with bone cells. Hence, we have functionalized polycaprolactone (PCL) films by peptides derived from the bone sialoprotein containing RGD sequence (pRGD), to increase their ability to interact with murine MC3T3-E1 preosteoblasts, and favour cell response to recombinant human bone morphogenetic protein-2 (rhBMP-2). RGE peptides (pRGE) were used as negative controls. The PCL films were hydrolyzed with NaOH and then carboxylic acid groups were activated to allow chemisorption of the peptides. Alkaline treatment increased the hydrophilicity of PCL films without significantly change their roughness. Peptide immobilization on PCL was checked by X-ray photoelectron spectroscopy. Hydrolyzed PCL films (Hydro PCL), which adsorbed fibronectin and vitronectin from serum after 1 h incubation, prevented the spreading of MC3T3-E1 preosteoblasts, while films bearing pRGD or pRGE did not. In contrast, MC3T3-E1 preosteoblasts attached to pRGD and incubated for 1 h in serum-free medium spread better than cells on Hydro PCL or pRGE. Only cells on pRGD had organized cytoskeleton, phosphorylated focal adhesion kinase on Y(397) and responded to rhBMP-2 by activating Smad pathway. Thus, pRGD PCL may be used to favour bone cell cytoskeletal organization and response to rhBMP-2.

Effect of BMP-2 and/or BMP-9 on preosteoblasts attached to polycaprolactone functionalized by adhesive peptides derived from bone sialoprotein

Biomaterials functionalized by adhesive peptides improve the cell-substratum interaction. However, their influence on the response of cells to growth factors is still poorly understood. We have shown that bone morphogenetic protein (BMP) 2 activates the Smad pathway only in murine MC3T3-E1 preosteoblasts attached to polycaprolactone (PCL) film functionalized by RGD peptides derived from bone sialoprotein (pRGD). We have now analysed the way recombinant human BMP-2 and/or BMP-9 (0.38 nM) influence the signal transduction and differentiation of MC3T3-E1 preosteoblasts attached to PCL-pRGD. While kinetics of MAPK activation were similar in cells treated by BMP-2 and BMP-9, different kinetics of Smad activation and β-catenin stabilization were observed. BMP-2 induced Smad1/5/8 phosphorylation within 0.5 and BMP-9 within 4 h, while the β-catenin was lower at 2 h only in cells treated with BMP-9. However, both BMPs induced the translocation of phosphorylated Smad1/5/8 to the nucleus at 4 h and increased Dlx5, osterix and osteocalcin transcripts as well as alkaline phosphatase activity at 72 h. A BMP-2/BMP-9 combination that maintained the β-catenin amount constant but reduced that of phosphorylated Smad within 4 h had quite similar effect than BMP-2 alone. It is therefore important to determine how biomimetic materials influence the response of cells to BMPs.

Modulation of MAPK signalling by immobilized adhesive peptides: Effect on stem cell response to BMP-9-derived peptides.

UNLABELLED Biomimetic materials were developed to regulate stem cell behaviour. We have analyzed the influence of polycaprolactone (PCL) films, functionalized with adhesive peptides derived from fibronectin (pFibro) or bone sialoprotein (pBSP), on the response of murine multipotent C3H10T1/2 cells to bone morphogenetic protein-9 (BMP-9) and its derived peptides (pBMP-9 and SpBMP-9). PCL-pFibro promoted better cell cytoskeleton organization and faster focal adhesion kinase activation than did PCL-pBSP. PCL-pFibro also promoted MAPK signalling to improve the cell response to BMP-9 by inactivating ERK1/2 and stimulating p38 and JNK. BMP-9, pBMP-9 and SpBMP-9 induced greater phosphorylation of Smad1/5/8 in cells attached to PCL-pFibro than in cells on PCL-pBSP. These phosphorylated Smad1/5/8 were translocated to the nucleus. BMP-9 and its derived peptides restored the phosphorylation of JNK in cells on PCL-pBSP, but it remained less phosphorylated than in cells on PCL-pFibro stimulated with pBMP-9 and SpBMP-9. Cells attached to PCL-pFibro contained more Runx2, essential for stem cell commitment to become osteoblasts, than did cells on PCL-pBSP when incubated with BMP-9 and its derived peptides. Runx2 was no longer detected when the cells were pre-treated with JNK inhibitor. Therefore pFibro plus BMP-9 and its derived peptides may be a promising strategy to develop biomimetic materials. STATEMENT OF SIGNIFICANCE Biomaterials functionalized with adhesive peptides to favour bone repair have generated a great interest over the past decade. However, the effect of these materials on the ability of cells to respond to growth factors remains poorly known. One major growth factor subfamily involved in bone formation is the bone morphogenetic protein (BMP). However, these BMPs are expensive. We therefore developed less costly derived molecules. We showed how adhesive peptides derived from bone matrix proteins grafted onto polymer films affect the intracellular signalling and thus the ability of stem cells to be activated by BMP and its derived molecules. We have therefore identified a combination of bioactive polymers and BMP molecules that direct the stem cells towards bone forming cells.

Preventing Mek1 activation influences the responses of human osteosarcoma cells to bone morphogenetic proteins 2 and 9

It was recently suggested that bone morphogenetic protein (BMP)-2 may be useful for treating osteosarcoma cells. BMP-9, which has been patented to treat breast and prostate cancers, has a higher osteoinductive potential than BMP-2. Peptides derived from the knuckle epitope of BMPs (pBMPs) also induced osteogenic differentiation. However, the effect of BMP-9 and pBMPs on osteosarcoma cells is unclear. We analyzed the effects of BMP-2, BMP-9, pBMP-2, and pBMP-9 on the behavior of human MG-63 and SaOS-2 osteosarcoma cells. An inhibitor of MEK1 activation (PD98059) that prevents downstream extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and a specific inhibitor of p38 were also used as mitogen activated protein kinase-targeting therapy is being investigated as a treatment modality for osteosarcoma. BMP-2 and BMP-9 (1.92 nmol/l) induced the phosphorylation of Smad1/5/8 in both osteosarcoma cells within 1 h but had different effects on mitogen activated protein kinase pathways. Whereas BMP-2 mainly activated ERK1/2, BMP-9 phosphorylated p38 within 1 h. pBMP-2 did not activate either the Smad or ERK/p38, whereas pBMP-9, like BMP-9, induced both Smad1/5/8 and p38 phosphorylation. p38 activation by BMP-9 or pBMP-9 was also enhanced by PD98059. However, BMP-2 or BMP-9 increased the amounts of distal-less homeobox 5 and Osterix mRNAs in SaOS-2 cells within 6 h, whereas pBMP-9 had no effect. PD98059 promoted the highest level of Osterix mRNA in SaOS-2 cells incubated with BMP-2 or BMP-9, whereas p38 inhibitor had no effect. Furthermore, PD98059 induced the lowest proliferation of MG-63 cells incubated with BMP-2, whereas p38 inhibitor did not affect the proliferation of either osteosarcoma cell line. Therefore a combination of BMP-2 or BMP-9 and an inhibitor of MEK1 may be a promising tool for regulating osteosarcoma cell behavior.

Peptides derived from the knuckle epitope of BMP-9 induce the cholinergic differentiation and inactivate GSk3beta in human SH-SY5Y neuroblastoma cells

The incidence of brain degenerative disorders like Alzheimer’s disease (AD) will increase as the world population ages. While there is presently no known cure for AD and current treatments having only a transient effect, an increasing number of publications indicate that growth factors (GF) may be used to treat AD. GFs like the bone morphogenetic proteins (BMPs), especially BMP-9, affect many aspects of AD. However, BMP-9 is a big protein that cannot readily cross the blood-brain barrier. We have therefore studied the effects of two small peptides derived from BMP-9 (pBMP-9 and SpBMP-9). We investigated their capacity to differentiate SH-SY5Y human neuroblastoma cells into neurons with or without retinoic acid (RA). Both peptides induced Smad 1/5 phosphorylation and their nuclear translocation. They increased the number and length of neurites and the expression of neuronal markers MAP-2, NeuN and NSE better than did BMP-9. They also promoted differentiation to the cholinergic phenotype more actively than BMP-9, SpBMP-9 being the most effective as shown by increases in intracellular acetylcholine, ChAT and VAchT. Finally, both peptides activated the PI3K/Akt pathway and inhibited GSK3beta, a current AD therapeutic target. BMP-9-derived peptides, especially SpBMP-9, with or without RA, are promising molecules that warrant further investigation.

Identification of a growth factor mimicking the synergistic effect of fetal bovine serum on BMP-9 cell response.

The bone morphogenetic proteins (BMPs) are potent osteogenic molecules that are used for bone repair in delivery systems and in regenerative medicine. We studied the responses of murine MC3T3-E1 preosteoblasts to doses of recombinant human (rh)BMP-9 with and without fetal bovine serum (FBS). rhBMP-2 was used as a control since it is currently approved by the Food and Drug Administration for bone application. We analyzed the major cell signaling pathways and the expression of osteogenic markers. Without FBS, BMP-9 had a similar effect on MC3T3-E1 preosteoblast differentiation in comparison to BMP-2. In contrast, FBS reduced the EC50 of BMP-9 fourfold to sixfold, as determined by osterix gene expression and alkaline phosphatase (ALP) activity, while it had no influence on EC50 of BMP-2. As suggested by MAPK inhibitor assays, FBS could induce an intracellular signaling environment that favors cell response to BMP-9 by inhibiting ERK1/2 activation and increasing p38 phosphorylation. Finally, IGF-2 (100 ng/mL) could mimic the effect of FBS on BMP-9 cell response in terms of MAPK signaling and ALP activity. Thus, the action of BMP-9 on preosteoblast differentiation can be greatly improved by IGF-2. This finding may well be critical for developing optimal growth factor delivery systems and bone tissue engineering strategies.

Effects of BMP-9 and BMP-2 on the PI3K/Akt Pathway in MC3T3-E1 Preosteoblasts.

The bone morphogenetic proteins (BMPs), which are involved in bone formation and repair, play an important role in tissue engineering. For example, BMP-9 and BMP-2, which are members of different BMP subfamilies, are osteoinductive factors. However, several studies have recently shown that BMP-9 is more osteogenic than BMP-2. We have previously shown that fetal bovine serum (FBS) strongly enhances the osteoblast differentiation of murine preosteoblasts (MC3T3-E1) to BMP-9 but not to BMP-2. This effect is mimicked by IGF-2, which primarily activates the PI3K/Akt pathway, but how Akt phosphorylation sites are implicated in such differentiation is unclear. The effects of BMP-9 and BMP-2 with or without FBS or IGF-2 on Akt phosphorylation sites and subsequent osteoblastic differentiation were determined, respectively, by western blot analysis and alkaline phosphatase activity measurements. The involvement of phosphorylated Akt at Thr308 and/or Ser473 on BMP-mediated osteoblast differentiation was further studied using specific inhibitors. In MC3T3-E1 incubated with or without FBS, BMP-9 and BMP-2 activate Akt on Ser473 and Thr308 very differently in a time and dose-dependent manner. Using inhibitors specific to each Akt phosphorylation site, we showed that both Ser473 and Thr308 must be phosphorylated for BMP-9 and/or IGF-2-induced osteoblast differentiation, whereas BMP-2 requires phosphorylation of only Ser473. Furthermore, cells stimulated with BMP-2 in the presence of FBS require the phosphorylation of Akt at Ser473 and the dephosphorylation of Akt at Thr308 to increase the osteoblast differentiation with alkaline phosphatase activity similar to that of BMP-9 plus FBS. These results provide a better understanding into how BMP-9 induces osteoblast differentiation and its synergy with IGF-2 at the signaling level. This knowledge is essential for preparing the serum-free osteogenic media required for bone tissue engineering or developing growth factor delivery systems to improve bone formation.