pH Stimuli-Responsive, Rapidly Self-healable Coatings Enhanced the Corrosion Resistance and Osteogenic Differentiation of Mg-1Ca Osteoimplant.

Mg-Ca alloys have emerged as a promising research direction for biomedical implants in the orthopedic field. However, their clinical use is deterred by their fast corrosion rate. In this work, a pH stimuli-responsive silk-halloysite (HNT)/phytic acid (PA) self-healing coating (Silk-HNT/PA) is constructed to slow down the corrosion rate of Mg-1Ca alloy and its cell viability and osteogenic differentiation ability are enhanced. The Silk-HNT/PA coating exhibits appealing active corrosion protection, by eliciting pH-triggerable self-healing effects, while simultaneously affording superior biocompatibility and osteogenic differentiation ability. Moreover, in vivo studies by histological analysis also demonstrate better osseointegration for the Silk-HNT/PA coated Mg-1Ca alloy. In summary, the Silk-HNT/PA coating in the present study has great potential in enhancing the biomedical utility of Mg alloys.

Catecholamine-Copper Redox as a Basis for Site-Specific Single-Step Functionalization of Material Surfaces.

Realization of robust and facile surface functionalization processes is critical to biomaterials and biotechnology yet remains a challenge. Here, we report a new chemical approach that enables operationally simple and site-specific surface functionalization. The mechanism involves a catechol-copper redox chemistry, where the oxidative polymerization of an alkynyl catecholamine reduces Cu(II) to Cu(I), which in situ catalyzes a click reaction with azide-containing molecules of interest (MOIs). This process enables drop-coating and grafting of two- and three-dimensional solid surfaces in a single operation using as small as sub-microliter volumes. Generalizability of the method is shown for immobilizing MOIs of diverse structure and chemical or biological activity. Biological applications in anti-biofouling, cellular adhesion, scaffold seeding, and tissue regeneration are demonstrated, in which the activities or fates of cells are site-specifically manipulated. This work advances surface chemistry by integrating simplicity and precision with multipurpose surface functionalization.

Mussel bioinspired morphosynthesis of substrate anchored core-shell silver self-assemblies with multifunctionality for bioapplications.

Core-shell nanoparticles (CSNs) have numerous intriguing properties for advanced device applications, while it remains challenging to directly grow them from a solid substrate. Here, we report a simple mussel-bioinspired solid chemistry strategy for in-situ synthesis of CSNs that are substrate anchored and morphologically tunable for wide-ranging biotechnological applications. Briefly, silver titanate was hydrothermally grown on template titanium and subjected to reaction with mussel-derived dopamine. The synergistic reactivity between silver titanate and dopamine prompted nanosilver/polydopamine (nAg/PD) CSNs to spontaneously assemble and grow on substrate. These CSNs possessed reaction time-dependent dimensions and morphologies, which were related to differing physiochemical properties and biological behaviors. Specifically, the CSNs-modified substrates demonstrated enhanced protein affinity and durable radical scavenging properties. In addition, they manifested remarkable yet robust release-killing and anti-biofilm activities against pathogenic Staphylococcus aureus bacteria. More delightedly, the surface-engineered substrates guaranteed the victory of the anti-infective battle of osteoblastic cells during cell/bacteria coculture, promising applications in implantable medical devices. The adaptability of this strategy was demonstrated by modifying complicated 3D-printed macroporous tissue engineering scaffolds. Intriguingly, the CSNs-modified scaffolds exhibited photothermal performances that bode well for phototherapy. To sum, our strategy combines the simplicity of synthesis modality, the controllability of core-shell silver structures, and the versatility of material functions. The resulting assemblies can enrich the library of nAg-based core-shell engineered nanomaterials.

Controllable biodegradation and enhanced osseointegration of ZrO2-nanofilm coated Zn-Li alloy: In vitro and in vivo studies.

Zinc and its alloys have emerged as a new research direction of biodegradable metals (BMs) due to the significant physiological functions of Zn2+ ions in human body. However, low inhibitory concentration threshold value to cause cytotoxicity by Zn2+ ions during in vitro study and delayed osseointegration in vivo are two key flaws for the bulk Zn-based BMs. To combat these issues, we constructed a barrier layer of ZrO2 nanofilm on the surface of Zn-0.1(wt.%) Li alloy via atomic layer deposition (ALD). A decreased release of Zn2+ ions accompanied with accelerated release of Li+ ions was observed on account of galvanic coupling between the coating compositions and Zn-0.1Li alloy substrate. Cytocompatibility assay reflected that ZrO2 nanofilm coated Zn-0.1Li alloy exhibited improved cell adhesion and viability. Histological analysis also demonstrated better in vivo osseointegration for the ZrO2 nanofilm coated Zn-0.1Li alloy. Hence, the present study elucidated that the ALD of ZrO2 nanofilm on Zn-based BMs can effectively promote osseointegration and control their biodegradation behavior. STATEMENT OF SIGNIFICANCE: Zn-Li binary alloy was reported recently to be the promising biodegradable metals with ultimate tensile strength over 500 MPa, yet the low inhibitory concentration threshold value to cause cytotoxicity by Zn2+ ions is the obstacle needed to be overcome. As a pilot study, a systematic investigation on the ZrO2 nanofilm coated Zn-Li alloy, prepared by atomic layer deposition (ALD) technique, was conducted in the present study, which involved in the formation process, in vitro and in vivo degradation behavior as well as biocompatibility evaluation. We found a controllable corrosion rate and better in vivo osseointegration can be achieved by ZrO2 nanofilm coating on Zn-Li alloy, which provides new insight into the surface modification on biodegradable Zn alloys for usage within bone.

Osteogenic and pH stimuli-responsive self-healing coating on biomedical Mg-1Ca alloy.

Various coatings have been used to slow down the corrosion rate of biomedical magnesium alloys. However, these coatings usually act only as passive barriers. It is much more desirable to endow such coatings with active, biocorrosion-responsive self-repairing capacity. In the present work, a self-healing coating system (denoted as "silk-PA") was constructed in the form of a sandwich architecture of fluoride precoating (bottom), silk-phytic acid (PA) coating (middle), and silk fibroin coating (top). Here, PA was loaded in the middle coating as a corrosion inhibitor by harnessing its strong chelating ability toward dissolving Mg2+ and Ca2+ ions. The self-healing property was evaluated by scratch and SVET tests, and the corrosion resistance was evaluated by in vitro immersion and electrochemical measurements. The results showed that the silk-PA manifested intriguing self-healing capacity with pH responsiveness, hence profiting the corrosion resistance of the Mg-1Ca alloy. The biocompatibility and osteogenic activity of the coating system were further evaluated using MC3T3-E1 cells, and it demonstrated favorable responses in multiple cellular behaviors, i.e., adherence, spreading, proliferation, and differentiation. These findings open new opportunities in the study of self-healing coatings for protection against corrosion in biomedical Mg alloys. STATEMENT OF SIGNIFICANCE: In the present study, a self-healing coating system with pH stimuli-responsiveness and osteogenic activity was fabricated on Mg-1Ca alloy by integrating a silk fibroin barrier coating, a silk fibrin/phytic acid composite coating, and a fluoride precoating. This coating system demonstrated interesting self-healing ability as compared to traditional surface modification layers. Furthermore, the self-healing ability enhanced the corrosion resistance of biomedical magnesium alloys, while effective compositions of the coating system endowed the substrate with osteogenic activity. This work provides some new insights into smart surface modification for biomedical Mg alloys.

Triple-Bioinspired Burying/Crosslinking Interfacial Coassembly Strategy for Layer-by-Layer Construction of Robust Functional Bioceramic Self-Coatings for Osteointegration Applications.

Coating bioceramics of inherent bioactivity onto biometallic implants is a straightforward yet promising solution to address poor osteointegration of the latter. One step further, it would be a nontrivial accomplishment to develop a mild, cheap, and universal route to firmly stabilizing, in principle, any ceramics onto any implant substrate, while imparting expectedly versatile biofunctional performances. Herein, we describe a triple-bioinspired burying/cross-linking interfacial coassembly strategy for enabling such ceramic coatings, which ingeniously fuses bioinspiration from sea rocks (burying assisted particle immobilization), marine mussels (universal adhesion and versatile chemical reactivity), and reef-building oysters (cross-linking rendered cohesion). Specifically, surface functionalized, aqueous dispersed ceramic particles were buried within an substrate-anchored organic matrix of polyelectrolyte multilayers (i.e., (poly(ether imide) (PEI)/poly(sodium-p-styrenesulfonate) (PSS)) n), through a new inorganic-organic hybrid layer-by-layer (LBL) coassembly scheme wherein mussel (oyster) inspired adhesive (cohesive) chemistries were exquisitely orchestrated. As a conceptual demonstration, bioactive baghdadite (Ca3ZrSi2O9) was synthesized as model ceramics, with which we constructed on medical titanium robust, biomimetic, and cross-linkable LBL self-assemblies harnessing the said strategy. Intimate substrate contacts and well-defined buried inorganic-organic interfaces were evidently seen, together with good structural and chemical stabilities, especially after cross-linking. Sustained bioactive ion releasing and appreciable biomineralization activity were confirmed in vitro. Subsequently, biological performances of the assemblies were systematically investigated with respect to surface hydrophilicity, protein adsorption, and osteoblast functions. Additionally, nanosilver deposition, which imparted the surfaces with added antibacterial potencies, was used to exemplify the strategy's versatility in allowing multifunctionality. What's more, the flexibility of our approach was testified through modifying clinically relevant complicated 3D porous scaffolds. Overall, our strategy basically met the design expectations, boding well for future medical adoption. This study offers the promise of an alternative broadly useful avenue to bioactive and functional surface design of bone implants. It may also provide insights into other multiple-bioinspired materials/interfaces for biological and other applications.

A pH-sensitive self-healing coating for biodegradable magnesium implants.

Self-healing coatings have attracted attention on surface modification of magnesium alloys, as it can recover the barrier ability of the coatings from corrosion attack. Nevertheless, previous works on this aspect are not suitable for biomedical magnesium alloys owing to the lack of biocompatibility. In this study, we fabricated a self-healing coating on biomedical Mg-1Ca alloy by compositing silk fibroin and K3PO4. PO43- ions act as corrosion inhibitor, while K3+ ions help to regulate the secondary structures of silk fibroin. The scratch test, scanning vibrating electrode technique (SVET), and electrochemical impedance spectroscopy (EIS) provide comprehensive results, confirming the pH-sensitive self-healing capacity of the composite coating. Moreover, cells' (MC3T3-E1) multiple responses including spreading, adhesion, proliferation, and differentiation illustrate the preferable biocompatibility as well as the osteogenic activity of the coating. These primary findings might open new opportunities in the exploration of self-healing coatings on biomedical magnesium alloys. STATEMENT OF SIGNIFICANCE: Biomedical magnesium alloys surface modifications have been studied for years, which however the biomedical self-healing coatings were rarely involved. In this work, silk fibroin and phosphate (K3PO4) were composited to fabricate coating on biomedical magnesium alloys. The coating not only owned the self-healing ability with pH sensitivity, but also endowed the substrate preferable corrosion resistance as well as osteogenic activity. This work gives a new insight into surface modification for biomedical Mg alloys.

Endowing polyetheretherketone with synergistic bactericidal effects and improved osteogenic ability.

Biomedical associated infections (BAI) are difficult to treat and may even lead to amputation and death, especially after the emergence of drug-resistant bacteria. The aim of this study was to harness the potential synergistic effects of multiple bactericidal agents to endow polyetheretherketone (PEEK) with the ability of achieving full eradication of planktonic and adherent bacteria while maintaining acceptable biocompatibility. In this work, a mussel inspired, silver nanoparticles (AgNPs) incorporated silk fibroin (SF)/gentamicin sulfate (GS) coating was constructed upon porous PEEK surface. The obtained coating greatly enhanced the bactericidal efficiency to Gram-positive bacteria and Gram-negative bacteria. The number of bacteria survived in the culture medium after treated with this coating was 106 fold lower than that survived after treated with PEEK sample, while the number of viable bacteria adhered to this coating was 105 lower than that adhered to PEEK sample. Furthermore, release of Ag+ and GS increased with decreasing pH, indicating great potential of this coating to be a "smart" bacteria-triggered self-defensive coating. Meanwhile, this functional coating shows favorable cytocompatibility and osteogenic ability. The mechanism behind this dual function is also partially revealed. Expectedly, this "smart" dual function coating can give a promise for PEEK to become a solution to increasingly deteriorated BAI. STATEMENT OF SIGNIFICANCE: In this study, a mussel inspired, silver nanoparticles (AgNPs) incorporated silk fibroin (SF)/gentamicin sulfate (GS) coating was constructed upon porous polyetheretherketone (PEEK) surface. This design was aimed to provide a solution to the increasingly deteriorated biomedical associated infections (BAI). Actually, this design endowed PEEK with dual function: bacteria-triggered synergistic bactericidal effect and improved osteogenic ability. The combination of silver and GS exhibited synergistic bacteria killing effect on both Gram-positive and Gram-negative bacteria, which showed 106 times higher in releasing-killing and 105 times higher in anti-adhesion than that of untreated PEEK. Furthermore, release of bactericidal agents increased with decreasing pH, indicating great potential of this coating to be a bacteria-triggered self-defensive coating. More interestingly, this study revealed the mechanism of synergistic effect between silver and GS.

Novel pH-responsive tobramycin-embedded micelles in nanostructured multilayer-coatings of chitosan/heparin with efficient and sustained antibacterial properties.

To endow orthopaedic implants with satisfactory antibacterial properties, the design and development of antibiotic coating on the surface of implants is highly desired. In this work a novel and facile strategy was developed to form pH-responsive layer-by-layer (LbL) films implanted with polymeric micelles as nano-vehicles loaded with charge-weak antibiotic drugs, enabling high drug loading efficiency. Negatively charged tobramycin (Tob)-embeded heparin miscells (HET) and positively charged chitosan (CHT) were exploited as a pH-responsive LBL multilayer building block, respectively. The formation mechanism and pH-stimulated release behavior of the Tob-contained heparin micelles were studied. The characterization on the morphologies, chemical compositions and hydrophilicity of the modified surface confirmed the successuful deposition of the Tob-loaded CHT/HET multilayers coatings on the polydopamine-modified Ti surface. The drug release profiles displayed fast release at pH 7.4 and slow release after exposure to weakly acidic environments. Antibacterial tests indicated that the Tob-embed CHT/HET nanostructured multilayers not only strongly inhibited initial bacterial adhesion, but also disruptted biofilm formation. Particularly, this functional coatings showed "long-term antibacterial" pattern in acid condition. Meanwhile, MC3T3 cells showed acceptable adhesion, spread and proliferation on the multilayer coatings in cytocompatible studies. In a word, these multilayer coatings incorporated with a wide variety of antibiotics show promisiong applications in preventing postoperative infection and resolving unmet clinical need.

Effect of vanadium released from micro-arc oxidized porous Ti6Al4V on biocompatibility in orthopedic applications.

MAO-treated porous Ti6Al4V holds enormous potential for use in orthopedic implants due to their excellent biocompatibility and favourable mechanical strength. However, the effects on the V ion accumulation and release following the MAO-treated Ti6Al4V remain undetermined. The aim of the present study was to assess the effects of Vanadium on biocompatibility. In this study, the surface features and chemical compositions were characterized by scanning electron microscopy (SEM), energy-dispersive X-ray spectrometry (EDS) and X-ray photoelectron spectroscopy (XPS). The ion release of Ti, Al and V was quantitatively measured by inductively-coupled plasma mass spectroscopy (ICP-MS) after immersion in Hanks' solution. To probe the mechanism of V release, the corrosion resistance of porous Ti6Al4V before and after the MAO process was evaluated by electrochemical tests. Thereafter, the effects on the biocompatibility were tested in vitro by cell culture assays and then in vivo by subcutaneous embedment. Finally, the bone tissue response and in vivo release profile of V ions were characterized by intra-osseous implantation. Therefore, this study suggests that the effect of V released from MAO-treated porous Ti6Al4V on biocompatibility and application safety is small and preventable.

Bioinspired and Biomimetic AgNPs/Gentamicin-Embedded Silk Fibroin Coatings for Robust Antibacterial and Osteogenetic Applications.

With the progressively increasing demand for orthopedic Ti implants, the balance between two primary complications restricting implant applications is needed to be solved: the lack of bone tissue integration and biomedical device-associated infections (BAI), where emergence of multiresistance bacteria make it worse. Notably, a combination of silver nanoparticles (AgNPs) and a kind of antibiotic can synergistically inhibit bacterial growth, where a low concentration of AgNPs has been confirmed to promote the proliferation and osteogenesis of osteoblasts. In this work, we built AgNPs/gentamicin (Gen)-embedded silk fibroin (SF)-based biomimetic coatings on orthopedic titanium by a facile dipping-drying circular process and with the assistance of polydopamine (PD). Ag+ was reduced to AgNPs by SF under ultraviolet (UV) irradiation, and then they were detected by transmission electron microscope (TEM) images and UV-visible (UV-vis) analyses. Intriguingly, the addition of Gen highly improved the reduction efficiency of Ag+. The antibacterial efficiency of SF-based coatings was examined by challenging them with pathogenic Staphylococcus aureus (S. aureus) bacteria which produced biofilms, and consequently, we found that low concentration loading, durable release of Ag+ (28 days), and 10-fold improvement of antibacterial efficiency were achieved for our novel AgNPs- and Gen-embeded silk fibroin coatings. In bacteria and a cells cocultured system, AgNPs/Gen-embedded coatings strongly inhibited adhesion and proliferation of S. aureus, simultaneously improving cell adhesion and growth. To investigate cytocompatibility and osteogenic potential, different coatings were cultured with MC3T3 cells; AgNPs/Gen-embedded coatings showed generally acceptable biocompatibility (cell adhesion, proliferation, and viability) and accelerated osteoblast maturation (alkaline phosphatase production, matrix secretion, and calcification). Expectantly, this novel biofunctional coating will have promising applications in orthopedic and dental titanium implants thanks to its excellently antibacterial, biocompatible, and osteogenic activities.

Bioinspired anchoring AgNPs onto micro-nanoporous TiO2 orthopedic coatings: Trap-killing of bacteria, surface-regulated osteoblast functions and host responses.

The therapeutic applications of silver nanoparticles (AgNPs) against biomedical device-associated infections (BAI), by local delivery, are encountered with risks of detachment, instability and nanotoxicity in physiological milieus. To firmly anchor AgNPs onto modified biomaterial surfaces through tight physicochemical interactions would potentially relieve these concerns. Herein, we present a strategy for hierarchical TiO2/Ag coating, in an attempt to endow medical titanium (Ti) with anticorrosion and antibacterial properties whilst maintaining normal biological functions. In brief, by harnessing the adhesion and reactivity of bioinspired polydopamine, silver nanoparticles were easily immobilized onto peripheral surface and incorporated into interior cavity of a micro/nanoporous TiO2 ceramic coating in situ grown from template Ti. The resulting coating protected the substrate well from corrosion and gave a sustained release of Ag(+) up to 28 d. An interesting germicidal effect, termed "trap-killing", was observed against Staphylococcus aureus strain. The multiple osteoblast responses, i.e. adherence, spreading, proliferation, and differentiation, were retained normal or promoted, via a putative surface-initiated self-regulation mechanism. After subcutaneous implantation for a month, the coated specimens elicited minimal, comparable inflammatory responses relative to the control. Moreover, this simple and safe functionalization strategy manifested a good degree of flexibility towards three-dimensional sophisticated objects. Expectedly, it can become a prospective bench to bedside solution to current challenges facing orthopedics.

Tailored Surface Treatment of 3D Printed Porous Ti6Al4V by Microarc Oxidation for Enhanced Osseointegration via Optimized Bone In-Growth Patterns and Interlocked Bone/Implant Interface.

3D printed porous titanium (Ti) holds enormous potential for load-bearing orthopedic applications. Although the 3D printing technique has good control over the macro-sturctures of porous Ti, the surface properties that affect tissue response are beyond its control, adding the need for tailored surface treatment to improve its osseointegration capacity. Here, the one step microarc oxidation (MAO) process was applied to a 3D printed porous Ti6Al4V (Ti64) scaffold to endow the scaffold with a homogeneous layer of microporous TiO2 and significant amounts of amorphous calcium-phosphate. Following the treatment, the porous Ti64 scaffolds exhibited a drastically improved apatite forming ability, cyto-compatibility, and alkaline phosphatase activity. In vivo test in a rabbit model showed that the bone in-growth at the untreated scaffold was in a pattern of distance osteogenesis by which bone formed only at the periphery of the scaffold. In contrast, the bone in-growth at the MAO-treated scaffold exhibited a pattern of contact osteogenesis by which bone formed in situ on the entire surface of the scaffold. This pattern of bone in-growth significantly increased bone formation both in and around the scaffold possibly through enhancement of bone formation and disruption of bone remodeling. Moreover, the implant surface of the MAO-treated scaffold interlocked with the bone tissues through the fabricated microporous topographies to generate a stronger bone/implant interface. The increased osteoinetegration strength was further proven by a push out test. MAO exhibits a high efficiency in the enhancement of osteointegration of porous Ti64 via optimizing the patterns of bone in-growth and bone/implant interlocking. Therefore, post-treatment of 3D printed porous Ti64 with MAO technology might open up several possibilities for the development of bioactive customized implants in orthopedic applications.

Additively Manufactured Macroporous Titanium with Silver-Releasing Micro-/Nanoporous Surface for Multipurpose Infection Control and Bone Repair - A Proof of Concept.

Restoring large-scale bone defects, where osteogenesis is slow while infections lurk, with biomaterials represents a formidable challenge in orthopedic clinics. Here, we propose a scaffold-based multipurpose anti-infection and bone repairing strategy to meet such restorative needs. To do this, personalized multifunctional titanium meshes were produced through an advanced additive manufacturing process and dual "TiO2-poly(dopamine)/Ag (nano)" post modifications, yielding macroporous constructs with micro-/nanoporous walls and nanosilver bullets immobilized/embedded therein. Ultrahigh loading capacity and durable release of Ag+ were accomplished. The scaffolds were active against planktonic/adherent bacteria (Gram-negative and positive) for up to 12 weeks. Additionally, they not only defended themselves from biofilm colonization but also helped destroy existing biofilms, especially in combination with antibiotics. Further, the osteoblasts/bacteria coculture study displayed that the engineered surfaces aided MG-63 cells to combat bacterial invasion. Meanwhile, the scaffolds elicited generally acceptable biocompatibility (cell adhesion, proliferation, and viability) and hastened osteoblast differentiation and maturation (alkaline phosphatase production, matrix secretion, and calcification), by synergy of micro-/nanoscale topological cues and bioactive catecholamine chemistry. Although done ex vivo, these studies reveal that our three-in-one strategy (infection prophylaxis, infection fighting, and bone repair) has great potential to simultaneously prevent/combat infections and bridge defected bone. This work provides new thoughts to the use of enabling technologies to design biomaterials that resolve unmet clinical needs.

Polydopamine-induced nanocomposite Ag/CaP coatings on the surface of titania nanotubes for antibacterial and osteointegration functions.

The initial implant-associated infections and post aseptic loosening are the major obstacles for the clinical applications of titanium-based dental and orthopedic implants. To tackle these issues, the implant surface is engineered to possess combined osteointegration and antibacterial properties. Therefore, a mussel-inspired novel nano silver/calcium phosphate (CaP) composite coating was prepared on anodized Ti, in the expectation of its surface maintaining preferable biological performance and possessing long-term antibacterial ability. This approach involves three steps: (i) the anodic oxidation of Ti to enable it to have a TiO2 nanotubular (TNT) surface structure, (ii) the self-polymerization of dopamine on TNT and the reduction of Ag and (iii) the modification of the Ag nanoparticles using polydopamine and further being immersed in SBF for the biomimetic mineralization of CaP. The surface morphology and microstructure of this novel coating was fully characterized. The Ag/CaP coatings displayed obvious antibacterial effects to S. aureus bacteria and relatively good in vitro cytocompatibility to MG63 cells. Compared with the pristine Ti, the cells cultured on the coated Ti showed enhanced ALP activities.

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.