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Bone infections are still a major problem in surgery. To avoid severe side effects of systemically administered antibiotics, local antibiotic therapy is increasingly being considered. Using a pressure-based method developed in our group, microporous β-TCP ceramics, which had previously been characterized, were loaded with 2% w/v alginate containing 50 mg/mL clindamycin and 10 µg/mL rhBMP-2. Release experiments were then carried out over 28 days with changes of liquid at defined times (1, 2, 3, 6, 9, 14, 21 and 28d). The released concentrations of clindamycin were determined by HPLC and those of rhBMP-2 by ELISA. Continuous release (anomalous transport) of clindamycin and uniform release (Fick's diffusion) of BMP-2 were determined. The composites were biocompatible (live/dead, WST-I and LDH) and the released concentrations were all antimicrobially active against Staph. aureus. The results were very promising and clindamycin was detected in concentrations above the MIC as well as a constant rhBMP-2 release over the entire study period. Biocompatibility was also not impaired by either the antibiotic or the BMP-2. This promising approach can therefore be seen as an alternative to the common treatment with PMMA chains containing gentamycin, as the new composite is completely biodegradable and no second operation is necessary for removal or replacement., (© 2024. The Author(s).)

This study evaluated the bone regeneration capacity and mechanical properties of block-type hydroxyapatite (HA)/tricalcium phosphate (TCP) scaffolds in response to different concentrations of polydeoxyribonucleotide (PDRN) and recombinant human bone morphogenic protein 2 (rhBMP-2). Thirty-two male white rabbits were used as a model of calvarial bone defect and classified into eight groups according to type and concentration of growth factor administered, viz., control group (only HA/TCP scaffold), scaffold + PDRN (0.1, 1, 5, and 10 mg/mL each) and scaffold + rhBMP-2 (0.01, 0.05, and 0.1 mg/mL each). The specimens were evaluated using histomorphometric and radiological analyses. Histomorphometric analyses indicated that the administration of PDRN did not increase bone formation. However, significant increases in bone formation were observed with the administration of rhBMP-2 at 0.05 and 0.10 mg/mL on week 8 compared to the control (p < 0.05). Radiological analyses revealed a significant increase in bone formation at week 8 with the administration of PDRN at 5 mg/mL and 10 mg/mL, and rhBMP-2 at 0.05 or 0.10 mg/mL compared to the control (p < 0.05). Our findings show that block-type HA/TCP scaffolds possess sufficient mechanical strength and bone regeneration capacity when used with optimal concentrations of growth factors.

Polycaprolactone (PCL) fiber mats with different surface modifications were functionalized with a chitosan nanogel coating to attach the growth factor human bone morphogenetic protein 2 (BMP-2). Three different hydrophilic surface modifications were compared with regard to the binding and in vitro release of BMP-2. The type of surface modification and the specific surface area derived from the fiber thickness had an important influence on the degree of protein loading. Coating the PCL fibers with polydopamine resulted in the binding of the largest BMP-2 quantity per surface area. However, most of the binding was irreversible over the investigated period of time, causing a low release in vitro. PCL fiber mats with a chitosan-graft-PCL coating and an additional alginate layer, as well as PCL fiber mats with an air plasma surface modification boundless BMP-2, but the immobilized protein could almost completely be released. With polydopamine and plasma modifications as well as with unmodified PCL, high amounts of BMP-2 could also be attached directly to the surface. Integration of BMP-2 into the chitosan nanogel functionalization considerably increased binding on all hydrophilized surfaces and resulted in a sustained release with an initial burst release of BMP-2 without detectable loss of bioactivity in vitro., (© 2020 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.)

Background: Current common techniques for repairing calvarial defects by autologous bone grafting and alloplastic implants have significant limitations. In this study, the authors investigated a novel alternative approach to bone repair based on peptide amphiphile nanofiber gels that are engineered to control the release of vascular endothelial growth factor (VEGF) to recruit circulating stem cells to a site of bone regeneration and facilitate bone healing by bone morphogenetic protein-2 (BMP-2)., Methods: VEGF release kinetics from peptide amphiphile gels were evaluated. Chemotactic functional scaffolds were fabricated by combining collagen sponges with peptide amphiphile gels containing VEGF. The in vitro and in vivo chemotactic activities of the scaffolds were evaluated by measuring mesenchymal stem cell migration, and angiogenic capability of the scaffolds was also evaluated. Large-scale rodent cranial bone defects were created to evaluate bone regeneration after implanting the scaffolds and other control materials., Results: VEGF was released from peptide amphiphile in a controlled-release manner. In vitro migration of mesenchymal stem cells was significantly greater when exposed to chemotactic functional scaffolds compared to control scaffolds. In vivo chemotaxis was evidenced by migration of tracer-labeled mesenchymal stem cells to the chemotactic functional scaffolds. Chemotactic functional scaffolds showed significantly increased angiogenesis in vivo. Successful bone regeneration was noted in the defects treated with chemotactic functional scaffolds and BMP-2., Conclusions: The authors' observations suggest that this bioengineered construct successfully acts as a chemoattractant for circulating mesenchymal stem cells because of controlled release of VEGF from the peptide amphiphile gels. The chemotactic functional scaffolds may play a role in the future design of clinically relevant bone graft substitutes for large-scale bone defects., Competing Interests: Disclosure:The authors have no commercial associations or financial disclosures that might pose a conflict of interest with any information presented in this article., (Copyright © 2020 by the American Society of Plastic Surgeons.)

The extent to which biologic payloads can be effectively delivered to cells is a limiting factor in the development of new therapies. Limitations arise from the lack of pharmacokinetic stability of biologics in vivo. Encapsulating biologics in a protective delivery vector has the potential to improve delivery profile and enhance performance. Coacervate microdroplets are developed as cell-mimetic materials with established potential for the stabilization of biological molecules, such as proteins and nucleic acids. Here, the development of biodegradable coacervate microvectors (comprising synthetically modified amylose polymers) is presented, for the delivery of biologic payloads to cells. Amylose-based coacervate microdroplets are stable under physiological conditions (e.g., temperature and ionic strength), are noncytotoxic owing to their biopolymeric structure, spontaneously interacted with the cell membrane, and are able to deliver and release proteinaceous payloads beyond the plasma membrane. In particular, myoglobin, an oxygen storage and antioxidant protein, is successfully delivered into human mesenchymal stem cells (hMSCs) within 24 h. Furthermore, coacervate microvectors are implemented for the delivery of human bone morphogenetic protein 2 growth factor, inducing differentiation of hMSCs into osteoprogenitor cells. This study demonstrates the potential of coacervate microdroplets as delivery microvectors for biomedical research and the development of new therapies., (© 2020 Wiley-VCH GmbH.)

Bone morphogenic protein-2 (BMP-2) knuckle epitope peptide has been recently discovered and known to activate chondrogenesis. However, the applications of this soluble peptide remain very limited due to rapid diffusion resulting in poor cellular uptake into target cells. We herein designed nanoparticles made from hyaluronic acid functionalized gold nanorods (GNRs) to conjugate with thiolated BMP-2 knuckle epitope peptide via a two-step reaction. Hyaluronic acid was modified to have thiol functional groups to replace the cetyl trimethylammonium bromide ligands on the surface of GNRs. The thiolated peptides were subsequently reacted with hyaluronic acid on the surface on GNRs via a maleimide-hydrazide crosslinker. The conjugation was confirmed by the change of surface charge of GNRs and the plasmon shift. A colorimetric peptide assay suggested more than 69% of the thiolated peptides were conjugated with the hyaluronic acid coated gold nanorods. Moreover, in vitro cell viability showed that BMP-2 conjugated hyaluronic acid functionalized gold nanorods (B2HGR) were cytocompatible and did not cause cytotoxicity to fibroblast cells. The B2HGRs also significantly promote cellular uptake of the BMP-2 peptides in both human mesenchymal stem cells and porcine chondrocytes due to multivalent ligand binding to the BMP receptors on the cell surface resulting in receptor-mediated endocytosis. The enhanced cellular uptake was clearly observed under a confocal microscope resulting in the significant activation of type II collagen gene expression and glucosaminoglycan secretion in those cells. Furthermore, our delivery system is a proof-of-concept of using scaffolds in combination with nanodelivery platform to enhance cartilaginous repair. The peptide loading capacity and the release is not limited by the scaffolds. Therefore, our delivery platform has potential applications for cartilage regeneration in a preclinical and clinical setting in the future.

Treatment aiming to enhance bone tissue regeneration can benefit from multiple growth factor or small molecule delivery. In the present study, the objective was to evaluate the feasibility of using nanocomposite fibrous scaffold to deliver prostacyclin I 2 agonist ONO-1301 in combination with BMP2 for treating critical sized bone defect. For this, ONO-1301 at three different concentrations (1.67 μg, 5 μg, 15 μg) and a fixed dose of BMP2 (5 μg) was loaded on the scaffold via physical adsorption. The results showed fast release of ONO-1301 for two weeks, whereas BMP2 exhibited slow and sustained release for four weeks. The scaffold with dual factors promoted the migration and osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro when compared to the scaffold with BMP2 alone. It also augmented bone tissue regeneration in critical sized rat calvarial defect at 12 weeks; mainly with lower dose of ONO-1301. However, synergistic effect on osteogenic differentiation and bone regeneration were not obtained through the concurrent release of BMP-2 and ONO-1301., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In experiments on Wistar rats, a simulated defect in the flat bones of the skull was filled with a collagen sponge of animal origin impregnated with BMP-2 or pure sponge; in control rats, the defect was left open. During follow-up, X-ray density of the collagen sponge in the experimental groups differed significantly. The results attest to the absence of spontaneous remodeling of the bone tissue under conditions modeled focal defect. Moreover, stimulation of reparative processes by the collagen matrix did not lead to positive dynamics. Saturation of the collagen sponge with BMP-2 in a concentration of 0.05 mg/ml allowed increasing Xray density of the bone starting from week 4.

Medication-related osteonecrosis of the jaw (MRONJ) poses an ongoing challenge for clinicians and researchers. Currently, there is a lack of preventative measures available for at-risk patients undergoing tooth extractions, especially those with prior bisphosphonate treatment due to osteoporosis or bone metastasis diagnoses. Here, these issues are addressed using a preventative tissue engineering strategy against MRONJ development. This study evaluates the efficacy of a poly(ethylene glycol)-heparin hydrogel as a tool for the delivery of arginylglycylaspartic acid (RGD) and recombinant human bone morphogenic protein-2 (rhBMP-2). Three groups of skeletally mature rats each receive two doses of intravenous zoledronic acid prior to surgery and undergo extraction of the right first mandibular molar with gingival closure. Experimental groups either have the sockets left empty, filled with hydrogel minus rhBMP-2, or filled with hydrogel plus rhBMP-2. Eight weeks postoperatively specimens are analyzed using radiological, histological, and scanning electron microscopy (SEM) techniques. µCT analysis shows increased bone formation with hydrogel/rhBMP-2 delivery compared to the empty socket. Hydrogel-treated groups display increased presence of osteocytes and increased osteoclastic action compared to the empty sockets. These results represent the first step toward improved delivery of rhBMP-2 and a potential MRONJ preventative for patients undergoing bisphosphonate treatment., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Traumatic musculoskeletal injuries that result in bone defects or fractures often affect both bone and the surrounding soft tissue. Clinically, these types of multi-tissue injuries have increased rates of complications and long-term disability. Vascular integrity is a key clinical indicator of injury severity, and revascularization of the injury site is a critical early step of the bone healing process. Our lab has previously established a pre-clinical model of composite bone-muscle injury that exhibits impaired bone healing; however, the vascularization response in this model had not yet been investigated. Here, the early revascularization of a bone defect following composite injury is shown to be impaired, and subsequently the therapeutic potential of combined vascularization and osteoinduction was investigated to overcome the impaired regeneration in composite injuries. A decorin (DCN)-supplemented collagen hydrogel was developed as a biomaterial delivery vehicle for the co-delivery microvascular fragments (MVF), which are multicellular segments of mature vasculature, and bone morphogenetic protein-2 (BMP-2), a potent osteoinductive growth factor. We hypothesized that collagen + DCN would increase BMP-2 retention over collagen alone due to DCN's ability to sequester TGF-ß growth factors. We further hypothesized that MVF would increase both early vascularization and subsequent BMP-2-mediated bone regeneration. Contrary to our hypothesis, BMP + MVF decreased the number of blood vessels relative to BMP alone and had no effect on bone healing. However, collagen + DCN was demonstrated to be a BMP-2 delivery vehicle capable of achieving bridging in the challenging composite defect model that is comparable to that achieved with a well-established alginate-based delivery system. STATEMENT OF SIGNIFICANCE: We have previously established a model of musculoskeletal trauma that exhibits impaired bone healing. For the first time, this work shows that the early revascularization response is also significantly, albeit modestly, impaired. A decorin-supplemented collagen hydrogel was used for the first time in vivo as a delivery vehicle for both a cell-based vascular therapeutic, MVF, and an osteoinductive growth factor, BMP-2. While MVF did not improve vascular volume or bone healing, collagen + DCN is a BMP-2 delivery vehicle capable of achieving bridging in the challenging composite defect model. Based on its support of robust angiogenesis in vitro, collagen + DCN may be extended for future use with other vascular therapeutics such as pre-formed vascular networks., (Copyright © 2019 Acta Materialia Inc. All rights reserved.)

Poly-lactic-co-glycolic acid (PLGA) microcarriers (0.8 ± 0.2 μm) have been fabricated with a load of 20 μg/g PLGA by an emulsion-based-proprietary technology to sustained deliver human bone morphogenetic protein 2 (hBMP2), a growth factor largely used for osteogenic induction. hBMP2 release profile, measured in vitro, showed a moderate "burst" release of 20% of the load in first 3 days, followed by a sustained release of 3% of the load along the following 21 days. PLGA microbeads loaded with fluorescent marker (8 mg/g PLGA ) and hydroxyapatite (30 mg/g PLGA ) were also fabricated and successfully dispersed within three-dimensional (3D) alginate scaffold (Ca-alginate 2% wt/wt) in a range between 50 and 200 mg/cm 3 ; the presence of microcarriers within the scaffold induced a variation of its stiffness between 0.03 and 0.06 MPa; whereas the scaffold surface area was monitored always in the range of 190-200 m 2 /g. Uniform microcarriers dispersion was obtained up to 200 mg/cm 3 ; higher loading values in the 3D scaffold produced large aggregates. The release data and the surface area were, then, used to simulate by finite element modeling the hBMP2 mass transfer within the 3D hydrogel bioengineered with stem cells, in dynamic and static cultivations. The simulation was developed with COMSOL Multiphysics® giving a good representation of hBMP2 mass balances along microbeads (bulk eroded) and on cell surface (cell binding). hBMP2 degradation rate was also taken into account in the simulations. hBMP2 concentration of 20 ng/cm 3 was set as a target because it has been described as the minimum effective value for stem cells stimulation versus the osteogenic phenotype. The sensitivity analysis suggested the best microbeads/cells ratio in the 3D microenvironment, along 21 days of cultivations in both static and dynamic cultivation (perfusion) conditions. The simulated formulation was so assembled experimentally using human mesenchymal stem cells and an improved scaffold stiffness up to 0.09 MPa (n = 3; p ≤ 0.01) was monitored after 21 days of cultivation; moreover a uniform extracellular matrix deposition within the 3D system was detected by Von Kossa staining, especially in dynamic conditions. The results indicated that the described tool can be useful for the design of 3D bioengineered microarchitecture by quantitative understanding., (© 2019 Wiley Periodicals, Inc.)

Large scale cortical allografts suffer from poor incorporation and healing and often end in graft failure 5-10 years after implantation. To reduce these failures we have developed a growth-factor loaded cortical allograft capable of delivering one or two factors with a degree of temporal control and precision that permits the early release of one growth factor followed by the later and more sustained release of the other. We have loaded vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2 (BMP-2), both critical components of bone formation and repair, onto cortical long bone allografts such that the VEGF is released first and followed shortly by BMP-2. Coated and factor-loaded allografts were placed into a critical sized rat femoral segmental defect and allowed to heal for either 4 or 8 weeks. Healing at each time point was compared to allografts loaded with only BMP-2 and allografts containing no growth factors. Results indicate statistically significant increases in new bone formation from 4 to 8 weeks around allografts loaded with both VEGF and BMP-2 over allografts with no growth factor, suggesting that factor-loaded polymer-coated allografts delivering multiple factors with temporal precision may provide a new off-the-shelf tool to the orthopedic surgeon for management of large-scale orthopedic bone defects. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1002-1010, 2019., (© 2018 Wiley Periodicals, Inc.)

Integrating proteins with chitosan (CS) can not only improve the biocompatibility of CS, but also enable the sustained release of proteins. The deacetylation degree (DD) and pH conditions are considered as the two most important factors affecting the interaction between CS and proteins. In this study, the effects of CS with different DDs and pH conditions on bone morphogenetic protein-2 (BMP-2) adsorption were evaluated using the molecular dynamics (MD) method. The results showed that the interaction energy first increased and then decreased, with the increase of the DD in the range of 0-100%; the strongest interaction was found between BMP-2 and 75% DD-CS surface. Furthermore, decreasing pH values favor the interaction between BMP-2 and CS surfaces. These results are useful to gain a better understanding of the interaction mechanism between polysaccharides and proteins and provide a meaningful reference for the application of CS with different DDs and pH conditions. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 769-779, 2019., (© 2018 Wiley Periodicals, Inc.)

We present a nanoengineered system for sustained and prolonged delivery of protein therapeutics, which has the potential to impact current orthopedic regeneration strategies. Specifically, we introduce two-dimensional nanosilicates with a high surface area and charged characteristics for delivery of active proteins for more than 30 days. The nanosilicates show high binding efficacy without altering the protein conformation and bioactivity. The released proteins are able to maintain high activity as demonstrated by enhanced differentiation of human mesenchymal stem cells at 10-fold lower concentration compared to the exogenous control. Utilizing the nanosilicates as a delivery vehicle could minimize the negative side effects observed because of the use of supraphysiological dosages of protein therapeutics for orthopedic regeneration strategies.

Impact Statement: 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.

Tissue engineering of bone requires the delivery of growth factors in a localized, sustained manner. Here, chitosan is used as polycation, while heparin and chondroitin sulfate are employed either as native or oxidized polyanions for formation of multilayers by layer-by-layer technique. The use of oxidized heparin and oxidized chondroitin sulfate permits additional stabilization by cross-linking through imine bond formation between amino groups of polycations and aldehydes of oxidized glycosaminoglycans (oGAGs). Since these multilayers are highly hydrophilic, adhesion of C2C12 myoblasts is improved either by the use of a specific 4 + 9 pH regime with native glycosaminoglycans or a terminal collagen I layer in case of oGAGs. Adhesion and proliferation studies with C2C12 myoblasts, seeded either on bone morphogenetic protein (BMP-2) loaded or non-loaded multilayers, show that intrinsic cross-linking in oGAG-based multilayers supports cell adhesion, spreading, proliferation, and subsequent cell differentiation into osteoblasts. This is related to higher thickness and roughness of multilayers made of oGAGs compared to their native counterparts studied by ellipsometry and atomic force microscopy. Taken together, oGAG multilayer systems provide stable surface coatings and are useful as biocompatible reservoirs for sustained release of BMP-2, paving the way for coating implants and scaffolds for repair and regeneration of bone., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Vertical bone augmentation of the jaws is required when the height of bone is insufficient at the site of dental implant placement. In this proof of concept study, we investigated the potential of a biphasic polycaprolactone construct combined with a hyaluronic acid based hydrogel loaded with recombinant human bone morphogenetic growth factor-2 (BMP-2) for vertical bone regeneration. The biphasic scaffold consisted of an outer shell manufactured by fused deposition modelling, mimicking native cortical bone and providing mechanical and space maintenance properties essential for bone formation. Within this shell, a 90% porous melt electrospun microfibrous mesh mimicking the architecture of cancellous bone was incorporated in order to facilitate hydrogel loading and subsequent osteogenesis and angiogenesis. The in vitro performances of the biphasic construct demonstrated that BMP-2 was released in a sustained manner over several weeks and that cell viability was maintained in the hydrogel over 21 days. qRT-PCR demonstrated the upregulation of bone markers such as osteopontin, osteocalcin and collagen 1A1 at day 3 and 14 in the constructs loaded with BMP2. In vivo assessment of the biphasic scaffold was performed using a dose of 30 μg of BMP-2 in a rabbit calvarial vertical bone augmentation model. The histology and micro-CT analysis of the elevated space demonstrated that the hydrogel and the presence of BMP-2 enabled bone formation. However, this was limited to the immediate vicinity of the calvarial bone. The amount of newly formed bone was relatively small which was likely due to poor vascularisation of the extraskeletal space. The utilisation of this biomimetic biphasic construct with excellent space maintenance properties can be of interest in dentistry although the in vivo model requires refinement to demonstrated appropriate efficacy., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Tissue engineering bones take great advantages in massive bone defect repairing; under the induction of growth factors, seed cells differentiate into osteoblasts, and the scaffold materials gradually degrade and are replaced with neogenetic bones, which simulates the actual pathophysiological process of bone regeneration. However, mechanism research is required and further developed to instruct elements selection and optimization. In the present study, we prepared vascular endothelial growth factor/bone morphogenetic protein-2- nanohydroxyapatite/collagen (VEGF/ BMP-2- nHAC/ PLGAs) scaffolds and inoculated mouse MC3T3-E1 preosteoblasts to detect osteogenic indexes and activation of related signaling pathways. The hypothesis is to create a three-dimensional environment that simulates bone defect repairing, and p38 mitogen-activated kinase (p38) inhibitor was applied and osterix shRNA was transferred into mouse MC3T3-E1 preosteoblasts to further investigate the molecular mechanism of crosstalk between BMP-2 and VEGF. Our results demonstrated the following: (1) BMP-2 and VEGF were sustainably released from PLGAs microspheres. (2) nHAC/PLGAs scaffold occupied a three-dimensional porous structure and has excellent physical properties. (3) MC3T3-E1 cells proliferated and differentiated well in the scaffold. (4) Osteogenic differentiation related factors expression of VEGF/BMP-2 loaded scaffold was obviously higher than that of other groups; p38 inhibitor SB203580 decreased the nucleus/cytoplasm ratio of osterix expression. To conclude, the active artificial bone we prepared could provide a favorable growth space for MC3T3-E1 cells, and osteogenesis and maturation reinforced by simultaneous VEGF and BMP-2 treatment may be mainly through the activation of the p38 MAPK pathway to promote nuclear translocation of osterix protein.

Bone morphogenetic proteins (BMP's) are vital for bone and cartilage formation, where bone morphogenetic protein-2 (BMP-2) is acknowledged as a growth factor in osteoblast differentiation. However, uncontrolled delivery may result in adverse clinical effects. In this study we investigated the possibility for longitudinal and non-invasive monitoring of implanted [ 125 I]BMP-2 retention and its relation to ossification at the site of implantation. A unilateral critically sized femoral defect was produced in the left limb of rats while the right femur was retained intact as a paired reference control. The defect was filled with a hyaluronan hydrogel with 25% hydroxyapatite alone (carrier control; n = 2) or combined with a mixture of [ 125 I]BMP-2 (150 μg/ml; n = 4). Bone formation was monitored using micro computed tomography (μCT) scans at 1, 3, 5, 7, 9 and 12 weeks. The retention of [ 125 I]BMP-2 was assessed with single photon emission computed tomography (SPECT), and the bone healing process was followed with sodium fluoride (Na 18 F) using positron emission tomography (PET) at day 3 and at week 2, 4, and 6. A rapid burst release of [ 125 I]BMP-2 was detected via SPECT. This was followed by a progressive increase in uptake levels of [ 18 F]fluoride depicted by PET imaging that was confirmed as bone formation via μCT. We propose that this functional, non-invasive imaging method allows tri-modal visualisation of the release of BMP-2 and the following in vivo response. We suggest that the potential of this novel technique could be considered for preclinical evaluation of novel smart materials on bone regeneration., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Local sustained delivery of bioactive molecules from biomaterials is a promising strategy to enhance bone regeneration. To optimize delivery vehicles for bone formation, the design characteristics are tailored with consequential effect on bone morphogenetic protein-2 (BMP-2) release and bone regeneration. Complying with the 3R principles (Replacement, Reduction, and Refinement), the growth factor release is often investigated in vitro using several buffers to mimic the in vivo physiological environment. However, this remains an unmet need. Therefore, this study investigates the in vitro-in vivo correlation (IVIVC) of BMP-2 release from complex delivery vehicles in several commonly used in vitro buffers: cell culture model, phosphate buffered saline, and a strong desorption buffer. The results from this study showed that the release environment affected the BMP-2 release profiles, creating distinct relationships between release versus time and differences in extent of release. According to the guidance set by the U.S. Food and Drug Administration (FDA), IVIVC resulted in level A internal predictability for individual composites. Since the IVIVC was influenced by the BMP-2 loading method and composite surface chemistry, the external predictive value of the IVIVCs was limited. These results show that the IVIVCs can be used for predicting the release of an individual composite. However, the models cannot be used for predicting in vivo release for different composite formulations since they lack external predictability. Potential confounding effects of drug type, delivery vehicle formulations, and application site should be added to the equation to develop one single IVIVC applicable for complex delivery vehicles. Altogether, these results imply that more sophisticated in vitro systems should be used in bone regeneration to accurately discriminate and predict in vivo BMP-2 release from different complex delivery vehicles.

In this study, an injectable and thermo-sensitive alginate/β-tricalcium phosphate hydrogel (TSAH/β-TCP) was prepared for aspirin release to a bone defect. Aspirin was dissolved into a mixture of poly(N-isopropylacrylamide) (PNIPAAm), an aminated alginate-g-PNIPAAm co-polymer, and β-TCP powders. Scanning electron microscopy showed that TSAH/β-TCP had an interconnected porous microstructure with a porosity of 86.78%. The cross-linked hydrogel released approximately 40% of the aspirin in the first 3 days and then slowly released the remainder. At a low concentration (≤100 μg/mL), aspirin did not promote cell proliferation, but enhanced the alkaline phosphatase activity, and osteocalcin (OCN) and collagen I expression of human bone marrow-derived mesenchymal stem cells. The TSAH/β-TCP/aspirin hydrogel was injected into the periosteum of the rat cranial bone, and its in vivo bone-forming ability was evaluated at 12 weeks. A bone morphogenetic protein 2 (BMP-2)-loaded TSAH/β-TCP hydrogel was injected as a control. Micro-computed tomography showed that the percentage of mineralized tissue in the TSAH/β-TCP/BMP-2 and TSAH/β-TCP/aspirin groups were similar and significantly higher than that in the TSAH/β-TCP group. Immunohistochemical staining showed considerable expression of OCN, especially in the TSAH/β-TCP/BMP-2 and TSAH/β-TCP/aspirin groups. These results suggest that the injectable TSAH/β-TCP/aspirin hydrogel has great potential for bone regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1739-1751, 2018., (© 2017 Wiley Periodicals, Inc.)

The bone-induction capacity of a porous biphasic calcium phosphate (pBCP) using heterotopic implantation in mouse (mHI-model) and its efficacy as substitute for autograft in mandibular critical-size defect in rabbit (rabMCSD-model) was investigated. In mHI-model, pBCP was implanted into the thigh muscles and bone formation was histomorphometrically and immunohistochemically evaluated. In rabMCSD-model, 13 mm bone defects were treated with pBCP or autograft and bone repair comparatively evaluated by radiographic and histomorphometric methods. In mHI-model, formed bone and immunolabeling for bone morphogenetic protein-2 and osteopontin were observed in 90% of pBCP implanted samples after 12 weeks. In rabMCSD-model neither statistically significant difference was found in newly formed bone between pBCP and autograft groups at 4 weeks (18.8 ± 5.5% vs 27.1 ± 5.6%), 8 weeks (22.3 ± 2.7% vs 26.2 ± 5.1), and 12 weeks (19.6 ± 4.7% vs 19.6 ± 2.3%). At 12 weeks, the stability and contour of the mandible were restored in both treatments. Near tooth remaining, pBCP particles were covered by small amount of mineralized tissue exhibiting perpendicular attachments of collagen fiber bundles with histological characteristic of acellular cementum. Within the limitations of this study, it was concluded that pBCP is osteoinductive and able to stimulate the new formation of bone and cementum-like tissues in rabMCSD-model, suggesting that it may be an alternative to treatment of large bone defect and in periodontal regenerative therapy. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1546-1557, 2018., (© 2017 Wiley Periodicals, Inc.)

Localization of recombinant human bone morphogenetic protein-2 (rhBMP-2) with continuous and effective osteogenic stimulation is still a great challenge in the field of bone regeneration. To achieve this aim, rhBMP-2 was tethered on chondroitin sulfate (CS)-functionalized calcium phosphate cement (CPC) scaffolds through specific noncovalent interactions. CS, one of the core glycosaminoglycans, was covalently conjugated onto CPC scaffolds with the assistance of polydopamine (PDA) and further immobilized rhBMP-2 in a biomimetic form. The CPC-PDA-CS scaffolds not only controlled the release kinetics and presentation state of rhBMP-2 but also effectively increased the expression levels of bone morphogenetic protein receptors (BMPRs) and enhanced the recognitions of the remaining rhBMP-2 to BMPRs. Strikingly, the rhBMP-2-loaded CPC-PDA-CS significantly promoted the cellular surface translocation of BMPRs (especially BMPR-IA). In vivo studies demonstrated that, compared with the rhBMP-2 upon CPC and CPC-PDA, the rhBMP-2 upon CPC-PDA-CS exhibited sustained release and induced high quality and more ectopic bone formation. Collectively, these results suggest that rhBMP-2 can be localized within CS-functionalized CPC scaffolds and exert continuous, long-term, and effective osteogenic stimulation. Thus, this work could provide new avenues in mimicking bone extracellular matrix microenvironment and localizing growth factor activity for enhanced bone regeneration., Statement of Significance: A bioinspired chondroitin sulfate (CS)-functionalized calcium phosphate cement (CPC) platform was developed to tether recombinant human bone morphogenetic protein-2 (rhBMP-2), which could exhibit continuous, long-term, and effective osteogenic stimulation in bone tissue engineering. Compared with rhBMP-2-loaded CPC, the rhBMP-2-loaded CPC-polydopamine-CS scaffolds induced higher expression of bone morphogenetic protein receptors (BMPRs), greater cellular surface translocation of bone morphogenetic protein receptor-IA, higher binding affinity of BMPRs/rhBMP-2, and thus higher activation of the drosophila gene mothers against decapentaplegic protein-1/5/8 (Smad1/5/8) and extracellular-regulated protein kinases-1/2 (ERK1/2) signaling. This work can provide new guidelines for the design of BMP-2-based bioactive materials for bone regeneration., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Graphene oxide (GO) attracts considerable attention for biomedical applications owing to its unique nanostructure and remarkable physicochemical characteristics. However, it is challenging to uniformly deposit GO on chemically inert Ti scaffolds, which have good biocompatibility and wide applications in bone engineering. In this study, a GO-functionalized Ti porous scaffold (GO/Ti scaffold) was prepared by depositing GO onto polydopamine (PDA) modified Ti scaffolds. The mussel-inspired PDA modification facilitated the interaction between GO and Ti surfaces, leading to a uniform coverage of GO on Ti scaffolds. BMP2 and vancomycin (Van) were separately encapsulated into gelatin microspheres (GelMS). Then, drug-containing GelMS were assembled on GO/Ti scaffolds and anchored by the functional groups of GO. The modified scaffold independently delivered multiple biomolecules with different physiochemical properties, without interfering with each other. Thus, the GO/Ti scaffold has the dual functions of inducing bone regeneration and preventing bacterial infection. In summary, this mussel-inspired GO/Ti hybrid scaffold combined the good mechanical properties of Ti scaffolds and the advantages of GO nanosheets. GO nanosheets with their unique nanostructure and functional groups, together with GelMS on Ti scaffolds, are suitable carriers for drug delivery and provide adhesive sites for cell adhesion and create nanostructured environments for bone regeneration.

Bone morphogenetic protein 2 (BMP-2) is a potent inducer of bone formation that is currently used in a limited number of clinical indications to treat extensive bone loss. Extending the field of applications of this molecule requires design of the delivery system to protect the protein from early degradation and allow a slow long-term release. This study describes the use of a non-polymer hydrogel, based on the self-assembly of small amphiphilic glycosyl-nucleolipids into micellar structures, as a new type of delivery system for BMP-2. BMP-2 was readily encapsulated in glycosyl-nucleosyl-fluorinated (GNF)-based gels and slowly released in vitro, while maintaining its osteogenic activity. When hydrogel pieces containing fluorophore-tagged BMP-2 were deposited onto a calvaria defect in mouse, the signal detected in living mice gradually decreased and was still detectable at 3 weeks. Gel-embedded protein promoted significant calvarial bone defect regeneration at 8 weeks after surgery. In contrast, when a solution of BMP-2 without hydrogel was injected into the defects, the fluorescence signal decreased rapidly and no significant bone formation was observed. The unique property of the GNF-based hydrogel as an injectable delivery system for low doses of BMP-2 was revealed in a subcutaneous model of ectopic bone formation. Injected BMP-2-laden GNF hydrogel blocks elicited the formation of cancellous bone, showing all the typical features of remodeling bone that contains bone marrow. These results show that this GNF-based hydrogel is an easy-to-use, efficient delivery system for BMP-2 and osteogenic material to support bone regeneration.

To investigate the effect of sustained bone morphogenetic protein-2 (BMP-2) release kinetics on bone formation in poly(propylene fumarate) (PPF) scaffolds, different poly(lactic-co-glycolic acid) (PLGA) microspheres were used as delivery vehicles. All PPF scaffolds had the same 75% porous structure, while the degradation rate of the embedded PLGA microspheres was changed to tailor BMP-2 release by varying the lactic-to-glycolic acid (L:G) ratio in the copolymer. Four PLGA microsphere formulations with 50/50, 65/35, 75/25, and 85/15 L:G ratios and varying in vivo degradation rates were fabricated. The in vitro and in vivo BMP-2 release kinetics were determined by analyzing radiolabeled 125 I-BMP-2. Biological activity of released BMP-2 was tested using a W20-17 cell culture model in vitro and a subcutaneous rat model in vivo. Corresponding outcome parameters were defined as capacity to increase the in vitro AP activity in weekly consecutive cell cultures over 14 weeks and the in vivo bone formation after 7 and 14 weeks. The PLGA/PPF composites showed similar biological activity and BMP-2 release profiles in vitro. In vivo, PPF/PLGA 85:15 composite released significantly less BMP-2 per time point in the first weeks. Micro-CT and histological analysis after 7 and 14 weeks of implantation showed bone formation, which significantly increased over time for all composites. No significant differences were seen between the composites. Overall, the results of this study show that small differences in BMP-2 sustained release had no significant effect on BMP-2 osteogenic efficacy in PPF/PLGA composites. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 477-487, 2018., (© 2017 Wiley Periodicals, Inc.)

A promising strategy to accelerate bone generation is to deliver a combination of certain growth factors to the integration site via a controlled spatial and temporal delivery mode. Here, a composite hydrogel incorporating poly(lactide-co-glycolide) (PLGA) microspheres was accordingly prepared to load and deliver the osteogenic rhBMP-2 and angiogenic rhVEGF 165 in the required manner. In addition, 2-N,6-O-sulphated chitosan (26SCS), which is a synergetic factor of growth factors, was incorporated in the composite hydrogel as well. The system showed a similar release behaviour of the two growth factors regardless of 26SCS inclusion. RhBMP-2 loaded in PLGA microspheres showed a sustained release over a period of 2 weeks, whereas rhVEGF 165 loaded in hydrogel eluted almost completely from the hydrogel over the first 16 days. Both growth factors retained their efficacy, as quantified with relevant in vitro assays. Moreover, an enhanced cell response was achieved upon the delivery of dual growth factors, compared to that obtained with a single factor. Furthermore, in the presence of 26SCS, the system revealed significantly upregulated alkaline phosphatase activity, human umbilical vein endothelial cell proliferation, sprouting, nitric oxide secretion, and angiogenic gene expression. This study highlighted that the composite hydrogel incorporated with 26SCS appears to constitute a promising approach to deliver multiple growth factors. From our findings, we could also conclude that rhBMP-2 can promote angiogenesis and that the mechanism is worthy of further study in subsequent research.

PLGA (poly(lactic-co-glycolic) acid)-PEG (polyethylene glycol)-PLGA synthesis conditions have an impact on the physicochemical features of the copolymer and its usefulness as biomaterial. This study reports on an analysis of the composition and structural properties of PLGA-PEG-PLGA copolymers applying a variety of analytical techniques. Viscoelastic properties and particularly the temperature-responsive behavior of PLGA-PEG-PLGA showed a marked dependence on copolymer structural features. Physicochemical and biological properties, such as bioadhesion, biocompatibility and cell viability, of the raw copolymers and their gels were also evaluated. The most promising copolymer was chosen to formulate the osteoinductive protein bone morphogenetic protein-2 ( 125 I-BMP-2), and the ability of its gels to sustain the release both in vitro and in vivo was monitored in situ using a gamma counter. In vitro diffusion studies were carried out using a bioinspired set-up that included a biorelevant receptor medium. In vivo release tests after implantation in a critical-size calvarial defect model showed an important burst, but then the release fitted well to the square-root kinetics. Importantly, the release rate constants recorded in vitro and in vivo matched each other suggesting close in vitro-in vivo correlation. Overall, the information gathered opens new perspectives in the biomedical application of these temperature-sensitive materials., (Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Synthetic polymeric scaffolds are commonly used in bone tissue engineering (BTE) due to their biocompatibility and adequate mechanical properties. However, their hydrophobicity and the lack of specific cell recognition sites confined their practical application. In this study, to improve the cell seeding efficiency and osteoinductivity, an injectable thermo-sensitive chitosan hydrogel (CSG) was incorporated into a 3D-printed poly(ε-caprolactone) (PCL) scaffold to form a hybrid scaffold. To demonstrate the feasibility of this hybrid system for BTE application, rabbit bone marrow mesenchymal stem cells (BMMSCs) and bone morphogenetic protein-2 (BMP-2) were encapsulated in CSG. Pure PCL scaffolds were used as controls. Cell proliferation and viability were investigated. Osteogenic gene expressions of BMMSCs in various scaffolds were determined with reverse transcription polymerase chain reaction (RT-PCR). Growth factor releasing profile and mechanical tests were performed. CCK-8 assay confirmed greater cell retention and proliferation in chitosan and hybrid groups. Confocal microscopy showed even distribution of cells in the hybrid system. After 2-week osteogenic culture in vitro, BMMSCs in hybrid and chitosan scaffolds showed stronger osteogenesis and bone-matrix formation. To conclude, chitosan/PCL hybrid scaffolds are a favorable platform for BTE due to its capacity to carry cells and drugs, and excellent mechanical strength.

Different materials have been used for vital dental pulp treatment. Preferably a pulp capping agent should show appropriate biological performance, excellent handling properties, and a good imaging contrast. These features can be delivered into a single material through the combination of therapeutic and diagnostic agents (i.e. theranostic). Calcium phosphate based composites (CPCs) are potentially ideal candidate for pulp treatment, although poor imaging contrast and poor dentino-inductive properties are limiting their clinical use. In this study, a theranostic dental pulp capping agent was developed. First, imaging properties of the CPC were improved by using a core-shell structured dual contrast agent (csDCA) consisting of superparamagnetic iron oxide (SPIO) and colloidal gold, as MRI and CT contrast agent respectively. Second, biological properties were implemented by using a dentinogenic factor (i.e. bone morphogenetic protein 2, BMP-2). The obtained CPC/csDCA/BMP-2 composite was tested in vivo, as direct pulp capping agent, in a male Habsi goat incisor model. Our outcomes showed no relevant alteration of the handling and mechanical properties (e.g. setting time, injectability, and compressive strength) by the incorporation of csDCA particles. In vivo results proved MRI contrast enhancement up to 7weeks. Incisors treated with BMP-2 showed improved tertiary dentin deposition as well as faster cement degradation as measured by µCT assessment. In conclusion, the presented theranostic agent matches the imaging and regenerative requirements for pulp capping applications., Statement of Significance: In this study, we combined diagnostic and therapeutic agents in order to developed a theranostic pulp capping agent with enhanced MRI and CT contrast and improved dentin regeneration ability. In our study we cover all the steps from material preparation, mechanical and in vitro characterization, to in vivo study in a goat dental model. To the best of our knowledge, this is the first time that a theranostic pulp capping material have been developed and tested in an in vivo animal model. Our promising results in term of imaging contrast enhancement and of induction of new dentin formation, open a new scenario in the development of innovative dental materials., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Biologic augmentation for rotator cuff repair is a challenging treatment in patients with chronic large, massive, and irreparable rotator cuff injuries. Particularly, the use of an extracellular matrix (ECM) patch such as dermal tissue offered improved biomechanical properties in previous studies. Cytokines induce cell chemotaxis, proliferation, matrix synthesis, and cell differentiation. Moreover, osteoinductive growth factors such as bone morphogenetic protein-2 (BMP-2) affect the formation of new bone and fibrocartilage in lesions. However, the effects of using a dermal patch in combination with BMP-2 have not been evaluated to date, although many researchers have recognized the importance thereof. In this study, rhBMP-2-coated dermal patch (1 cm × 2 cm) isolated from human cadaveric donor was inserted in a rabbit model of chronic rotator cuff injury for in vivo evaluation. Bone mineral density and biomechanical strength were tested and histological and histomorphometric analyses were performed. The results showed that insertion of an rhBMP-2-coated acellular dermal patch not only significantly ameliorated new bone formation, it also improved biomechanical properties such as ultimate tensile strength. Thus, the use of this combination may improve the chronic rotator cuff injury-healing rate and clinical outcomes after rotator cuff repair. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1840-1846, 2017., (© 2016 Wiley Periodicals, Inc.)

Bone morphogenetic protein-2 (BMP-2) is an osteoinductive growth factor used clinically to induce bone regeneration and fusion. Some complications associated with BMP-2 treatment have been attributed to rapid release of BMP-2 from conventional collagen scaffolds, motivating the development of tunable sustained-release strategies. We incorporated BMP-2-binding heparin microparticles (HMPs) into a hydrogel scaffold to improve spatiotemporal control of BMP-2 delivery to large bone defects. HMPs pre-loaded with BMP-2 were mixed into alginate hydrogels and compared to hydrogels containing BMP-2 alone. BMP-2 release from scaffolds in vitro, BMP-2 retention within injury sites in vivo, and bone regeneration in a critically sized femoral defect were evaluated. Compared to hydrogel delivery alone, BMP-2-loaded HMPs reduced BMP-2 release in vitro and increased early BMP-2 retention in the bone defect. BMP-2-loaded HMPs induced bone formation at both ectopic and orthotopic sites; however, the volume of induced bone was lower for defects treated with BMP-2-loaded HMPs compared to hydrogel delivery. To better understand the effect of HMPs on BMP-2 release kinetics, a computational model was developed to predict BMP-2 release from constructs in vivo. The model suggested that HMPs limited BMP-2 release into surrounding tissues, and that changing the HMP density could modulate BMP-2 release. Taken together, these experimental and computational results suggest the importance of achieving a balance of BMP-2 retention within the bone defect and BMP-2 release into surrounding soft tissues. HMP delivery of BMP-2 may provide a method of tuning BMP-2 release in vivo that can be further investigated to improve current methods of bone regeneration., Statement of Significance: The development of effective biomaterials for sustained protein delivery is a crucial component of tissue engineering strategies. However, in most applications, including bone repair, the optimal balance between protein presentation in the injury site and protein release into the surrounding tissues is unknown. Herein, we introduced heparin microparticles (HMPs) into a tissue engineered construct to increase in vivo retention of bone morphogenetic protein-2 (BMP-2) and enhance healing in femoral defects. Although HMPs induced bone regeneration, no increase in bone volume was observed, leading to further experimental and computational analysis of the effect of HMP-BMP-2 interactions on protein retention and release. Ultimately, this work provides insight into designing tunable protein-material interactions and their implications for controlling BMP-2 delivery., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The performance of bone tissue engineering scaffolds can be assessed through cell responses to scaffolds, including cell attachment, infiltration, morphogenesis, proliferation, differentiation, etc, which are determined or heavily influenced by the composition, structure, mechanical properties, and biological properties (e.g. osteoconductivity and osteoinductivity) of scaffolds. Although some promising 3D printing techniques such as fused deposition modeling and selective laser sintering could be employed to produce biodegradable bone tissue engineering scaffolds with customized shapes and tailored interconnected pores, effective methods for fabricating scaffolds with well-designed hierarchical porous structure (both interconnected macropores and surface micropores) and tunable osteoconductivity/osteoinductivity still need to be developed. In this investigation, a novel cryogenic 3D printing technique was investigated and developed for producing hierarchical porous and recombinant human bone morphogenetic protein-2 (rhBMP-2)-loaded calcium phosphate (Ca-P) nanoparticle/poly(L-lactic acid) nanocomposite scaffolds, in which the Ca-P nanoparticle-incorporated scaffold layer and rhBMP-2-encapsulated scaffold layer were deposited alternatingly using different types of emulsions as printing inks. The mechanical properties of the as-printed scaffolds were comparable to those of human cancellous bone. Sustained releases of Ca 2+ ions and rhBMP-2 were achieved and the biological activity of rhBMP-2 was well-preserved. Scaffolds with a desirable hierarchical porous structure and dual delivery of Ca 2+ ions and rhBMP-2 exhibited superior performance in directing the behaviors of human bone marrow-derived mesenchymal stem cells and caused improved cell viability, attachment, proliferation, and osteogenic differentiation, which has suggested their great potential for bone tissue engineering.

Bone reconstruction is a challenging issue in the regeneration of surgically removed bone and disease-related bone defects. Although bone morphogenetic protein-2 (BMP-2) has received considerable attention as a bone regeneration inducer, a high dose of BMP-2 is typically required due to its short life-time under in vivo conditions. We have proposed a method to enhance the osteogenetic differentiation ability of BMP-2 in vitro that is based on supramolecular polyelectrolyte complexation with sulfonated polyrotaxanes (PRXs) consisting of sulfopropyl ether (SPE)-modified α-cyclodextrins threaded along a poly(ethylene glycol) chain capped with terminal bulky stopper molecules. In this study, we evaluated the in vivo bone regeneration ability of the SPE-PRX/BMP-2 complexes in a mouse calvarial defect model in comparison to free BMP-2 and heparin/BMP-2 complexes. The regenerated bone area was determined by X-ray computed microtomography, and the mice implanted with sulfonated PRX/BMP-2 complexes exhibited rapid and significant bone regeneration compared to those implanted with free BMP-2 and heparin/BMP-2 complexes. We concluded that the sulfonated PRX/BMP-2 complexes are a promising candidate for clinical bone regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1355-1363, 2017., (© 2017 Wiley Periodicals, Inc.)

Bone morphogenetic protein-2 (BMP-2) has unique bone regeneration property. The powerful osteoinductive nature makes it considered as second line of therapy in nonunion bone defect. A large number of carriers and delivery systems made up of different materials have been investigated for controlled and sustained release of BMP-2. The delivery systems are in the form of hydrogel, microsphere, nanoparticles, and fibers. The carriers used for the delivery are made up of metals, ceramics, polymers, and composites. Implantation of these protein-loaded carrier leads to cell adhesion, degradation which eventually releases the drug/protein at site specific. But, problems like ectopic growth, lesser protein delivery, inactivation of the protein are reported in the available carrier systems. Therefore, it is need of an hour to modify the available carrier systems as well as explore other biomaterials with desired properties. In this review, all the reported carrier systems made of metals, ceramics, polymers, composites are evaluated in terms of their processing conditions, loading capacity and release pattern of BMP-2. Along with these biomaterials, the attempts of protein modification by adding some functional group to BMP-2 or extracting functional peptides from the protein to achieve the desired effect, is also evaluated. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 904-925, 2017., (© 2016 Wiley Periodicals, Inc.)

Biodegradable PLA-PEG-PLA block copolymers were synthesized with desired backbone structures and molecular weights using PEG20000. Rectangular scaffolds were prepared by freeze drying with or without using NaCl particles. Bone morphogenetic protein (BMP)-2 was loaded to the matrix after the scaffold formation for sustained release while vascular endothelial growth factor (VEGF) was loaded within the pores with gelatin solution. VEGF release was quite fast and almost 60% of it was released in 2 d. However, sequential - sustained released was observed for BMP-2 in the following few months. Corporation of VEGF/BMP-2 couple into the scaffolds increased the cell adhesion and proliferation. Neither significant cytotoxicity nor apoptosis/necrosis were observed.

Growth factor-based therapeutics using bone morphogenetic protein 2 (BMP-2) presents a promising strategy to reconstruct craniofacial bone defects such as mandible. However, clinical applications require supraphysiological BMP doses that often increase inappropriate adipogenesis, resulting in well-documented, cyst-like bone formation. Here we reported a novel complementary strategy to enhance osteogenesis and mandibular bone repair by using small-molecule phenamil that has been shown to be a strong activator of BMP signaling. Phenamil synergistically induced osteogenic differentiation of human bone marrow mesenchymal stem cells with BMP-2 while suppressing their adipogenic differentiation induced by BMP-2 in vitro. The observed pro-osteogenic and antiadipogenic activity of phenamil was mediated by expression of tribbles homolog 3 (Trb3) that enhanced BMP-smad signaling and inhibited expression of peroxisome proliferator-activated receptor gamma (PPARγ), a master regulator of adipogenesis. The synergistic effect of BMP-2+phenamil on bone regeneration was further confirmed in a critical-sized rat mandibular bone defect by implanting polymer scaffolds designed to slowly release the therapeutic molecules. These findings indicate a new complementary osteoinductive strategy to improve clinical efficacy and safety of current BMP-based therapeutics.

Chronic back pain is related to intervertebral disc (IVD) degeneration and dogs are employed as animal models to develop growth factor- and cell-based regenerative treatments. In this respect, the differential effects of transforming growth factor beta-1 (TGF-β 1 ) and bone morphogenetic protein-2 (BMP2) on canine and human chondrocyte-like cells (CLCs) derived from the nucleus pulposus of degenerated IVDs were studied. Human and canine CLCs were cultured in 3D microaggregates in basal culture medium supplemented with/without TGF-β 1 (10 ng/mL) or BMP2 (100 or 250 ng/mL). Both TGF-β 1 and BMP2 increased proliferation and glycosaminoglycan (GAG) deposition of human and canine CLCs. TGF-β 1 induced collagen type I deposition and fibrotic (re)differentiation, whereas BMP2 induced more collagen type II deposition. In dogs, TGF-β 1 induced Smad1 and Smad2 signaling, whereas in humans, it only tended to induce Smad2 signaling. BMP2 supplementation increased Smad1 signaling in both species. This altogether indicates that Smad1 signaling was associated with collagen type II production, whereas Smad2 signaling was associated with fibrotic CLC (re)differentiation. As a step toward preclinical translation, treatment with BMP2 alone and combined with mesenchymal stromal cells (MSCs) was further investigated. Canine male CLCs were seeded in albumin-based hydrogels with/without female bone marrow-derived MSCs (50:50) in basal or 250 ng/mL BMP2-supplemented culture medium. Although the results indicate that a sufficient amount of MSCs survived the culture period, total GAG production was not increased and GAG production per cell was even decreased by the addition of MSCs, implying that MSCs did not exert additive regenerative effects on the CLCs.

Combination of tissue-engineered bone scaffolds with cell-adhesive, osteoconductive, or osteoinductive biomolecules is a critical strategy to improve their properties that significantly influence cellular behaviors, such as adhesion, proliferation, and differentiation, which is beneficial for critical-sized bone defects repairing. However, the traditional surface modification techniques, such as physical adsorption, coating, and plasma treatment, et al, have great limitations for immobilization of bioactive molecules due to undesirable controlled delivery performance or overly complex multistep procedures. In this study, we functionalized the chitosan/hydroxyapatite (CS/HA) biomimetic composite scaffold for controlled delivery of BMP2-derived peptide (P24) by the chemical grafting modification technique: firstly, P24 was conjugated with a thiolated chitosan, chitosan-4-thiobutylamidine (CS-TBA); secondly, the resultant CS-P24 was then combined with HA to prepare CS-P24/HA scaffolds. The effect of CS-P24/HA scaffolds on bone regeneration was evaluated, along with the underlying biological mechanisms responsible in vitro and in vivo . In vitro , the controlled and sustained release of bioactive P24 could last up to 90 days, furthermore, the release profiles of CS-5%P24/HA and CS-10%P24/HA were linear and could be fitted according to zero-order kinetic model (R 2 =0.9929; R 2 =0.9757); P24 on the scaffold significantly promoted cell adhesion, proliferation, osteodifferentiation, and mineralization with synergistic effects. Bone marrow stromal cells (BMSCs) revealed spindle-shaped surface morphology, indicating the CS-P24/HA scaffolds supported cell adhesion and possessed a high proliferation rate that varied according to the P24 concentration levels. Furthermore, mRNA levels for OCN, Runx2, and collagen I were significantly up-regulated on CS-P24/HA scaffolds compared with cells grown on CS/HA scaffolds in vitro ( p < 0.05). Similarly, the BMSCs exhibited a higher ALP expression and calcium deposition level on CS-P24/HA scaffolds compared with CS/HA scaffolds ( p < 0.05). In vivo , osteoinductive studies revealed a significantly higher ectopic osteogenesis level of CS-10%P24/HA scaffolds in rat dorsal muscle pockets compared with that of CS/HA scaffolds. Finally, CS-P24/HA scaffolds showed superior performance in the reconstruction of rat calvarial bone defects. This novel CS-P24/HA scaffold is deemed a strong potential candidate for the repair of bone defects in human bone tissue engineering., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Objective: To construct a novel biomimetic calcium phosphate (BioCaP) scaffold loaded with bone morphogenetic protein-2 (BMP-2), and to investigate its role in the osteogenesis of human adipose-derived stem cells (hASCs) in vitro and in vivo., Methods: The BioCaP scaffold coprecipitated with BMP-2 (BMP-2-BioCaP) was constructed in this study. Field emission scanning electron microscopy (SEM) was used to analyze the morphology of the surfaces. The release kinetics was measured to evaluate the slow-release characteristics in vitro. BMP-2-BioCaP was immersed in proliferation medium (PM) or osteogenic medium (OM), respectively. The supernatants were collected and used to culture hASCs in vitro. Cell numbers were determined using the cell-counting kit-8 (CCK-8) to assess the cell proliferation. After 7 and 14 days, alkaline phosphatase (ALP) staining and quantification were performed to test the activity of ALP. After 14 and 21 days, the calcification deposition was determined by alizarin red S (ARS) staining and quantification. The expressions of the osteoblast-related genes were tested on day 4 and day 14. In the in vivo study, 6 nude mice were used and implanted subcutaneously into the back of the nude mice for 4 groups: (1) BioCaP scaffold only, (2) BioCaP scaffold+hASCs, (3) BMP-2-BioCaP scaffold, (4) BMP-2-BioCaP scaffold+hASCs (test group). After 4 weeks of implantation, hematoxylin-eosin (HE) staining was performed to evaluate the in vivo osteogenesis of hASCs., Results: SEM observations showed that BioCaP and BMP-2-BioCaP scaffold were entirely composed of straight, plate-like and sharp-edged crystal units, and the length of the crystal units varied between 5 and 10 μm. Release kinetics analysis demonstrated that BMP-2 incorporated with BioCaP could be released at certain concentration and last for more than 21 days, and the accumulative protein release could reach 20%. CCK-8 assays showed that cell proliferation was not significantly affected by BMP-2-BioCaP. ALP activity was higher by the induction of OM+BMP-2-BioCaP than of the other groups (P<0.01). More mineralization deposition and more expressions of osteoblast-related genes such as Runt-related transcription factor 2 (RUNX2), ALP, osteopontin (OPN) and osteocalcin (OC) were determined in the OM+BMP-2-BioCaP group at different time points (P<0.01). HE staining showed that, in the test group and BMP-2-BioCaP scaffold group, the extracellular matrix (ECM) with eosinophilic staining were observed around hASCs, and newly-formed bone-like tissues could be found in ECM around the scaffold materials. Moreover, compared with the BMP-2-BioCaP scaffold group, more bone-like tissues could be observed in ECM with typical structure of bone tissue in the test groups. No obvious positive results were found in the other groups., Conclusion: BMP-2-BioCaP scaffold could achieve slow-release of BMP-2 and promote the osteogenic differentiation of hASCs in vitro and in vivo. The novel tissue-engineered bone composed of hASCs and BMP-2-BioCaPis promising for the repair of bone defect.

In this study we report on the effectiveness of click chemistry-enhanced zirconium dioxide (ZrO2-3) for the immobilization of biomolecules, and the enhancement of osteoblastic differentiation of MC3T3-E1 cells by bone morphogenetic protein-2 (BMP-2) immobilized on ZrO2-6. The surfaces of ZrO2-1 through 6 were characterized by scanning electron microscopy (SEM), static contact angles, and X-ray photoelectron spectroscopy (XPS) measurements. The results from these tests indicated that ZrO2-1 was successfully surface-modified via click chemistry (ZrO2-3). Through quantitative analysis of heparin immobilized on ZrO2-5, we found that ZrO2-3 was a useful tool for immobilizing biomolecules such as heparin. Release tests of BMP-2 from ZrO2-6 showed well-controlled release kinetics over a period of 28 days. MC3T3-E1 cell proliferation tests indicated that ZrO2-6 was highly biocompatible with these cells. Through In Vitro tests such as alkaline phosphatase (ALP) activity, calcium deposition, and real-time polymerase chain reaction (real-time PCR), we found that ZrO2-6 was a useful tool for enhancing osteoblastic differentiation of MC3T3-E1 cells.

Objective: The chitosan microspheres encapsulated with bone morphogenetic protein-2 (BMP-2) were prepared by the emulsion cross-linking method. Then the chitosan microspheres were loaded in the ceramic bovine bone (CBB) to achieve the drug delivery system., Methods: The chemical structure and surface morphology of the drug delivery system were investigated by Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscope (SEM) observation. Characterization preserved the loading capacity and encapsulation efficiency of the BMP-2. The dynamic immersion method was used to examine the in vitro release characteristic of BMP-2., Results: The chitosan microspheres were successfully encapsulated BMP-2 by cross-linking method. The microspheres were micron-sized (5.982 μm) and spherical in shape with smooth surface morphology. From the release experiments, it was found that 5 mg chitosan/BMP-2 soaked for 21 days with a gradual release of BMP-2. The concentration of BMP-2 was (239.1±20.0) mg/L on Day 21. The in vitro experiment showed that this novel drug delivery system could accelerate MC3T3-E1 cells proliferation and osteogenic differentiation., Conclusion: The drug delivery system achieves the biological function of BMP-2 and sustaining slow release in bone repair parts. Also it can provide the basis for repair of bone tissue defect treatment and the selection of bone tissue engineering scaffolds.

Tissue engineering promises therapies ideal for treating conventional large bone injuries and defects. In the present study, we developed a novel Si-HA scaffold loaded with a synthetic BMP-2-related peptide, P28, and tested its ability to repair a critical-sized calvarial defect. We created a calvarial defect (5 mm in diameter) in the parietal bone of 32 rats and implanted one of the following biomaterials: No implant (control), Si-HA, P28/Si-HA, or rhBMP-2/Si-HA. As assessed by micro CT imaging and histological evaluations, the P28/Si-HA scaffold promoted bone recovery to a similar degree as the rhBMP-2/Si-HA scaffold. In addition, both P28/Si-HA and rhBMP-2/Si-HA promoted recovery better than Si-HA alone. The novel P28/Si-HA scaffold might represent a promising biomaterial for future bone tissue engineering applications. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1874-1882, 2016., (© 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Sustained release of bone morphogenetic protein (BMP)-2 by heparin-contained biomaterials is advantageous for bone tissue regeneration using low-dose BMP-2. However, its effect with high-dose BMP-2 is still unclear and should be clarified considering the clinical use of a high dose of BMP-2 in spine and oral surgery. This study aimed to evaluate the efficacy of a heparin-conjugated collagen sponge (HCS) with high-dose BMP-2 delivery by investigating in vivo initial osteogenic regulation and bone healing over 12 weeks in comparison with that of an absorbable collagen sponge (ACS). The in vitro BMP-2 release profile in the HCS exhibited a lower burst followed by a sustained release of BMP-2, whereas that of the ACS showed an initial burst phase only. As a result of a lower burst, the HCS-BMP group showed higher expression of bone-forming/resorbing markers and enhanced activation of osteoclasts than the ACS-BMP group within the scaffold of defect after 7 days, which is presumed to be because of retention of relatively higher amounts of BMP-2. However, the surrounding calvariae were less resorbed in the HCS-BMP group, compared with the aggressive resorptive response in the ACS-BMP group. Microcomputed tomography and histology revealed that HCS-BMP guided more effective bone regeneration of central defect over time inducing minor ossification at the defect exterior, whereas ACS-BMP exhibited excessive ossification at the defect exterior. These results showed that HCS-mediated BMP-2 delivery at a high dose has advantages over ACS, including less early resorption of surrounding bone tissue and higher efficacy in compact bone regeneration over a longer period, highlighting a clinical feasibility of this technology.

Recently, 3D printing as effective technology has been highlighted in the biomedical field. Previously, a porous hydroxyapatite (HA) scaffold with the biocompatibility and osteoconductivity has been developed by this method. However, its osteoinductivity is limited. The main purpose of this study was to improve it by the introduction of recombinant human bone morphogenetic protein-2 (rhBMP-2). This scaffold was developed by coating rhBMP-2-delivery microspheres with collagen. These synthesized scaffolds were characterized by Scanning Electron Microscopy (SEM), a delivery test in vitro, cell culture, and the experiments in vivo by a Micro-computed tomography (μCT) scan and histological evaluation of VanGieson staining. SEM results indicated the surface of scaffolds were more fit for the adhesion of hMSCs to coat collagen/rhBMP-2 microspheres. Biphasic release of rhBMP-2 could continue for more than 21 days, and keep its osteoinductivity to induce osteogenic differentiation of hMSCs in vitro. In addition, the experiments in vivo showed that the scaffold had a good bone regeneration capacity. These findings demonstrate that the HA/Collagen/Chitosan Microspheres system can simultaneously achieve localized long-term controlled release of rhBMP-2 and bone regeneration, which provides a promising route for improving the treatment of bone defects., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: To compare the direct osteogenic effect between placental growth factor-2 (PlGF-2) and bone morphogenic protein-2 (BMP-2)., Methods: Three groups of PlGF-2/BMP-2-loaded heparin-N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (HTCC) nanocomplexes were prepared: those with 0.5 μg PlGF-2; with 1.0 μg BMP-2; and with 0.5 μg PlGF-2 combined with 1.0 μg BMP-2. The loading efficiencies and release profiles of these growth factors (GFs) in this nanocomplex system were quantified using enzyme-linked immunosorbent assay, their biological activities were evaluated using cell counting kit-8, cell morphology, and cell number counting assays, and their osteogenic activities were quantified using alkaline phosphatase and Alizarin Red S staining assays., Results: The loading efficiencies were more than 99% for the nanocomplexes loaded with just PlGF-2 and for those loaded with both PlGF-2 and BMP-2. For the nanocomplex loaded with just BMP-2, the loading efficiency was more than 97%. About 83%-84% of PlGF-2 and 89%-91% of BMP-2 were stably retained on the nanocomplexes for at least 21 days. In in vitro biological assays, PlGF-2 exhibited osteogenic effects comparable to those of BMP-2 despite its dose in the experiments being lower than that of BMP-2. Moreover, the results implied that heparin-based nanocomplexes encapsulating two GFs have enhanced potential in the enhancement of osteoblast function., Conclusion: PlGF-2-loaded heparin-HTCC nanocomplexes may constitute a promising system for bone regeneration. Moreover, the dual delivery of PlGF-2 and BMP-2 appears to have greater potential in bone tissue regeneration than the delivery of either GFs alone.

Bone morphogenetic protein 2 (BMP-2) has been known for decades as a strong osteoinductive factor and for clinical applications is combined solely with collagen as carrier material. The growing concerns regarding side effects and the importance of BMP-2 in several developmental and physiological processes have raised the need to improve the design of materials by controlling BMP-2 presentation. Inspired by the natural cell environment, new material surfaces have been engineered and tailored to provide both physical and chemical cues that regulate BMP-2 activity. Here we describe surfaces designed to present BMP-2 to cells in a spatially and temporally controlled manner. This is achieved by trapping BMP-2 using physicochemical interactions, either covalently grafted or combined with other extracellular matrix components. In the near future, we anticipate that material science and biology will integrate and further develop tools for in vitro studies and potentially bring some of them toward in vivo applications., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Heparin has a high affinity for bone morphogenetic protein-2 (BMP-2), which is a key growth factor in bone regeneration. The aim of this study was to investigate how the rate of release of BMP-2 was affected when adsorbed to nanosized hydroxyapatite (HAP) particles functionalized with heparin by different methods. Heparin was attached to the surface of HAP, either via adsorption or covalent coupling, via a 3-aminopropyltriethoxysilane (APTES) layer. The chemical composition of the particles was evaluated using X-ray photoelectron spectroscopy and elemental microanalysis, revealing that the heparin grafting densities achieved were dependent on the curing temperature used in the fabrication of APTES-modified HAP. Comparable amounts of heparin were attached via both covalent coupling and adsorption to the APTES-modified particles, but characterization of the particle surfaces by zeta potential and Brunauer-Emmett-Teller measurements indicated that the conformation of the heparin on the surface was dependent on the method of attachment, which in turn affected the stability of heparin on the surface. The release of BMP-2 from the particles after 7 days in phosphate-buffered saline found that 31% of the loaded BMP-2 was released from the APTES-modified particles with heparin covalently attached, compared to 16% from the APTES-modified particles with the heparin adsorbed. Moreover, when heparin was adsorbed onto pure HAP, it was found that the BMP-2 released after 7 days was 5% (similar to that from unmodified HAP). This illustrates that by altering the mode of attachment of heparin to HAP the release profile and total release of BMP-2 can be manipulated. Importantly, the BMP-2 released from all the heparin particle types was found by the SMAD 1/5/8 phosphorylation assay to be biologically active.

In this study, three systems containing BMP-2 were fabricated, including two electrospun sandwich-like-systems of PLGA 75:25 and PLGA 50:50 and a microsphere system of PLGA 50:50 to be implanted in a critical size defect in rat calvaria. The in vivo BMP-2 release profiles of the three systems were similar. The total dose was released during the first two weeks. To evaluate the nano and microstructure of the regenerated bone a multi-technique analysis was used, including stereo microscope, X-Ray; AFM, micro-CT, and histological analyses. The progression of bone regeneration was followed at 4, 8, and 12 weeks after the microsphere system implantation whereas the two electrospun systems were evaluated at fixed 12 weeks. All the techniques applied showed high bone regeneration. The average values of bone volume density, bone mineral density, Young's modulus, and the percent of bone repair were ∼70% of the values of the native bone. Besides, SEM-EDX analysis indicated that the main chemical elements in the new bone were oxygen, calcium, and phosphorus in a ratio similar to that of native bone. In comparison, the micro-CT may provide an alternative to histology for the evaluation of bone formation at the defect size., (© 2015 Wiley Periodicals, Inc.)

Preserving bioactivity of bone morphogenetic protein 2 (BMP-2) still remains a challenge in protein-based therapy. It is not known how Ca(2+) released from extracellular matrix or existing in physiological environment influences bioactivity in situ till now. Here, effects of extracellular Ca(2+) on conformation and osteogenic bioactivity of recombinant human BMP-2 (rhBMP-2) were investigated systematically. In vitro results indicated that Ca(2+) could bind rhBMP-2 rapidly and had no obvious effect on cell behaviors. Low concentration of Ca(2+) (0.18 mM) enhanced rhBMP-2-induced osteogenic differentiation, while high Ca(2+) concentration (>1.80 mM) exerted negative effect. In vivo ectopic bone formation exhibited similar trend. Further studies by circular dichroism spectroscopy, fluorescence spectroscopy, together with cell culture experiments revealed at low concentration, weak interaction of Ca(2+) and rhBMP(-)2 slightly increased β-sheet/-turn content and facilitated recognition of BMP-2 and BMPRIA. But, high Ca(2+) concentration (>1.8 mM) induced formation of Ca-rhBMP-2 complex and markedly increased content of β-sheet/-turn, which led to inhibition binding of rhBMP-2 and BMPRIA and thus suppression of downstream Smad1/5/8, ERK1/2 and p38 mitogen-associated protein kinase signaling pathways. Our work suggests osteogenic bioactivity of BMP-2 can be adjusted via extracellular Ca(2+), which should provide guide and assist for development of BMP-2-based materials for bone regeneration.