Our objective is to reveal the molecular mechanism of the anti-inflammatory action of low-molecular-weight heparin (LMWH) based on its influence on the activity of two key cytokines, IFNγ and IL-6. The mechanism of heparin binding to IFNγ and IL-6 and the resulting inhibition of their activity were studied by means of extensive molecular-dynamics simulations. The effect of LMWH on IFNγ signalling inside stimulated WISH cells was investigated by measuring its antiproliferative activity and the translocation of phosphorylated STAT1 in the nucleus. We found that LMWH binds with high affinity to IFNγ and is able to fully inhibit the interaction with its cellular receptor. It also influences the biological activity of IL-6 by binding to either IL-6 or IL-6/IL-6Rα, thus preventing the formation of the IL-6/IL-6Rα/gp130 signalling complex. These findings shed light on the molecular mechanism of the anti-inflammatory action of LMWH and underpin its ability to influence favourably conditions characterised by overexpression of these two cytokines. Such conditions are not only associated with autoimmune diseases, but also with inflammatory processes, in particular with COVID-19. Our results put forward heparin as a promising means for the prevention and suppression of severe CRS and encourage further investigations on its applicability as an anti-inflammatory agent.

Heparin has been proven to enhance bone resorption and induce bone loss. Since osteoclasts play a pivotal role in bone resorption, the effect of heparin on osteoclastogenesis needs to be clarified. Since osteocytes are the key modulator during osteoclastogenesis, we evaluated heparin's effect on osteoclastogenesis in vitro by co-culturing an osteocyte cell line (MLO-Y4) and pre-osteoclasts (RAW264.7). In this co-culture system, heparin enhanced osteoclastogenesis and osteoclastic bone resorption while having no influence on the production of RANKL (receptor activator of NFκB ligand), M-CSF (macrophage colony-stimulating factor), and OPG (osteoprotegerin), which are three main regulatory factors derived from osteocytes. According to previous studies, heparin could bind specifically to OPG and inhibit its activity, so we hypothesized that this might be a possible mechanism of heparin activity. To test this hypothesis, osteoclastogenesis was induced using recombinant RANKL or MLO-Y4 supernatant. We found that heparin has no effect on RANKL-induced osteoclastogenesis (contains no OPG). However, after incubation with OPG, the capacity of MLO-Y4 supernatant for supporting osteoclast formation was increased. This effect disappeared after OPG was neutralized and reappeared after OPG was replenished. These results strongly suggest that heparin promotes osteocyte-modulated osteoclastogenesis in vitro, at least partially, through inhibiting OPG activity.

Ischemic stroke is the most common type of stroke and thrombolytic therapy is the only approved treatment. However, current thrombolytic therapy with tissue plasminogen activator (tPA) is often hampered by the increased risk of hemorrhage. Plasmin, a direct fibrinolytic, has a significantly superior hemostatic safety profile; however, if injected intravenously it becomes rapidly inactivated by anti-plasmin. Nanoformulations have been shown to increase drug stability and half-life and hence could be applied to increase the plasmin therapeutic efficacy. Here in this paper, we report a novel heparin and arginine-based plasmin nanoformulation that exhibits increased plasmin stability and efficacy. In vitro studies revealed significant plasmin stability in the presence of anti-plasmin and efficient fibrinolytic activity. In addition, these particles showed no significant toxicity or oxidative stress effects in human brain microvascular endothelial cells, and no significant blood brain barrier permeability. Further, in a mouse photothrombotic stroke model, plasmin nanoparticles exhibited significant efficacy in reducing stroke volume without overt intracerebral hemorrhage (ICH) compared to free plasmin treatment. The study shows the potential of a plasmin nanoformulation in ischemic stroke therapy.

Heparin is a polysaccharide expressed in animal connective tissue-type mast cells. Owing to the special pentasaccharide sequence, heparin specifically binds to antithrombin (AT) and increases the inhibitory activity of AT towards coagulation enzymes. Heparin isolated from porcine intestinal mucosa has an average molecular weight of 15 kDa, while heparins recovered from rat skin and the peritoneal cavity were 60-100 kDa and can be fragmented by the endo-glucuronidase heparanase in vitro. In this study, we have examined heparin isolated from in vitro matured fetal skin mast cells (FSMC) and peritoneal cavity mast cells (PCMC) collected from wildtype (WT), heparanase knockout (Hpa-KO), and heparanase overexpressing (Hpa-tg) mice. The metabolically 35S-labeled heparin products from the mast cells of WT, Hpa-KO, and Hpa-tg mice were compared and analyzed for molecular size and AT-binding activity. The results show that PCMC produced heparins with a size similar to heparin from porcine intestinal mast cells, whilst FSMC produced much longer chains. As expected, heparanase overexpression resulted in the generation of smaller fragments in both cell types, while heparins recovered from heparanase knockout cells were slightly longer than heparin from WT cells. Unexpectedly, we found that heparanase expression affected the production of total glycosaminoglycans (GAGs) and the proportion between heparin and other GAGs but essentially had no effect on heparin catabolism.

The cell-cell/cell-matrix interactions between myoblasts and their extracellular microenvironment have been shown to play a crucial role in the regulation of in vitro myogenic differentiation and in vivo skeletal muscle regeneration. In this study, by harnessing the heparin-mimicking polymer, poly(sodium-4-styrenesulfonate) (PSS), which has a negatively charged surface, we engineered an in vitro cell culture platform for the purpose of recapitulating in vivo muscle atrophy-like phenotypes. Our initial findings showed that heparin-mimicking moieties inhibited the fusion of mononucleated myoblasts into multinucleated myotubes, as indicated by the decreased gene and protein expression levels of myogenic factors, myotube fusion-related markers, and focal adhesion kinase (FAK). We further elucidated the underlying molecular mechanism via transcriptome analyses, observing that the insulin/PI3K/mTOR and Wnt signaling pathways were significantly downregulated by heparin-mimicking moieties through the inhibition of FAK/Cav3. Taken together, the easy-to-adapt heparin-mimicking polymer-based in vitro cell culture platform could be an attractive platform for potential applications in drug screening, providing clear readouts of changes in insulin/PI3K/mTOR and Wnt signaling pathways.

Previous studies reported on the broad-spectrum antiviral function of heparin. Here we investigated the antiviral function of magnesium-modified heparin and found that modified heparin displayed a significantly enhanced antiviral function against human adenovirus (HAdV) in immortalized and primary cells. Nuclear magnetic resonance analyses revealed a conformational change of heparin when complexed with magnesium. To broadly explore this discovery, we tested the antiviral function of modified heparin against herpes simplex virus type 1 (HSV-1) and found that the replication of HSV-1 was even further decreased compared to aciclovir. Moreover, we investigated the antiviral effect against the new severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and measured a 55-fold decreased viral load in the supernatant of infected cells associated with a 38-fold decrease in virus growth. The advantage of our modified heparin is an increased antiviral effect compared to regular heparin.

Chemokines are critically involved in controlling directed leukocyte migration. Spatiotemporal secretion together with local retention processes establish and maintain local chemokine gradients that guide directional cell migration. Extracellular matrix proteins, particularly glycosaminoglycans (GAGs), locally retain chemokines through electrochemical interactions. The two chemokines CCL19 and CCL21 guide CCR7-expressing leukocytes, such as antigen-bearing dendritic cells and T lymphocytes, to draining lymph nodes to initiate adaptive immune responses. CCL21-in contrast to CCL19-is characterized by a unique extended C-terminus composed of highly charged residues to facilitate interactions with GAGs. Notably, both chemokines can trigger common, but also ligand-biased signaling through the same receptor. The underlying molecular mechanism of ligand-biased CCR7 signaling is poorly understood. Using a series of naturally occurring chemokine variants in combination with newly designed site-specific chemokine mutants, we herein assessed CCR7 signaling, as well as GAG interactions. We demonstrate that the charged chemokine C-terminus does not fully confer CCL21-biased CCR7 signaling. Besides the positively charged C-terminus, CCL21 also possesses specific BBXB motifs comprising basic amino acids. We show that CCL21 variants where individual BBXB motifs are mutated retain their capability to trigger G-protein-dependent CCR7 signaling, but lose their ability to interact with heparin. Moreover, we show that heparin specifically interacts with CCL21, but not with CCL19, and thereby competes with ligand-binding to CCR7 and prevents signaling. Hence, we provide evidence that soluble heparin, but not the other GAGs, complexes with CCL21 to define CCR7 signaling in a ligand-dependent manner.

Blood sampling with different anticoagulants alters matrix metalloproteinase (MMP-) 9 expression, thus influencing its concentration and diagnostic validity. Here, we aimed to evaluate the effects of different anticoagulants on MMP-9 regulation. MMP-9 expression was assessed in response to ethylenediaminetetraacetic acid, citrate, and high-/low-molecular-weight heparin (HMWH, LMWH) in co-culture experiments using THP-1, Jurkat, and HT cells (representing monocytes, T, and B cells). Triple and double cell line co-culture experiments revealed that HMWH treatment of THP-1 and Jurkat led to a significant MMP-9 induction, whereas other anticoagulants and cell type combinations had no effect. Supernatant of HMWH-treated Jurkat cells also induced MMP-9 in THP-1 suggesting monocytes as MMP-9 producers. HMWH-induced cytokine/chemokine secretion was assessed in co-culture supernatant, and the influence of cytokines/chemokines on MMP-9 production was analyzed. These experiments revealed that Jurkat-derived IL-16 and soluble intercellular adhesion molecule (sICAM-) 1 are able to induce MMP-9 and IL-8 production by THP-1. As a consequence, the increased MMP-9 expression found in HMWH blood samples may be influenced by HMWH-dependent secretion of IL-16 and sICAM-1 by T cells resulting in an increased production of MMP-9 and IL-8 by monocytes. IL-8, in turn, may support MMP-9 and its own expression in a positive autocrine feedback loop.

Heparin, a class of glycosaminoglycans (GAGs), is widely used to induce sperm capacitation and fertilization. How heparin induces sperm capacitation remains unclear. Olfactory receptors (ORs) which are G protein-coupled receptors, have been proposed to be involved in sperm capacitation. However, the interaction between ORs and odor molecules and the molecular mechanism of ORs mediating sperm capacitation are still unclear. The present study aimed to explore the underlying interaction and mechanism between heparin and ORs in carrying out the boar sperm capacitation. The results showed that olfactory receptor 2C1 (OR2C1) is a compulsory unit which regulates the sperm capacitation by recognizing and binding with heparin, as determined by Dual-Glo Luciferase Assay and molecular docking. In addition, molecular dynamics (MD) simulation indicated that OR2C1 binds with heparin via a hydrophobic cavity comprises of Arg3, Ala6, Thr7, Asn171, Arg172, Arg173, and Pro287. Furthermore, we demonstrated that knocking down OR2C1 significantly inhibits sperm capacitation. In conclusion, we highlighted a novel olfactory receptor, OR2C1, in boar sperm and disclosed the potential binding of heparin to Pro287, a conserved residue in the transmembrane helices region 7 (TMH7). Our findings will benefit the further understanding of ORs involved in sperm capacitation and fertilization.

CXCL7, a chemokine highly expressed in platelets, orchestrates neutrophil recruitment during thrombosis and related pathophysiological processes by interacting with CXCR2 receptor and sulfated glycosaminoglycans (GAG). CXCL7 exists as monomers and dimers, and dimerization (~50 μM) and CXCR2 binding (~10 nM) constants indicate that CXCL7 is a potent agonist as a monomer. Currently, nothing is known regarding the structural basis by which receptor and GAG interactions mediate CXCL7 function. Using solution nuclear magnetic resonance (NMR) spectroscopy, we characterized the binding of CXCL7 monomer to the CXCR2 N-terminal domain (CXCR2Nd) that constitutes a critical docking site and to GAG heparin. We found that CXCR2Nd binds a hydrophobic groove and that ionic interactions also play a role in mediating binding. Heparin binds a set of contiguous basic residues indicating a prominent role for ionic interactions. Modeling studies reveal that the binding interface is dynamic and that GAG adopts different binding geometries. Most importantly, several residues involved in GAG binding are also involved in receptor interactions, suggesting that GAG-bound monomer cannot activate the receptor. Further, this is the first study that describes the structural basis of receptor and GAG interactions of a native monomer of the neutrophil-activating chemokine family.

A self-nanoemulsifying drug delivery system (SNEDDS) was developed to enhance the absorption of heparin after oral administration, in which heparin was compounded with phospholipids to achieve better fat solubility in the form of heparin-phospholipid (HEP-Pc) complex. HEP-Pc complex was prepared using the solvent evaporation method, which increased the solubility of heparin in n-octanol. The successful preparation of HEP-Pc complex was confirmed by differential scanning calorimetry (DSC), Fourier-transform infrared (FT-IR) spectroscopy, NMR, and SEM. A heparin lipid microemulsion (HEP-LM) was prepared by high-pressure homogenization and characterized. HEP-LM can enhance the absorption of heparin after oral administration, significantly prolong activated partial thromboplastin time (APTT) and thrombin time (TT) in mice, and reduce fibrinogen (FIB) content. All these outcomes indicate that HEP-LM has great potential as an oral heparin formulation.

Uncontrolled bleeding after enoxaparin (ENX) is rare but may be life-threatening. The only registered antidote for ENX, protamine sulfate (PS), has 60% efficacy and can cause severe adverse side effects. We developed a diblock copolymer, heparin-binding copolymer (HBC), that reverses intravenously administered heparins. Here, we focused on the HBC inhibitory activity against subcutaneously administered ENX in healthy mice. BALB/c mice were subcutaneously injected with ENX at the dose of 5 mg/kg. After 110 min, vehicle, HBC (6.25 and 12.5 mg/kg), or PS (5 and 10 mg/kg) were administered into the tail vein. The blood was collected after 3, 10, 60, 120, 360, and 600 min after vehicle, HBC, or PS administration. The activities of antifactors Xa and IIa and biochemical parameters were measured. The main organs were collected for histological analysis. HBC at the lower dose reversed the effect of ENX on antifactor Xa activity for 10 min after antidote administration, whereas at the higher dose, HBC reversed the effect on antifactor Xa activity throughout the course of the experiment. Both doses of HBC completely reversed the effect of ENX on antifactor IIa activity. PS did not reverse antifactor Xa activity and partially reversed antifactor IIa activity. HBC modulated biochemical parameters. Histopathological analysis showed changes in the liver, lungs, and spleen of mice treated with HBC and in the lungs and heart of mice treated with PS. HBC administered in an appropriate dose might be an efficient substitute for PS to reverse significantly increased anticoagulant activity that may be connected with major bleeding in patients receiving ENX subcutaneously.

Long-term delivery of growth factors and immunomodulatory agents is highly required to support the integrity of tissue in engineering constructs, e.g., formation of vasculature, and to minimize immune response in a recipient. However, for proteins with a net positive charge at the physiological pH, controlled delivery from negatively charged alginate (Alg) platforms is challenging due to electrostatic interactions that can hamper the protein release. In order to regulate such interactions between proteins and the Alg matrix, we propose to complex proteins of interest in this study - CXCL12, FGF-2, VEGF - with polyanionic heparin prior to their encapsulation into Alg microbeads of high content of α-L-guluronic acid units (high-G). This strategy effectively reduced protein interactions with Alg (as shown by model ITC and SPR experiments) and, depending on the protein type, afforded control over the protein release for at least one month. The released proteins retained their in vitro bioactivity: CXCL12 stimulated the migration of Jurkat cells, and FGF-2 and VEGF induced proliferation and maturation of HUVECs. The presence of heparin also intensified protein biological efficiency. The proposed approach for encapsulation of proteins with a positive net charge into high-G Alg hydrogels is promising for controlled long-term protein delivery under in vivo conditions.

Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction characterized by thrombocytopenia and a high risk for venous or arterial thrombosis. HIT is caused by antibodies that recognize complexes of platelet factor 4 and heparin. The pathogenic mechanisms of this condition are not fully understood. In this study, we used flow cytometry, fluorimetry, and Western blot analysis to study the direct effects of pathogenic immune complexes containing platelet factor 4 on human platelets isolated by gel-filtration. HIT-like pathogenic immune complexes initially caused pronounced activation of platelets detected by an increased expression of phosphatidylserine and P-selectin. This activation was mediated either directly through the FcγRIIA receptors or indirectly via protease-activated receptor 1 (PAR1) receptors due to thrombin generated on or near the surface of activated platelets. The immune activation was later followed by the biochemical signs of cell death, such as mitochondrial membrane depolarization, up-regulation of Bax, down-regulation of Bcl-XL, and moderate activation of procaspase 3 and increased calpain activity. The results show that platelet activation under the action of HIT-like immune complexes is accompanied by their death through complex apoptotic and calpain-dependent non-apoptotic pathways that may underlie the low platelet count in HIT.

Titanium alloy (Ti6Al4V) is one of the most prominent biomaterials for bone contact because of its ability to bear mechanical loading and resist corrosion. The success of Ti6Al4V implants depends on bone formation on the implant surface. Hence, implant coatings which promote adhesion, proliferation and differentiation of bone-forming cells are desirable. One coating strategy is by adsorption of biomacromolecules. In this study, Ti6Al4V substrates produced by additive manufacturing (AM) were coated with whey protein isolate (WPI) fibrils, obtained at pH 2, and heparin or tinzaparin (a low molecular weight heparin LMWH) in order to improve the proliferation and differentiation of bone-forming cells. WPI fibrils proved to be an excellent support for the growth of human bone marrow stromal cells (hBMSC). Indeed, WPI fibrils were resistant to sterilization and were stable during storage. This WPI-heparin-enriched coating, especially the LMWH, enhanced the differentiation of hBMSC by increasing tissue non-specific alkaline phosphatase (TNAP) activity. Finally, the coating increased the hydrophilicity of the material. The results confirmed that WPI fibrils are an excellent biomaterial which can be used for biomedical coatings, as they are easily modifiable and resistant to heat treatments. Indeed, the already known positive effect on osteogenic integration of WPI-only coated substrates has been further enhanced by a simple adsorption procedure.

As a member of the HPSG family, heparin is often used as a specific probe of their role in cell physiology; indeed, we have previously shown a reduction in the tumorigenicity of breast cancer cells when cultured in its presence. However, a partial reversal of the anti-tumorigenic effect occurred when the treated cells were cultured in fresh medium without heparin, which led us to consider whether a more persistent effect could be achieved by treatment of the cells with small extracellular vesicles (sEV) from heparin-treated cells. The tumorigenicity was analyzed using sEV isolated from the culture medium of heparin-treated MCF-7 and MDA-MB231 breast cancer cells (sEV-HT) or from conditioned medium following the termination of treatment (heparin discontinued, sEV-HD). Tumorigenicity was reduced in cells cultured in the presence of sEV-HT compared to that of cells cultured in the presence of sEV from untreated cells (sEV-Ctrl). sEV-HD were also observed to exert an anti-tumorigenic effect on the expression of pro-tumorigenic and cell cycle regulatory proteins, as well as signaling activities when added to fresh cultures of MCF-7 and MDA-MB231 cells. The anti-tumorigenic activity of the heparin-derived sEV may arise from observed changes in the miRNA content or from heparin, which was observed to be bound to the sEV. sEV may constitute a relatively stable reservoir of circulating heparin, allowing heparin activity to persist in the circulation even after therapy has been discontinued. These findings can be considered as a special additional pharmacological characteristic of heparin clinical therapy.

Patients with sepsis frequently require mechanical ventilation (MV) to survive. However, MV has been shown to induce the production of proinflammatory cytokines, causing ventilator-induced lung injury (VILI). It has been demonstrated that hypoxia-inducible factor (HIF)-1α plays a crucial role in inducing both apoptotic and inflammatory processes. Low-molecular-weight heparin (LMWH) has been shown to have anti-inflammatory activities. However, the effects of HIF-1α and LMWH on sepsis-related acute lung injury (ALI) have not been fully delineated. We hypothesized that LMWH would reduce lung injury, production of free radicals and epithelial apoptosis through the HIF-1α pathway. Male C57BL/6 mice were exposed to 6-mL/kg or 30-mL/kg MV for 5 h. Enoxaparin, 4 mg/kg, was administered subcutaneously 30 min before MV. We observed that MV with endotoxemia induced microvascular permeability; interleukin-6, tumor necrosis factor-α, macrophage inflammatory protein-2 and vascular endothelial growth factor protein production; neutrophil infiltration; oxidative loads; HIF-1α mRNA activation; HIF-1α expression; bronchial epithelial apoptosis; and decreased respiratory function in mice (p < 0.05). Endotoxin-induced augmentation of VILI and epithelial apoptosis were reduced in the HIF-1α-deficient mice and in the wild-type mice following enoxaparin administration (p < 0.05). Our data suggest that enoxaparin reduces endotoxin-augmented MV-induced ALI, partially by inhibiting the HIF-1α pathway.

We report dual therapeutic effects of a synthetic heparin-binding peptide (HBP) corresponding to residues 15-24 of the heparin binding site in BMP4 in a collagen-induced rheumatic arthritis model (CIA) for the first time. The cell penetrating capacity of HBP led to improved cartilage recovery and anti-inflammatory effects via down-regulation of the iNOS-IFNγ-IL6 signaling pathway in inflamed RAW264.7 cells. Both arthritis and paw swelling scores were significantly improved following HBP injection into CIA model mice. Anti-rheumatic effects were accelerated upon combined treatment with Enbrel® and HBP. Serum IFNγ and IL6 concentrations were markedly reduced following intraperitoneal HBP injection in CIA mice. The anti-rheumatic effects of HBP in mice were similar to those of Enbrel®. Furthermore, the combination of Enbrel® and HBP induced similar anti-rheumatic and anti-inflammatory effects as Enbrel®. We further investigated the effect of HBP on damaged chondrocytes in CIA mice. Regenerative capacity of HBP was confirmed based on increased expression of chondrocyte biomarker genes, including aggrecan, collagen type II and TNFα, in adult human knee chondrocytes. These findings collectively support the utility of our cell-permeable bifunctional HBP with anti-inflammatory and chondrogenic properties as a potential source of therapeutic agents for degenerative inflammatory diseases.

The use of implants can be hampered by chronic inflammatory reactions, which may result in failure of the implanted device. To prevent such an outcome, the present study examines the anti-inflammatory properties of surface coatings made of either hyaluronic acid (HA) or heparin (Hep) in combination with chitosan (Chi) prepared as multilayers through the layer-by-layer (LbL) technique. The properties of glycosaminoglycan (GAG)-modified surfaces were characterized in terms of surface topography, thickness and wettability. Results showed a higher thickness and hydrophilicity after multilayer formation compared to poly (ethylene imine) control samples. Moreover, multilayers containing either HA or Hep dampened the inflammatory response visible by reduced adhesion, formation of multinucleated giant cells (MNGCs) and IL-1β release, which was studied using THP-1 derived macrophages. Furthermore, investigations regarding the mechanism of anti-inflammatory activity of GAG were focused on nuclear transcription factor-кB (NF-κB)-related signal transduction. Immunofluorescence staining of the p65 subunit of NF-κB and immunoblotting were performed that showed a significant decrease in NF-κB level in macrophages on GAG-based multilayers. Additionally, the association of FITC-labelled GAG was evaluated by confocal laser scanning microscopy and flow cytometry showing that macrophages were able to associate with and take up HA and Hep. Overall, the Hep-based multilayers demonstrated the most suppressive effect making this system most promising to control macrophage activation after implantation of medical devices. The results provide an insight on the anti-inflammatory effects of GAG not only based on their physicochemical properties, but also related to their mechanism of action toward NF-κB signal transduction.

In this study, we prepared human bone morphogenetic protein-2 (hBMP-2)/human growth and differentiation factor-5 (hGDF-5)-coated titanium (Ti) disc and screw types for controlled release of the growth factors (GFs). The two growth factors were coated onto Ti with a smooth surface using their specific interaction with heparin, because they have heparin binding sites in their molecular structures. Efficacy of the two growth factor-coated Ti for enhancement of bone formation and osseointegration was compared to pristine Ti, and hBMP-2- and hGDF-5-coated Ti in vivo. The surface chemical composition, surface morphology, and wettability characteristics of the metal samples were determined by X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and contact angle measurement, respectively. The initial burst of hBMP-2, hGDF-5, and their combination, occurred within one day of the release study, resulting in 12.5%, 4.5%, and 13.5%/3.2%, and then there was a sustained, even release of these two growth factors from the coated metal for 30 days. In vitro tests revealed that MC3T3-E1 cells cultured on the two growth factor-coated Ti had a higher proliferation rate and a higher activity for alkaline phosphatase (ALP), which led to a larger amount of calcium deposition and larger expressions of type I collagen (COL 1), ALP, and osteocalcin (OCN) mRNAs. In vivo animal tests using ten white New Zealand rabbits showed that the two growth factor-coated Ti enhanced bone formation and osseointegration at the interface between the implants and host bone. In addition, histological evaluation showed that bone remodeling, including bone formation by osteoblasts and bone resorption by osteoclasts, actively occurred between the two growth factor-coated Ti and host bone. Consequently, it is suggested that Ti surface modification with the combination of hBMP-2 and hGDF-5 for the two growth factor-coated Ti implants can improve the clinical properties of implants for orthopedic and dental applications.