Release Of Bone Morphogenic Protein-2 Research Articles

RhBMP-2-Conjugated Three-Dimensional-Printed Poly(L-lactide) Scaffold is an Effective Bone Substitute.

Bone growth factors, particularly bone morphogenic protein-2 (BMP-2), are required for effective treatment of significant bone loss. Despite the extensive development of bone substitutes, much remains to be desired for wider application in clinical settings. The currently available bone substitutes cannot sustain prolonged BMP-2 release and are inconvenient to use. In this study, we developed a ready-to-use bone substitute by sequential conjugation of BMP to a three-dimensional (3D) poly(L-lactide) (PLLA) scaffoldusing novel molecular adhesive materials that reduced the operation time and sustained prolonged BMP release. A 3D PLLA scaffold was printed and BMP-2 was conjugated with alginate-catechol and collagen. PLLA scaffolds were conjugated with different concentrations of BMP-2 and evaluated for bone regeneration in vitro and in vivo using a mouse calvarial model. The BMP-2 release kinetics were analyzed using ELISA. Histological analysis and micro-CT image analysis were performed to evaluate new bone formation. The 3D structure of the PLLA scaffold had a pore size of 400µm and grid thickness of 187-230µm. BMP-2 was released in an initial burst, followed by a sustained release for 14days. Released BMP-2 maintained osteoinductivity in vitro and in vivo. Micro-computed tomography and histological findings demonstrate that the PLLA scaffold conjugated with 2µg/ml of BMP-2 induced optimal bone regeneration. The 3D-printed PLLA scaffold conjugated with BMP-2 enhanced bone regeneration, demonstrating its potential as a novel bone substitute.

Controlled growth factor delivery system with osteogenic-angiogenic coupling effect for bone regeneration.

ObjectiveBone regeneration involves a coordinated cascade of events that are regulated by several cytokines and growth factors, among which bone morphogenic protein-2 (BMP-2), vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) play important roles. In this study, we investigated the effects of dual release of the three growth factors on bone regeneration in femur defects.MethodsA composite consisting of Gelatin microparticles loaded with VEGF/FGF-2 and poly(lactic-co-glycolic acid)-poly(ethylene glycol)-carboxyl (PLGA-PEG-COOH) microparticles loaded with BMP-2 encapsulated in a nano hydroxyapatite-poly actic-co-glycolic acid (nHA-PLGA) scaffold was prepared for the dual release of the growth factors.ResultsOn the 14th day, decreased release rate of BMP-2 compared with FGF-2 and VEGF was observed. However, after 14 days, compared to FGF-2 and VEGF, BMP-2 showed an increased release rate. Controlled dual release of BMP-2 and VEGF, FGF-2 resulted in a significant osteogenic differentiation of bone mesenchymal stem cells (BMSCs). Moreover, effects of the composite scaffold on functional connection of osteoblast-vascular cells during bone development were evaluated. The synergistic effects of dual delivery of growth factors were shown to promote the expression of VEGF in BMSCs. Increased secretion of VEGF from BMSCs promoted the proliferation and angiogenic differentiation of human umbilical vein endothelial cells (HUVECs) in the co-culture system. At 12 weeks after implantation, blood vessel and bone formation were analyzed by micro-CT and histology. The composite scaffold significantly promoted the formation of blood vessels and new bone in femur defects.ConclusionsThese findings demonstrate that dual delivery of angiogenic factors and osteogenic factors from Gelatin and PLGA-PEG-COOH microparticles-based composite scaffolds exerted an osteogenic-angiogenic coupling effect on bone regeneration. This approach will inform on the development of appropriate designs of high-performance bioscaffolds for bone tissue engineering.

Sequential dual-drug delivery of BMP-2 and alendronate from hydroxyapatite-collagen scaffolds for enhanced bone regeneration

The clinical use of bioactive molecules in bone regeneration has been known to have side effects, which result from uncontrolled and supraphysiological doses. In this study, we demonstrated the synergistic effect of two bioactive molecules, bone morphogenic protein-2 (BMP-2) and alendronate (ALN), by releasing them in a sequential manner. Collagen-hydroxyapatite composite scaffolds functionalized using BMP-2 are loaded with biodegradable microspheres where ALN is encapsulated. The results indicate an initial release of BMP-2 for a few days, followed by the sequential release of ALN after two weeks. The composite scaffolds significantly increase osteogenic activity owing to the synergistic effect of BMP-2 and ALN. Enhanced bone regeneration was identified at eight weeks post-implantation in the rat 8-mm critical-sized defect. Our findings suggest that the sequential delivery of BMP-2 and ALN from the scaffolds results in a synergistic effect on bone regeneration, which is unprecedented. Therefore, such a system exhibits potential for the application of cell-free tissue engineering.

Preparation of BMP-2/chitosan/hydroxyapatite antibacterial bio-composite coatings on titanium surfaces for bone tissue engineering.

In this paper, petaling hydroxyapatite (HA)/TiO2 composite coatings were firstly prepared on titanium (Ti) surface by one-step micro-arc oxidation (MAO), and then pure chitosan (CS) and bone morphogenic protein-2 (BMP-2)-encapsulated CS coatings were respectively loaded on the HA/TiO2 surfaces by dip-coating method to endow Ti with good antibacterial and biological properties. The bonding strength between coatings was studied by scratch method. The degradability of CS, BMP-2 release behavior, bioactivity, biocompatibility and antibacterial activity of the obtained (BMP-2)/CS/HA/TiO2 coatings were examined by in vitro tests. The results showed that, the thicker the HA layer, the larger the loaded BMP-2 and CS amount, resulting in better bonding strength between coatings, antibacterial activity and biocompatibility. In addition, with the increase of CS concentration, more CS was loaded on HA coatings, which benefited the increase of CS degrading amount, the prolonged CS degradation time and BMP-2 release time, resulting in improved antibacterial and biological property. All CS/HA/TiO2 coatings accelerated cell adhesion, spreading and proliferation, and promoted HA formation in simulated body fluids (SBF). After loading BMP-2 in CS, the BMP-2 can significantly improve cell adhesion, spreading and proliferation, and the loaded amount can also be controlled by the concentration of BMP-2 solution. The present study indicates that, by controlling the thickness of HA layers and concentrations of CS and BMP-2 solutions, the Ti implant material with excellent biological and antibacterial properties can be achieved.

Biomodulation of an implant for enhanced bone-implant anchorage

Aseptic loosening of implants is the major cause for revision surgery. By modulating the bone-implant interface, early bone-implant anchorage could be improved. Implant surface manipulation by the addition of osteopromotive molecules locally and systemically to promote implant integration has been described with limited success. This study describes a novel approach by making the implant capable of biologically modulating its surroundings. It was hypothesized that the early implant fixation would improve by filling the interior of the implant with a carrier providing spatio-temporal release of bone active drugs with known osteogenic effect. The implant consisted of a threaded polyether ether ketone (PEEK) hollow chamber with holes at the bottom. The implant was filled with a calcium sulphate (CaS)/hydroxyapatite (HA) carrier, delivering two bone active molecules; zoledronic acid (ZA) and bone morphogenic protein-2 (BMP-2). At first, a rat abdominal muscle pouch model indicated a sustained in-vivo release of both 125I-rhBMP-2 (57%) and 14C-ZA (22%) from the CaS/HA carrier over a period of 4-weeks. The biomodulated implant was then inserted in the proximal tibia in rats with the following experimental groups: G1) Empty implant, G2) Implant + CaS/HA, G3) Implant + CaS/HA + ZA and G4) Implant + CaS/HA + ZA + rhBMP-2. Significantly higher bone volume (BV) was seen around the implant in groups G3 (3.3 ± 0.7 mm3) and G4 (3.1 ± 0.7 mm3) compared to the control (1.3 ± 0.4 mm3) using micro-computed tomography and qualitative histology. Group G3, also exhibited significantly higher pull-out force and absorbed energy when compared to the control group G1. These findings indicate that a low dose of ZA alone, released in a controlled manner from within a fenestrated implant is enough to improve implant anchorage without the need of adding rhBMP-2. This simple method of using a fenestrated implant containing a ceramic carrier releasing bone active molecules improved bone anchorage and could clinically reduce prosthetic failure. Statement of SignificanceAseptic loosening remains as a major cause for implant revisions and early reaction of surrounding bone to the prosthesis is important for longevity. A novel approach to enhance early bone-implant anchorage is presented.The implant is filled with a carrier providing controlled release of bone active molecules. In an animal model, a calcium sulphate (CaS)/hydroxyapatite (HA) carrier was used to provide a spatio-temporal release of bone morphogenic protein-2 (BMP-2) and zoledronic acid (ZA). Significantly better bone-implant integration was achieved using ZA alone, thereby eliminating the need for adding BMP-2. The developed method of implant biomodulation holds potential to prevent implant loosening and is an alternative to prosthetic coatings or systemic drug treatment. Importantly, all constituents are approved for clinical use.

The Osteoinductive Effect of Controlled Bone Morphogenic Protein 2 Release Is Location Dependent.

The main challenge in bone morphogenic protein 2 (BMP-2)-based application lies in finding strategies to prolong its biologic activity as it has a short biological half-life. The present study uses a phosphate-modified oligo[(polyethylene glycol) fumarate] hydrogel that can be tuned to achieve differential release profiles of biologically active BMP-2 release. We demonstrate that this platform outperforms Infuse®, currently used in the clinic and that the osteoinductive effect of BMP-2 is location dependent. Altogether, this study stresses the importance of evaluating efficacy of bone tissue engineering strategies at an orthotopic location rather than subcutaneously. Even more so, it emphasizes the role of biomaterials as a scaffold to achieve proper bone tissue engineering.

Design of a 3D BMP-2-Delivering Tannylated PCL Scaffold and Its Anti-Oxidant, Anti-Inflammatory, and Osteogenic Effects In Vitro.

In this study, a novel three-dimensional (3D) bone morphogenic protein-2 (BMP-2)-delivering tannylated polycaprolactone (PCL) (BMP-2/tannic acid (TA)/PCL) scaffold with anti-oxidant, anti-inflammatory, and osteogenic activities was fabricated via simple surface coating with TA, followed by the immobilization of BMP-2 on the TA-coated PCL scaffold. The BMP-2/TA/PCL scaffold showed controlled and sustained BMP-2 release. It effectively scavenged reactive oxygen species (ROS) in cells, and increased the proliferation of MC3T3-E1 cells pre-treated with hydrogen peroxide (H2O2). Additionally, the BMP-2/TA/PCL scaffold significantly suppressed the mRNA levels of pro-inflammatory cytokines, including matrix metalloproteinases-3 (MMP-3), cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), in lipopolysaccharide (LPS)-induced MC3T3-E1 cells. Furthermore, it showed outstanding enhancement of the osteogenic activity of MC3T3-E1 cells through increased alkaline phosphatase (ALP) activity and calcium deposition. Our findings demonstrated that the BMP-2/TA/PCL scaffold plays an important role in scavenging ROS, suppressing inflammatory response, and enhancing the osteogenic differentiation of cells.

Soybean Lecithin-Mediated Nanoporous PLGA Microspheres with Highly Entrapped and Controlled Released BMP-2 as a Stem Cell Platform.

Injectable polymer microsphere-based stem cell delivery systems have a severe problem that they do not offer a desirable environment for stem cell adhesion, proliferation, and differentiation because it is difficult to entrap a large number of hydrophilic functional protein molecules into the core of hydrophobic polymer microspheres. In this work, soybean lecithin (SL) is applied to entrap hydrophilic bone morphogenic protein-2 (BMP-2) into nanoporous poly(lactide-co-glycolide) (PLGA)-based microspheres by a two-step method: SL/BMP-2 complexes preparation and PLGA/SL/BMP-2 microsphere preparation. The measurements of their physicochemical properties show that PLGA/SL/BMP-2 microspheres had significantly higher BMP-2 entrapment efficiency and controlled triphasic BMP-2 release behavior compared with PLGA/BMP-2 microspheres. Furthermore, the in vitro and in vivo stem cell behaviors on PLGA/SL/BMP-2 microspheres are analyzed. Compared with PLGA/BMP-2 microspheres, PLGA/SL/BMP-2 microspheres have significantly higher in vitro and in vivo stem cell attachment, proliferation, differentiation, and matrix mineralization abilities. Therefore, injectable nanoporous PLGA/SL/BMP-2 microspheres can be potentially used as a stem cell platform for bone tissue regeneration. In addition, SL can be potentially used to prepare hydrophilic protein-loaded hydrophobic polymer microspheres with highly entrapped and controlled release of proteins.

Tunable osteogenic differentiation of hMPCs in tubular perfusion system bioreactor

The use of bioreactors for bone tissue engineering has been widely investigated. While the benefits of shear stress on osteogenic differentiation are well known, the underlying effects of dynamic culture on subpopulations within a bioreactor are less evident. In this work, we explore the influence of applied flow in the tubular perfusion system (TPS) bioreactor on the osteogenic differentiation of human mesenchymal progenitor cells (hMPCs), specifically analyzing the effects of axial position along the growth chamber. TPS bioreactor experiments conducted with unidirectional flow demonstrated enhanced expression of osteogenic markers in cells cultured downstream from the inlet flow. We utilized computational fluid dynamic modeling to confirm uniform shear stress distribution on the surface of the scaffolds and along the length of the growth chamber. The concept of paracrine signaling between cell populations was validated with the use of alternating flow, which diminished the differences in osteogenic differentiation between cells cultured at the inlet and outlet of the growth chamber. After the addition of controlled release of bone morphogenic protein-2 (BMP-2) into the system, osteogenic differentiation among subpopulations along the growth chamber was augmented, yet remained homogenous. These results allow for greater understanding of axial bioreactor cultures, their microenvironment, and how well-established parameters of osteogenic differentiation affect bone tissue development. With this work, we have demonstrated the capability of tuning osteogenic differentiation of hMPCs through the application of fluid flow and the addition of exogenous growth factors. Such precise control allows for the culture of distinct subpopulation within one dynamic system for the use of complex engineered tissue constructs. Biotechnol. Bioeng. 2016;113: 1805-1813. © 2016 Wiley Periodicals, Inc.

Graphene oxide-bone morphogenic protein2 complex coating by electrophoretic deposition

Event Abstract Back to Event Graphene oxide-bone morphogenic protein2 complex coating by electrophoretic deposition Eun-Jung Lee1 and Hae-Won Kim1 1 Institute of Tissue Regeneration Engineering, Nanobiomedical Science, Korea Introduction: Surface modification of metallic implants through a coating method using bioactive and degradable compositions is very useful to improve the osteogenic properties of implants. Furthermore, drug deliverable coating can induce more effective tissue regeneration. Electrophoretic deposition (EPD) is one of the most effective coating methods available, due to its simplicity, uniformity, the short process time and low-temperature process to use in biomedical applications[1]. This study was aimed to produce graphene oxide (GO)-bone morphogenic protein2 (BMP2) complex coatings on Ti for osteogenic surface with sustained drug release. GO has been reported it has high capacity as a delivery carrier for biomolecules as well as supportive material for osteogenic improvement of stem cells[2]. The BMP2 incorporation is considered to enhance the osteo-conductivity. Therefore, it is expected that the GO-BMP2 coating is contributed to improve tissue-compatibility of metallic implants. Materials and Methods: The GO-BMP2 were deposited on Ti substrate (10 mm ´ 10 mm) through the EPD process. In order to prepare deposit materials, the BMP2 (100 ㎍/ml) was combined with GO (50 ㎍/ml) 10 % ethanol. The coating layers were observed by scanning electron microscopy (SEM, Hitachi S-3000H). The cellular behavior of the composite coating was investigated using mesenchymal stem cells (MSCs), and the result was compared with bare Ti as a control. The BMP2 release was monitored up to 20 days. Results and Discussion: Figs. 1 show the picture of GO-BMP2 coated Ti plates and the morphology of the composite coating surface generated through the EPD process, respectively. Fig. 1. Picture (A) and SEM images of the surface morphology of the EPD composite coating layer. The GO-BMP2 complex was strongly deposited on the Ti surface. We could obtaine uniform coating [ Fig 1(A)]. However, in detailed view through the SEM, the deposition of GO-BMP2 complex (black arrows) induced the surface morphology change slightly, compared to bare Ti surface. Fig. 2. Cumulative BMP2 released in PBS. BMP2 release profiles for 20 days are presented in fig 2. The composite coatings revealed continuous and long-term release patterns of BMP2, and the amount of BMP2 released from the coatings were approximately 65%. Fig 3. Differentiation levels of MSCs on samples after culturing for 10 days. The ALP activity was assessed to evaluate the effect of composite coating on the osteogenic properties of metallic implants. The ALP activity of the GO-BMP2 composite coating was improved compared with the bare Ti. Conclusion: The GO-BMP2 composite coatings were successfully produced via electrophoretic deposition (EPD) technique. The composite coatings showed a thin and uniform surface morphology. The composite coatings continuously released BMP2 for 20 days. The biological property of coatings, particularly ALP activity, was improved compared with that of the bare Ti.

Design of extracellular protein based particles for intra and extra-cellular targeting

In recent years, a significant amount of effort has been dedicated to the development of both nano- and micro-technologies as they offer endless possibilities with regards to their biological applications. At the forefront of these technologies is the synthesis of particles for various applications in targeted drug and growth factor delivery, gene therapy, medical imaging and tissue engineering. In this study we used the layer-by-layer self-assembly method to synthesis protein-based microparticles using extracellular matrix proteins, such as collagen and fibronectin, in a simple and scalable way. Particle characterisation was performed using fluorescence microscopy, zeta potential analysis and scanning electron microscopy. Furthermore, two different cell types were used to investigate microparticle toxicity, attachment and/or internalisation. The results obtained not only showed a significant reduction in the cytotoxicity of these protein-based particles but also a significant increase in their attachment and internalisation by cells compared to their polymeric counterparts. In addition, we provide evidence for use of such particles in achieving the sustained release of Bone Morphogenic Protein-2 which is widely used for bone tissue engineering. This study has implications in the development of functional, biocompatible and non-toxic particles for intra and extra-cellular targeting and sustained release of various drugs, growth factors and genetic materials for numerous applications in medicine.

Surface functionalization of microgrooved titanium with dual growth factor-releasing nanoparticles for synergistic osteogenic differentiation of human mesenchymal stem cells

We demonstrate that dual release of bone morphogenic protein-2 (BMP-2) and insulin-like growth factor-1 (IGF-1) by catechol-functionalized adhesive polymer nanoparticles on microgrooved titanium (Ti) surface enhances in vitro osteoblastic differentiation of human mesenchymal stem cells (MSCs). The nanoparticles consisted of three distinct domains, surface Ti-adhesive catechol groups, anionic poly(L-aspartic acid) (PAsp) shells, and hydrophobic poly(L-phenylalanine) (PPhe) cores. The immobilization of the adhesive nanoparticles onto microgrooved Ti surface was verified using various surface analytical tools, such as field-emission scanning electron microscopy (Fe-SEM), X-ray photoelectron spectroscopy (XPS), contact angle measurement. The nanoparticles were immobilized both on the groove bottom surface and the ridge top surface with a similar anchoring density. A fluorescence microscope visualized that BMP-2 and IGF-1 of positive charges were efficiently loaded onto the negatively charged PAsp shells of immobilized nanoparticles. We confirmed the enhanced osteoblastic differentiation of MSCs by presenting the expression results of major osteoblast marker genes and proteins. In addition, overall significant correlations between the experimental results verified the validity of our study. The proposed combined surface of microgrooves and growth factor-releasing nanoparticles can be used as a strong osteogenic promoter on various biomaterial surfaces.

Enhanced angiogenesis and osteogenesis in critical bone defects by the controlled release of BMP-2 and VEGF: implantation of electron beam melting-fabricated porous Ti6Al4V scaffolds incorporating growth factor-doped fibrin glue

Electron beam melting (EBM)-fabricated porous titanium implants possessing low elastic moduli and tailored structures are promising biomaterials for orthopedic applications. However, the bio-inert nature of porous titanium makes reinforcement with growth factors (GFs) a promising method to enhance implant in vivo performance. Bone-morphogenic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) are key factors of angiogenesis and osteogenesis. Therefore, the present study is aimed at evaluating EBM-fabricated porous titanium implants incorporating GF-doped fibrin glue (FG) as composite scaffolds providing GFs for improvement of angiogenesis and osteogenesis in rabbit femoral condyle defects. BMP-2 and VEGF were added into the constituent compounds of FG, and then this GF-doped FG was subsequently injected into the porous scaffolds. In five groups of implants, angiogenesis and osteogenesis were evaluated at 4 weeks post-implantation using Microfil perfusion and histological analysis: eTi (empty scaffolds), cTi (containing undoped FG), BMP/cTi (containing 50 μg rhBMP-2), VEGF/cTi (containing 0.5 μg VEGF) and Dual/cTi (containing 50 μg rhBMP-2 and 0.5 μg VEGF). The results demonstrate that these composite implants are biocompatible and provide the desired gradual release of the bioactive growth factors. Incorporation of GF delivery, whether a single factor or dual factors, significantly enhanced both angiogenesis and osteogenesis inside the porous scaffolds. However, the synergistic effect of the dual factors combination was observable on angiogenesis but absent on osteogenesis. In conclusion, fibrin glue is a biocompatible material that could be employed as a delivery vehicle in EBM-fabricated porous titanium for controlled release of BMP-2 and VEGF. Application of this method for loading a porous titanium scaffold to incorporate growth factors is a convenient and promising strategy for improving osteogenesis of critical-sized bone defects.

BMP2-encapsulated chitosan coatings on functionalized Ti surfaces and their performance in vitro and in vivo

Bone morphogenic protein-2 (BMP2)-encapsulated chitosan (CS) coatings were prepared to immobilize BMP2 on titanium (Ti) surfaces. The Ti substrates were functionalized through a three-step process: alkali treatment, silanization with 3-aminopropyltriethoxysilane and aldehydation with glutaraldehyde (GA). BMP2-encapsulated CS coatings (BMP2-CS) were bonded to Ti surfaces through reactions between the aldehyde groups of GA and the amine groups of CS. Direct BMP2 immobilization on aldehyde-treated Ti (BMP2-Ti) and pure CS coatings (CS-Ti) were used as controls. The release rate of BMP2-CS-Ti was half of that of BMP2-Ti at initial stage, which indicates that the CS coatings are suitable carriers for sustained BMP2 release. The osteoinductivities of BMP2-CS-Ti, BMP2-Ti, CS-Ti and pristine Ti were examined by both in vitro cell tests and in vivo experiments. Bone marrow stem cell (BMSC) culture indicated that BMP2-CS-Ti is more potent in stimulating the differentiation of the adhering BMSC than the three other groups. Rabbit femur implantation revealed the excellent osteoinductivity of BMP2-CS-coated Ti implants. These results demonstrate that the BMP2-encapsulated CS coatings are stable osteoinductive coatings that realize the sustained release of BMP2 and maintain the activity of the protein.

In vitro and in vivo evaluation of bone formation using solid freeform fabrication-based bone morphogenic protein-2 releasing PCL/PLGA scaffolds

The aim of this study was to develop novel polycaprolactone/poly(lactic-co-glycolic acid) (PCL/PLGA) scaffolds with a heparin–dopamine (Hep–DOPA) conjugate for controlled release of bone morphogenic protein-2 (BMP-2) to enhance osteoblast activity in vitro and also bone formation in vivo. PCL/PLGA scaffolds were prepared by a solid freeform fabrication method. The PCL/PLGA scaffolds were functionalized with Hep–DOPA and then BMP-2 was sequentially coated onto the Hep–DOPA/PCL/PLGA scaffolds. The characterization and surface elemental composition of all scaffolds were evaluated by scanning electron microscope and x-ray photoelectron spectroscopy. The osteoblast activities on all scaffolds were assessed by cell proliferation, alkaline phosphatase (ALP) activity and calcium deposition in vitro. To demonstrate bone formation in vivo, plain radiograph, micro-computed tomography (micro-CT) evaluation and histological studies were performed after the implantation of all scaffolds on a rat femur defect. Hep–DOPA/PCL/PLGA had more controlled release of BMP-2, which was quantified by enzyme-linked immunosorbent assay, compared with Hep/PCL/PLGA. The in vitro results showed that osteoblast-like cells (MG-63 cells) grown on BMP-2/Hep–DOPA/PCL/PLGA had significantly enhanced ALP activity and calcium deposition compared with those on BMP-2/Hep/PCL/PLGA and PCL/PLGA. In addition, the plain radiograph, micro-CT evaluation and histological studies demonstrated that the implanted BMP-2/Hep–DOPA/PCL/PLGA on rat femur had more bone formation than BMP-2/Hep/PCL/PLGA and PCL/PLGA in vivo.

Bone morphogenic protein-2 (BMP-2) loaded hybrid coating on porous hydroxyapatite scaffolds for bone tissue engineering

In this study, a silica xerogel-chitosan hybrid is utilized as a coating material to incorporate bone morphogenic protein-2 (BMP-2) on a porous hydroxyapatite (HA) scaffold for bone tissue engineering. BMP-2 is known as a therapeutic agent for improving bone regeneration and repair. Silica xerogel-chitosan hybrids have been used for the delivery of a growth factor as well as osteoconductive coatings. The biological properties of the hybrid coating incorporated with BMP-2 were evaluated in terms of the BMP-2 release behavior, osteoblastic cellular responses and in vivo performance. BMP-2 was continuously released from the hybrid coating layer on the porous HA scaffold for up to 6 weeks. The hybrid coating containing BMP-2 showed significantly enhanced osteoblastic cell responses in comparison with the hybrid coating and HA substrate. Consequently, new bone formation was significantly increased within the hybrid coating containing BMP-2. These results reveal that the hybrid coating containing BMP-2 has the potential to be used as a bone implant, whose osteogenic properties are promoted by the release of BMP-2 in a controlled manner for a prolonged period of time.

BMP‐2 and bFGF release and in vitro effect on human osteoblasts after adsorption to bone grafts and biomaterials

Combination of scaffolds and growth factors is a promising option for several clinical problems in bone biomaterials. Simplified growth factor loading by adsorption from aqueous solution is one important option for this technology. We evaluated the adsorption followed by PBS rinsing, release and biological effect of transient loading with basic fibroblast growth factor (bFGF) and bone morphogenic protein 2 (BMP-2) on fresh frozen bone, processed bone matrix, collagen, and a ceramic material with immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and qRT-PCR. The study consisted of three in vitro experiments (immunofluorescence, ELISA, and qRT-PCR) in human osteoblasts (HOB). The first evaluated the adsorption of the growth factors bFGF and BMP-2 to the biomaterials, analyzed by immunofluorescence assays. The second experiment used ELISA to analyze the release of the growth factors from the matrix. The biological effect of the growth factors on HOB was then studied with qRT-PCR experiments as the third step. Strongest sustained release peaks in ELISA were observed in bFGF loading on processed bone matrix (steam-resistant mineralized bone matrix, SMBM) with up to 553pg/ml medium. BMP-2 loading was less effective in ELISA peak release experiments with up to 257pg/ml medium in processed bone matrix (SMBM). bFGF showed also higher release peaks in collagen material (192pg/ml) compared with BMP-2 (101pg/ml). Cumulative release values 0-72h were estimated. The expression of runX2, osteocalcin, and alkaline phosphatase as markers for osteoblast activity was correlating. The results showed sustained release of BMP-2 and bFGF after transient loading on bone biomaterials with a stronger effect in biological scaffolds. This is interesting for therapeutic growth factor loading as well as insights in natural growth factor matrix deposition during bone healing.

Non-glycosylated BMP-2 can induce ectopic bone formation at lower concentrations compared to glycosylated BMP-2

Bone morphogenic protein-2 (BMP-2) is a well-known growth factor that can improve the biological performance of bone substitute materials. BMP-2 produced via bacterial expression systems are non-glycosylated (ng) whereas native and recombinant equivalents produced in mammalian cell expression systems are glycosylated (g) proteins. ngBMP-2 is less soluble, resulting in lower BMP-2 release from carriers as used as bone substitute materials. This seems promising for reducing the amount of included growth factor in bone substitute materials. Hence, it was hypothesized that ngBMP-2 would induce formation of the same amount of bone at an ectopic site at lower dosage as gBMP-2. To that end, gBMP-2 and ngBMP-2 were firstly in vitro comparatively evaluated for biological activity and release from a calcium phosphate (CaP) based bone substitute material. Thereafter, an ectopic implantation model in rats was used, in which gBMP-2 and ngBMP2 were loaded in various dosages (2–20μg/implant) on the CaP-based bone substitute material and implanted for 4 and 12weeks. The results revealed that both the in vitro biological activity of and the in vitro release of ngBMP-2 are lower compared to gBMP2. Upon ectopic implantation, however, ngBMP-2 loaded implants induced more bone formation at lower concentrations from 4-weeks onward compared to gBMP-2 equivalents, indicating the value of ngBMP-2 as a potential alternative for mammalian produced recombinant BMP-2 for bone regenerative therapies.

An analysis of bone regeneration at a segmental bone defect by controlled release of bone morphogenetic protein 2 from a biodegradable sponge composed of gelatin and β-tricalcium phosphate

To treat large bone defects is a clinically challenging problem and utilizing tissue engineering technology is an attractive approach for overcoming such a problem. Previously, a biodegradable sponge incorporating bone morphogenic protein-2 (BMP-2), which can control the release of BMP-2 for a prolonged time in an in vivo environment, was reported. In addition, a biodegradable sponge composed of gelatin and β-tricalcium phosphate (βTCP), gelatin-βTCP sponge to develop a more ideal scaffold for enhancing bone regeneration was also created and previously reported. The purpose of this study was to investigate the effectiveness of the gelatin-βTCP sponge for the promotion of bone regeneration in a critical-sized bone defect site in vivo. Apparent bone regeneration was induced by the gelatin sponge incorporating BMP-2 and the gelatin-βTCP sponge with BMP-2 incorporation. In contrast, no apparent bone formation was induced by either the gelatin sponge only or the gelatin-βTCP sponge without BMP-2. To investigate the quality of the regenerated bone, we conducted a biomechanical evaluation with a three-point bending test. We found no significant difference between the gelatin sponge incorporating BMP-2 and the gelatin-βTCP sponge incorporating BMP-2 groups. Incorporation of βTCP into the gelatin sponge was expected to enhance biomechanical strength during the initial bone regeneration. However, our observations showed that the gelatin-βTCP sponge did not significantly improve the quality of regenerated bone from the viewpoint of biomechanical assessment, even though it did not impair the effectiveness of the promotion of bone regeneration by BMP-2 in the bone defect.

The effect of the delivery of vascular endothelial growth factor and bone morphogenic protein-2 to osteoprogenitor cell populations on bone formation

Regenerating bone tissue involves complex, temporal and coordinated signal cascades of which bone morphogenic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF 165) play a prominent role. The aim of this study was to determine if the delivery of human bone marrow stromal cells (HBMSC) seeded onto VEGF 165/BMP-2 releasing composite scaffolds could enhance the bone regenerative capability in a critical sized femur defect. Alginate-VEGF 165/P DLLA-BMP-2 scaffolds were fabricated using a supercritical CO 2 mixing technique and an alginate entrapment protocol. Increased release of VEGF 165 (750.4 ± 596.8 ρg/ml) compared to BMP-2 (136.9 ± 123.4 ρg/ml) was observed after 7-days in culture. Thereafter, up till 28 days, an increased rate of release of BMP-2 compared to VEGF 165 was observed. The alginate-VEGF 165/P DLLA-BMP-2 + HBMSC group showed a significant increase in the quantity of regenerated bone compared to the alginate-VEGF 165/P DLLA-BMP-2 and alginate/P DLLA groups respectively in a critical sized femur defect study as indices measured by μCT. Histological examination confirmed significant new endochondral bone matrix in the HBMSC seeded alginate-VEGF 165/P DLLA-BMP-2 defect group in comparison to the other groups. These studies demonstrate the ability to deliver a combination of HBMSC with angiogenic and osteogenic factors released from biodegradable scaffold composites enhances the repair and regeneration of critical sized bone defects.