Influence of multilayer rhBMP-2 DNA coating on the proliferation and differentiation of MC3T3-E1 cells seeded on roughed titanium surface

For bone morphogenetic protein (BMP) gene therapy to be a viable approach for enhancing implant osseointegration clinically, requires the development of efficient nonviral delivery vectors that can coat the implant. This study evaluated a multilayer cationic liposome-DNA complex (LDc) coating as a delivery vehicle for recombinant human BMP-2 (rhBMP-2). Multilayered coatings, comprising hyaluronic acid (HA) and LDc, were fabricated onto titanium using a layer-by-layer (LBL) assembly technique. Preosteoblastic MC3T3-E1 cells were cultured on the roughened titanium surfaces coated with multilayers of HA/LDc, or on uncoated or HA/liposome only surfaces as controls. The amount of rhBMP-2 secreted by the MC3T3-E1 cells and the effect of the various surfaces on cell viability, proliferation, alkaline phosphatase (ALP) activity, osteocalcin (OC) secretion, and calcium deposition were evaluated. Messenger RNA levels of OC, ALP, Runx2, and Osx were also investigated. The results demonstrated that rhBMP-2 protein secreted into culture medium at 3 days was significantly higher than control groups. MC3T3-E1 cells cultured on the HA/LDc coating displayed significantly higher ALP activity and OC secretion at 7 days and 14 days culture, respectively. MC3T3-E1 cells cultured on HA/LDc upregulated expression of the osteoblast differentiation markers, especially on days 12 for OC and on days 6 and 12 for ALP and Osx. In conclusion, MC3T3-E1 cell cultured on the multilayer HA/LDc coating surface can secret rhBMP-2 protein and the protein levels were effective in inducing early osteogenic differentiation. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A 100A: 2766-2774, 2012.     

Interaction of TGF-beta1 and rhBMP-2 on human bone marrow stromal cells cultured in collagen gel matrix.

Transforming growth factor-beta1 (TGF-beta1) and bone morphogenetic protein-2 (BMP-2) are abundant proteins in the bone matrix. However, their interaction in controlling osteoblast differentiation is not clearly understood. In this study, HBMSCs were cultured in collagen gel matrix with different condition of exogenous rhBMP-2 and TGF-beta1 in order to determine the interaction of BMP-2 and TGF-beta1 on human bone marrow stromal cells (HBMSCs) differentiation. The cultured cells were analyzed for cell proliferation, alkaline phophatase (ALP) activity and mineralization staining with Von-Kossa. The cells treated with TGF-beta1 exhibited a higher rate of cell growth than those without. However, the cells cultured in collagen gel matrix showed a lower rate of cell growth than the cells cultured in a monolayer. To investigate the effects of both cytokines on osteoblast differentiation, the cells were treated with 0, 1, 5, 10 ng/ml of TGF-beta1 for 2 days. This was followed by culturing with 0, 1, 5, and 10 ng/ml of TGF-beta1 and 100 ng/ml of rhBMP-2 together for 3 days with the alkaline phosphatase (ALP) activity measured. The cells treated with 1 ng/ml of TGF-beta1 responded efficiently to rhBMP-2 and expressed ALP activity with a level equivalent to that exhibited by cells that were not treated with TGF-beta1. The cells treated with 5 and 10 ng/ml of TGF-beta1 showed a dramatic decrease in ALP activity. The cells treated with 10 ng/ml of TGF-beta1 followed by rhBMP-2 alone exhibited an intermediate ALP activity. The cells treated with 100 ng/ml of rhBMP-2 demonstrated Von-Kossa positive solid deposits after 3 weeks, while there were few Von-Kossa positive solid deposits when the cells treated with 10 ng/ml of TGF-beta1. These results show that TGF-beta1 inhibits the effects of rhBMP-2 on the osteoblast differentiation of HBMSCs in a dose dependant manner. Furthermore, the effects of TGF-beta1 on HBMSCs are reversible. This suggest that TGF-beta1 and rhBMP-2 are coordinately controlled during the osteoblast differentiation of HMBSCs.     

Bone morphogenetic protein 2 incorporated into biomimetic coatings retains its biological activity

We have previously shown that proteins can be incorporated into the latticework of calcium phosphate layers when biomimetically coprecipitated with the inorganic components, upon the surfaces of titanium-alloy implants. In the present study, we wished to ascertain whether recombinant human bone morphogenetic protein 2 (rhBMP-2) thus incorporated retained its bioactivity as an osteoinductive agent. Titanium alloy implants were coated biomimetically with a layer of calcium phosphate in the presence of different concentrations of rhBMP-2 (0.1-10 microg/mL). rhBMP-2 was successfully incorporated into the crystal latticework, as revealed by protein blot staining. rhBMP-2 was taken up by the calcium phosphate coatings in a dose-dependent manner, as determined by ELISA. Rat bone marrow stromal cells were grown directly on these coatings for 8 days. Their osteogenicity was then assessed quantitatively by monitoring alkaline phosphatase activity. This parameter increased as a function of rhBMP-2 concentrations within the coating medium. rhBMP-2 incorporated into calcium phosphate coatings was more potent in stimulating the alkaline phosphatase activity of the adhering cell layer than was the freely suspended drug in stimulating that of cell layers grown on a plastic substratum. This system may be of osteoinductive value in orthopedic and dental implant surgery.     

Osteogenic media and rhBMP-2-induced differentiation of umbilical cord mesenchymal stem cells encapsulated in alginate microbeads and integrated in an injectable calcium phosphate-chitosan fibrous scaffold

The need for bone tissue engineering has increased as the world population ages. The objectives of this study were to (1) develop a novel human umbilical cord mesenchymal stem cell (hUCMSC)-encapsulating, fiber-reinforced injectable calcium phosphate cement (CPCF) scaffold, and (2) investigate the effects of osteogenic media delivery, preosteodifferentiation, and bone morphogenetic protein-2 (BMP-2) delivery on hUCMSC osteodifferentiation inside CPCF for the first time. CPCF was developed using calcium phosphate powders, chitosan, and absorbable fibers. Four types of hUCMSC-encapsulating constructs were fabricated: control media in alginate hydrogel microbeads in CPCF; osteogenic media in microbeads; preosteodifferentiation; and recombinant human BMP-2 (rhBMP-2) in microbeads. The hUCMSCs inside CPCF maintained good viability, successfully differentiated into the osteogenic lineage, and synthesized bone minerals. The preosteodifferentiation method yielded high gene expressions of alkaline phosphatase, osteocalcin, collagen, and osterix, as well as alkaline phosphatase protein synthesis. The mineralization for the preosteodifferentiation constructs exceeded those of the rhBMP-2 group at 1-7 days, and was slightly lower than the rhBMP-2 group at 21 days. Mineralization of the rhBMP-2 group was 12-fold that of the control constructs at 21 days. In conclusion, although the BMP-2 delivery promoted osteodifferentiation, the preosteodifferentiation method and the ostegenic media method with hUCMSCs in CPCF were also promising for bone regeneration. hUCMSCs may be an effective alternative to the gold-standard bone marrow MSCs, which require an invasive procedure to harvest. The novel injectable stem cell-CPCF construct may be useful in minimally invasive and other orthopedic surgeries.     

The effects of recombinant human BMP-7, prepared from a COS-7 expression system,on the proliferation and differentiation of rat newborn calvarial osteoblasts

A family of proteins, the bone morphogenetic proteins (BMPs), which promote osteoblast differentiation and bone mineralization, have recently been identified. One, BMP-7, has shown the ability to induce cartilage and bone formation processes. In this report, the possibility that other cell lines, to CHO cells, may also be available as host cells for the expression of hBMP-7 was validated. Recombinant human BMP (rhBMP)-7 was produced in COS-7 cells, as a processed mature disulfide-linked homodimer, with an apparent molecular weight of 36,000. Examination of the expressions of the markers characteristic of osteoblast phenotypes showed that the rhBMP-7 specifically stimulated the inductions of alkaline phosphatase (ALP) (5-fold increase at 100 ng of rhBMP-7/ml), parathyroid hormone (PTH)-mediated intracellular cAMP production (4-fold increase at 100 ng of rhBMP-7/ml) and osteocalcin synthesis (5-fold increase at 100 ng of rhBMP- 7/ml). In summary, the in vitro mineralization assay results provide evidence that the rhBMP-7 peptide, produced by COS-7 expression system, possesses intact biological activity. A similar pattern of biological activity was observed for the BMP-7 in COS-7 cells compared to the corresponding CHO cell expression system. Thus, these findings can be experimentally utilized for the production of rhBMPs for in vitro or in vivo studies.     

Retention and activity of BMP-2 in hyaluronic acid-based scaffolds in vitro.

Bone morphogenetic protein-2 (BMP-2) delivered in a suitable implantable matrix has the potential to repair local skeletal defects by inducing new bone formation from undifferentiated pluripotent stem cells resident in host tissue. In this study, we examined in vitro the potential of a derivatized hyaluronic acid (Hyaff-11) scaffold as a delivery vehicle for recombinant human BMP-2 (rhBMP-2) in bone and cartilage repair therapies. Hyaff-11 scaffolds were fabricated using a phase inversion/particulate leaching method and soak-loaded with rhBMP-2. In vitro release kinetics of rhBMP-2, demonstrated using enzyme-linked immunosorbant assay and alkaline phosphatase (ALP) assay revealed a slow, sustained rhBMP-2 release during 28 days, with a cumulative release of 31.82% of the initial rhBMP-2 loaded. rhBMP-2 was released in bioactive form as demonstrated by ALP induction of pluripotent cell line, C3H10T1/2 (T1/2), down the osteoblast lineage when incubated with the release supernatants. rhBMP-2 retention in Hyaff-11 scaffolds was greater than that from collagen gels, which released most of the initially loaded rhBMP-2 by 14 days. rhBMP-2-loaded Hyaff-11 scaffolds were also seeded with T1/2 cells and evaluated at 3, 7, 14, and 28 days for viability and expression of osteoblast phenotype. Cells remained viable throughout the study and expressed a time- and dose-dependent ALP and osteocalcin expression in the rhBMP-2 groups. Based on these observations, Hyaff-11 scaffolds may be suitable delivery systems for rhBMP-2 in bone/cartilage repair because of their ability to retain rhBMP-2, release low levels of bioactive rhBMP-2 to the local environment in a sustained manner, and stimulate differentiation of pluripotent stem cells.     

The effect of rhBMP-2 on canine osteoblasts seeded onto 3D bioactive polycaprolactone scaffolds

Our strategy entails investigating the influence of varied concentrations (0, 10, 100 and 1000ng/ml) of human recombinant bone morphogenetic protein-2 (rhBMP-2) on the osteogenic expression of canine osteoblasts, seeded onto poly-caprolactone 20% tricalcium phosphate (PCL-TCP) scaffolds in vitro. Biochemical assay revealed that groups with rhBMP-2 displayed an initial burst in cell growth that was not dose-dependent. However, after 13 days, cell growth declined to a value similar to control. Significantly less cell growth was observed for construct with 1000ng/ml of rhBMP-2 from 20 days onwards. Confocal microscopy confirmed viability of osteoblasts and at day 20, groups seeded with rhBMP-2 displayed heightened cell death as compared to control. Phase contrast and scanning electron microscopy revealed that osteoblasts heavily colonized surfaces, rods and pores of the PCL-TCP scaffolds. This was consistent for all groups. Finally, Von Kossa and osteocalcin assays demonstrated that cells from all groups maintained their osteogenic phenotype throughout the experiment. Calcification was observed as early as four days after stimulation for groups seeded with rhBMP-2. In conclusion, rhBMP-2 seems to enhance the differentiated function of canine osteoblasts in a non-dose dependent manner. This resulted in accelerated mineralization, followed by death of osteoblasts as they underwent terminal differentiation. Notably, PCL-TCP scaffolds seeded only with canine osteoblasts could sustain excellent osteogenic expression in vitro. Hence, the synergy of PCL with bioactive TCP and rhBMP-2 in a novel composite scaffold, could offer an exciting approach for bone regeneration.     

骨髓基质干细胞成骨分化过程中Wnt、骨形态发生蛋白2信号通路相关因子与辛伐他汀的作用

背景：辛伐他汀可促进体外培养的人或鼠骨髓基质干细胞向成骨细胞分化,但作用机制尚不清楚。 目的：观察辛伐他汀对大鼠骨髓基质干细胞向成骨细胞分化过程中Wnt与骨形态发生蛋白2信号途径中相关因子表达的影响。 方法：取6周龄雌性SD大鼠双侧股骨、胫骨全骨髓进行体外成骨细胞诱导培养。实验分为对照组及SIM组。SIM组加入浓度为10-7 mol/L辛伐他汀,对照组加入等量无水乙醇和PBS。培养14 d,行碱性磷酸酶染色,28 d时,行von Kossa染色观察细胞外基质矿化情况;培养14,21 d,免疫荧光细胞化学染色观察成骨细胞中β-catenin,Smad1/5,Cbfa1的表达及分布。  结果与结论：大鼠骨髓基质干细胞经体外诱导后可分化为具有碱性磷酸酶活性和矿化细胞外基质能力的成熟成骨细胞。辛伐他汀可显著上调骨髓基质干细胞成骨分化过程中碱性磷酸酶的表达。同时,与对照组比较,SIM组β-catenin,Smad1/5,Cbfa1表达明显增多(P < 0.05),且呈现明显的核内聚集趋势。说明辛伐他汀促进骨髓基质干细胞向成骨细胞分化的作用可能与调控Wnt与骨形态发生蛋白2信号通路中相关因子的表达及细胞内分布有关。     

A reporter-cell assay for the detection of BMP-2 immobilized on porous and nonporous materials

Human recombinant bone morphogenetic protein-2 (rhBMP-2) immobilized on the surface of metal implants can facilitate osseointegration. Here, we describe a cell reporter assay useful for quantifying small amounts of immobilized rhBMP-2 on various materials. The peptide was dotted and heat-fixed on titanium, 316L stainless steel, nitrocellulose, or glass, and its distribution was monitored by in situ biotinylation followed by detection with the avidin-biotin method. Bioactivity of rhBMP-2 was demonstrated by means of a confluent layer of osteoblastic MC3T3-E1 cells that evenly covered rhBMP-2-free and rhBMP-2-loaded surface areas, as shown with epifluorescence microscopy of calcein acetoxymethyl (AM)-loaded cells. Expression of osteocalcin, fibronectin, actin, and vimentin increased where cells were located on rhBMP-2 dotted areas, but the signal:noise ratio was too low to bioassay the peptide. However, local pronounced expression of alkaline phosphatase was used to quantify BMP-2 in the range of 5-80 ng/dot by means of a cytochemical color reaction for alkaline phosphatase and image analysis of resulting dots. The lower detection limit was in the order nitrocellulose > glass > titanium > 316L steel. We conclude that the cell reporter assay is useful to assess biological activity of rhBMP-2 even after immobilization on three-dimensional implant materials.     

The role of recombinant human bone morphogenetic protein-2 in PLGA capsules at an extraskeletal site of the rat.

Bone morphogenetic protein-2 (BMP-2) is a member of the transforming growth factor-beta (TGF-beta) superfamily and has strong bone-inductive activity in vivo. To examine the role of BMP-2 in an extraskeletal site of rat using a controlled release system of peptides, we encapsulated the recombinant human BMP-2 (rhBMP-2) with poly(DL-lactide-co-glycolide) (PLGA) and implanted the rhBMP-2/PLGA capsules in the subcutaneous area of rats. Upon histochemical examination, it was found that bone-inducing cells having alkaline phosphatase (ALP) activity appeared around the capsules by the suitably released rhBMP-2. In addition, the temporal histological examination showed that direct bone formation without cartilage occurred in the process of this ectopic bone induction. These data indicate that the role of rhBMP-2 in the extraskeletal site of rats is to induce the differentiation of mesenchymal cells into the osteoblasts.Copyright 1999 John Wiley & Sons, Inc.     

Influence of rhBMP-2 on rat bone marrow stromal cells cultured on titanium fiber mesh

Titanium (Ti) fiber mesh is a candidate scaffold material for the creation of bone graft substitutes (BGS). Two densities (3.54 x 10(4) cells/cm(2) [LD or low density] and 3.54 x 10(5) cells/cm(2) [HD or high density]) of rat bone marrow stromal cells were seeded on Ti-fiber mesh discs. Cells were cultured for up to 16 days, 7 days of which the cells were in the presence of various concentrations of rhBMP-2 (0, 10, 100, and 1,000 ng/mL) in order to evaluate osteogenic expression. Scanning electron microscopy (SEM), light microscopy (LM), energy dispersive spectroscopy (EDS), DNA and calcium (Ca) content measurements, and x-ray diffraction (XRD) analysis were performed. SEM and EDS evaluation showed that a confluent layer of cells was present on top of the meshes together with collagen bundles and calcified globular accretions. Light microscopical evaluation showed a densely stained layer in the upper part of the mesh. SEM and Ca content measurement showed that calcification starts at 8 days. In addition, it was demonstrated that DNA content peaked at 8 days. LM, SEM, and Ca content evaluation revealed positive effects of increasing the cell seeding density, the rhBMP-2 concentration and the culture time on mineralization. Increasing the cell seeding density also showed a positive effect on DNA content. No effects of rhBMP-2 concentration were seen on DNA content. Finally, XRD revealed that the deposited matrix contained a precipitate of a stable calcium phosphate phase. We conclude that (1) titanium fiber mesh sustains excellent osteogenic expression in vitro, (2) increasing the cell seeding density has a positive effect on osteogenic expression in titanium mesh in vitro, and (3) in high density specimens, rhBMP-2 concentrations of 100 ng/mL and 1,000 ng/mL stimulate extracellular matrix calcification in a dose-responsive manner.     

Immobilized recombinant human bone morphogenetic protein-2 enhances the phosphorylation of receptor-activated Smads

Bone morphogenetic protein (BMP)-2 plays an important role in bone growth and regeneration; however, BMP-2 is easily lost by diffusion through body fluid and has some inhibitory pathways. To address this problem, we previously immobilized recombinant human BMP-2 (rhBMP-2) on succinylated type I atelocollagen. Here, we examined the effect of immobilized rhBMP-2 in vitro and vivo. In ST2, MC3T3-E1, and C2C12 cells, alkaline phosphatase activity, which is a marker of osteoblast differentiation, was enhanced more by immobilized than nonimmobilized rhBMP-2. In addition, the phosphorylation of receptor-activated Smads, part of the signaling pathway activated by BMP-2, was prolonged by immobilized rhBMP-2 in these cells. Furthermore, implantation of immobilized rhBMP-2 into the backs of rats promoted the formation of mature bone-like structure. These results demonstrate that immobilized rhBMP-2 has higher bioactivity than nonimmobilized rhBMP-2, and, therefore, immobilization of rhBMP-2 can prolong BMP signaling.     

The effect of different titanium and hydroxyapatite-coated dental implant surfaces on phenotypic expression of human bone-derived cells

Roughened titanium (Ti) surfaces have been widely used for dental implants. In recent years, there has been the tendency to replace Ti plasma-sprayed surfaces by sandblasted and acid-etched surfaces in order to enhance osseous apposition. Another approach has been the utilization of hydroxyapatite (HA)-coated implants. This study examines the effect of two roughened Ti dental implant surfaces on the osteoblastic phenotype of human bone-derived cells (HBDC) and compares this behavior to that for cells on an HA-coated surface. Test materials were an acid-etched and sandblasted Ti surface (Ti-DPS), a porous Ti plasma-sprayed coating (Ti-TPS), and a plasma-sprayed porous HA coating (HA). Smooth Ti machined surfaces served as control (Ti-ma). HBDC were grown on the substrata for 3, 7, 14, and 21 days, counted and probed for various bone-related mRNAs and proteins (type I collagen, osteocalcin, osteopontin, osteonectin, alkaline phosphatase, and bone sialoprotein). All dental implant surfaces significantly affected cellular growth and the temporal expression of an array of bone-related genes and proteins. HA-coated Ti had the most effect on osteoblastic differentiation inducing a greater expression of an array of osteogenic markers than recorded for cells grown on Ti-DPS and Ti-TPS, thus suggesting that the HA-coated surface may possess a higher potency to enhance osteogenesis. Furthermore, Ti-DPS surfaces induced greater osteoblast proliferation and differentiation than Ti-TPS.     

Implantation of hydrophilic and hydrophobic titanium discs in rat tibia: cellular reactions on the surfaces during the first 3 weeks in bone

In a previous study, a method for evaluation of short-time (1-8 days) healing of titanium implants in rat tibiae was described (J. Biomed. Mater. Res. 66A(3) (2003) 662). The implants were disc-shaped and cells and tissue on the surface were investigated, not the adjacent tissue. In this study healing during the first 3 weeks in bone was examined and the healing response between hydrophilic and hydrophobic titanium was compared. Immunofluorescence techniques were used to detect signs of bone formation on the surfaces. Cell viability, alkaline phosphatase (ALP) activity, presence of osteocalcin and cells positive for bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) were investigated. Both viable and non-viable cells were found on both surfaces during the first week. Only initially was there a difference between them; 4% viable cells on hydrophilic discs compared to 56% on hydrophobic ones. More BMP-2 positive cells were found on hydrophilic discs than on hydrophobic ones after 1 week. VEGF was detected after 8 days on both surfaces. Osteocalcin positive cells were found from 2 weeks. ALP positive cells were found after 8 days, while at 2-3 weeks ALP positive tissue was abundant on both surfaces. In conclusion, signs of bone formation were detected during the period investigated. Surface energy appeared to be of more importance initially, with higher surface energy resulting in more rapid cell activation and differentiation than lower.     

Titanium with surface-grafted dextran and immobilized bone morphogenetic protein-2 for inhibition of bacterial adhesion and enhancement of osteoblast functions

Failure of bone and joint implants has been attributed mainly to poor bonding of the implant to bone tissue, and to bacterial infection. The probability of successful osseointegration or implant infection depends on the race for the surface between tissue cells and bacteria. One promising strategy to enhance tissue integration is to develop a selective biointeractive surface that increases bone cell (osteoblast) function while decreasing bacterial adhesion. In this in vitro study, the surface of titanium alloy substrates was first functionalized by covalently grafted oxidized dextran, which is known to have activity against bacterial adhesion. Bone morphogenetic protein-2 (BMP-2) was then covalently linked to dextran-grafted surfaces through a chemical conjugation process. The composition and properties of the surface were investigated by X-ray photoelectron spectroscopy and by measuring the surface density of BMP-2 using an enzyme-linked immunosorbent assay. Bacterial adhesion was assayed with Staphylococcus aureus and Staphylococcus epidermidis. Bacterial adhesion on both the dextran and dextran-BMP-2-functionalized surfaces was significantly decreased compared to that on the pristine substrates. Further, the dextran-BMP-2 modified substrates with a surface protein density of >50 ng/cm(2) or higher significantly promoted osteoblast spreading, alkaline phosphatase activity, and calcium mineral deposition. Thus, the results from this study suggest that surface grafting of dextran in conjunction with the bone growth factor BMP-2 on metal surfaces can enhance tissue integration of implants through the dual functions of reducing bacterial adhesion and promoting osteoblast functions.     

Segmental bone regeneration using an rhBMP-2-loaded gelatin/nanohydroxyapatite/fibrin scaffold in a rabbit model

We aimed to develop a hybrid scaffold with a porous structure and similar composition as natural bone for the controlled release of bone morphogenetic protein-2 (BMP-2) to enhance bone regeneration. We fabricated a gelatin/nanohydroxypatite (nHAP) scaffold by glutaraldehyde chemical cross-linking a gelatin aqueous solution with nHAP granules at a 5:1 ratio (v/w). Then, fibrin glue (FG) mixed with recombinant human BMP-2 (rhBMP-2) was infused into the gelatin/nHAP scaffold and lyophilized to develop an rhBMP-2-loaded gelatin/nHAP/FG scaffold. On scanning electron microscopy, the composite had a 3-D porous structure. The rhBMP-2 release kinetics from the hybrid scaffold was sustained and slow, and release of rhBMP-2 was complete at 40 days. Immunohistochemistry, azo-coupling and alizarin S-red staining were used to study in vitro differentiation of human bone-marrow mesenchymal cells (hBMSCs). Strong positive staining results confirmed that rhBMP-2 released from the scaffold could improve osteocalcin (OCN) and alkaline phosphatase (ALP) expression and calcium deposition formation. RT-PCR results showed significantly high mRNA expression of ALP and OCN in hBM-MSCs cultured on the gelatin/nHAP/FG scaffold with rhBMP-2. DNA assay demonstrated that the scaffold was noncytotoxic and could promote hBMSC proliferation from the components of the hybrid scaffold, not released rhBMP-2. The hybrid scaffolds were then used to repair critical-size segmental bone defects of rabbit radius. Gross specimen, X-ray, bone histomorphology and bone mineral density assay demonstrated that the rhBMP-2-loaded gelatin/nHAP/FG scaffold had good osteogenic capability and could repair the segmental bone defect completely in 12 weeks.     

Comparison of platelet-rich plasma,bovine BMP,and rhBMP-4 on bone matrix protein expression in vitro

This study investigated the potential use of platelet-rich plasma (PRP) in conjunction with mRNA expression of bone matrix proteins using bioassay and RT-PCR comparing bovine bone morphogenetic proteins (BMP), recombinant human BMP-4 (rhBMP-4) during rat bone marrow stromal cell (Mesenchymal Stem Cell) differentiation at 14 days. The results showed that all three growth factors were associated with significantly elevated alkaline phosphatase activity. PRP and bovine BMP resulted in increased protein content. The mRNA of type I collagen was expressed with all three growth factors and remained consistently elevated. Osteopontin was observed with PRP from days 1 to 7; bone sialoprotein expression was detected on days 1 and 3. PRP, bovine BMP and rhBMP-4 enhanced the steady-state expression of PDGF-A as time-dependent to day 14 and in PRP was the strongest. PTHr was expressed at days 1 and 5. Vascular endothelial growth factor expression was the most highly expressed after day 3. These findings suggest that PRP increases mRNA expression of bone matrix protein, enchances osteogenesis and angiogenesis in vitro.     

Osteogenic Responses to Different Concentrations/Ratios of BMP-2 and bFGF in Bone Formation

Recombinant human bone morphogenetic protein-2 (rhBMP-2) and basic fibroblast growth factor (bFGF) are the focus of research pertaining to the stimulation of bone formation. We ascertained the effects of different concentrations rhBMP-2 on proliferation and differentiation of bone marrow stromal cells (BMSCs) in vitro and on ectopic bone formation in rats. BMSCs were obtained from beagle dogs and cultured in medium containing different concentrations rhBMP-2 and bFGF (0, 25, 50, 100, or 200 ng/mL). In a separate experiment, BMSCs were treated with different ratios (1:1, 2:1, 4:1, or 8:1) of rhBMP to bFGF (in each case the concentration of rhBMP was 100 ng/mL and the bFGF concentrations 100, 50, 25, or 12.5 ng/mL). Proliferation and differentiation of BMSCs were quantified by assessing methyl thiazole tetrazolium (MTT) and alkaline phosphatase (ALP) over 6 consecutive days. Von Kossa staining was performed on day 6. For the in vivo tests, porous calcium phosphate cement (CPC) was seeded with BMSCs (5 × 10⁴) in medium containing 100 ng/mL rhBMP-2, 50 ng/mL bFGF or combined 100 ng/mL rhBMP-2 and 50 ng/mL bFGF. These cells were then subcutaneously implanted in four sites in nude rats. Bone formation was detected by histology at weeks 4 and 12 and quantified using a KS400 computer based image analysis system. It was determined that combined rhBMP-2 and bFGF at a ratio of 2:1 (100:50 ng/mL) promoted significantly increased BMSC proliferation and differentiation of BMSCs compared to rhBMP-2 or bFGF alone (p < 0.05). CPC with combined 100 ng/mL rhBMP-2 and 50 ng/mL bFGF stimulated more bone formation than either 100 ng/mL rhBMP-2 or 100 ng/mL bFGF (p < 0.05). These results show that a combination of rhBMP-2 and bFGF effectively induces early BMSC proliferation and differentiation in vitro. When combined, rhBMP-2 and bFGF synergistically promote new bone formation.