Bone Tissue Engineering Research Articles

In-vitro biomineralization of magnesium and copper co-doped wollastonite

Calcium silicate-based ceramics, particularly wollastonite (CaSiO3), are gaining prominence in hard tissue engineering due to their biocompatibility and bioactivity. However, pure wollastonite faces challenges such as insufficient mechanical strength and susceptibility to bacterial colonization. This study addresses these issues by systematically synthesizing magnesium and copper co-doped wollastonite (MgCuW) using the sol-gel combustion technique, aiming to enhance its mechanical stability and antibacterial properties. The average crystalline size of the synthesized materials ranged from 25 to 47 nm. In vitro biomineralization studies showed significant hydroxyapatite deposition, confirming enhanced bioactivity. Antibacterial tests against Gram-positive (S. aureus, S. epidermidis) and Gram-negative (E. coli, P. aeruginosa) bacteria demonstrated superior antibacterial activity with increased copper doping. The results indicate that MgCuW is a promising biomaterial for bone tissue engineering, combining bioactivity and antibacterial efficacy.

Hybrid direct ink writing of bioglass-calcite-carbon composite scaffolds supported by novel silicone-based emulsions

70S30C (70 mol% SiO2, 30% CaO) bioglass is one of the most promising bioceramics for bone tissue engineering. We discuss the feasibility of 70S30C bioglass/calcite/carbon composites derived from novel silicone-based emulsions, leading to highly porous lattice scaffolds. These are produced by direct ink writing (DIW) 3D printing, followed by ceramic conversion at 700°C in flowing nitrogen. The emulsions consisted of droplets of concentrated calcium nitrate aqueous solution incorporated into blends of H44 commercial polysiloxane and photocurable acrylate resin. This formulation offered unprecedented opportunities in both synthesis and shaping. Specifically, the homogeneous dispersion of the CaO precursor in silicone enabled a uniform SiO2/CaO distribution, favoring the formation of a glass matrix. Additionally, the acrylate component and water content allowed for tuning of the microstructure both immediately after printing and upon firing. Photopolymerization of acrylates consolidated the printed bodies (configuring a 'hybrid DIW') after extrusion, while water evaporation enhanced gas evolution during ceramic conversion, promoting pore interconnectivity.

Effects of Ultrasonic Vibration on 3D Printing of Polylactic Acid/Akermanite Nanocomposite Scaffolds

The mechanical properties of 3D-printed scaffolds for load-bearing implantation are crucial. Although the addition of nanoparticles to polymeric matrix scaffolds can improve their mechanical and biological properties, due to certain limitations in printability, high amounts of reinforcement cannot be used. Therefore, in this study, an attempt was made to use ultrasonic (UT) vibration to inhibit nozzle clogging during fused filament fabrication (FFF) of polylactic acid (PLA) scaffolds including 0, 20, and 40 wt.% Akermanite (Ak). Nozzle clogging happened during the conventional 3D printing of PLA-40wt.%Ak, while that did not occur during UT-assisted 3D printing of PLA-40wt.%Ak. Results of X-ray Diffraction (XRD) analysis and Scanning Electron Microscopy (SEM) indicated that applying ultrasonic (UT) vibration had no negative effect on the phases and morphology of the scaffolds. The results obtained by the compression test indicated that applying UT during 3D-printing resulted in almost 27% increment of elastic modulus and almost 25% increase of the compressive strength of the scaffolds. As the conclusion, this study highlights the effectiveness of ultrasonic-assisted 3D printing in producing nanocomposite scaffolds with significantly higher nanoparticle loadings, as compared to conventional printing methods. By utilizing ultrasonic vibration during the printing process, the study showcases the possibility of overcoming viscosity limitations and optimizing the mechanical performance of scaffolds for various biomedical applications, including bone tissue engineering.

Chitosan/Bioglass Nanocomposites for Bone Tissue Engineering and Regenerative Medicine: An Overview of Promising Biomaterials

: Bioactive glass (BG) shows great potential as a biomaterial for bone regeneration. Chitosan enhances the biological characteristics of BG. Chitosan is the sole commonly utilized natural polysaccharide that may be chemically altered for various purposes and roles. Composite materials formed by combining chitosan bioactive glass (BG) nanoparticles and microparticles are used in this context. Integrating bioactive glasses enhances the mechanical characteristics, bioactivity, and regenerative capacity of the end product. Research indicates that chitosan/BG composites enhance angiogenesis, cell adhesion, and proliferation. Bioglass improves biomineralization and boosts bone extracellular matrix formation by osteoblasts. The current findings demonstrate that the chitosan-glass nanofiber composites can enhance both antibacterial capabilities and bone conductivity. This review examines novel techniques for creating chitosan-based materials for engineering purposes, as well as upcoming difficulties and outlooks.

Tissue-Engineered Bone Regeneration for Medium-to-Large Osteonecrosis of the Femoral Head in the Weight-Bearing Portion: An Observational Study.

Stem cell therapy for the treatment of osteonecrosis of the femoral head (ONFH) showed promising outcomes. However, ONFH with a large lesion in the weight-bearing portion is a poor prognostic factor and still challenging issue to be solved. We aimed to evaluate the effect of tissue-engineered bone regeneration for this challenging condition to preserve the femoral head. A total of 7 patients (9 hips) with ONFH who received osteoblasts expanded ex vivo from bone marrow-derived mesenchymal stem cells (BMdMSCs) and calcium metaphosphate (CMP) as scaffolds from March 2002 to March 2004 were retrospectively reviewed. The median age was 27.0 years (interquartile range [IQR], 23.0-34.0 years), and the median follow-up period was 20.0 years (IQR, 11.0-20.0 years). After culture and expansion of stem cells, we performed core decompression with BMdMSC implantation at a median number of 10.1 ×107 (IQR, 9.9-10.9 ×107). To evaluate radiographic outcomes, the Association Research Circulation Osseous (ARCO) classifications, the Japanese Investigation Committee (JIC) classification, and modified Kerboul combined necrotic angle (mKCNA) were evaluated preoperatively and during follow-up. Clinical outcomes were evaluated by a visual analog scale (VAS) and Harris Hip Score (HHS). The preoperative stage of ONFH was ARCO 2 in 5 hips and ARCO 3a in 4 hips. The ARCO staging was maintained in 3 hips of ARCO 2 and 4 hips of ARCO 3a. Two hips of ARCO 2 with radiographic progression underwent total hip arthroplasty. According to mKCNA, 2 hips showed medium lesions, and 7 hips showed large lesions. The size of necrotic lesion was decreased in 4 hips (2 were ARCO 2 and 2 were ARCO 3a). There were no significant changes in JIC classification in all hips (type C1: 3 hips and type C2: 6 hips) (p = 0.655). Clinically, there were no significant changes in the VAS and HHS between preoperative and last follow-up (p = 0.072 and p = 0.635, respectively). Tissue engineering technique using osteoblasts expanded ex vivo from BMdMSC and CMP showed promising outcomes for the treatment of pre-collapsed and early-collapsed stage ONFH with medium-to-large size, mainly located in weight-bearing areas.

REVIEW OF LITERATURE ON NANOSCALE TOPOGRAPHIC MODIFICATION, MAGNETIC NANOPARTICLES, AND THEIR ROLES IN OSSEOINTEGRATION AND BONE TISSUE ENGINEERING

Here, we will review together the roles of nanoscale modification of titanium substrates and use of magnetic nanoparticles in osseointegration and bone tissue engineering. The technological advances in nanotechnology that has been achieved create new measures to increase the implants-biological systems for an optimal osseointegration through improving important cell-specific functions. We systematically review nanoscale surface modification techniques of titanium implants, covering their physicochemical properties, influence on cellular behaviors and clinical relevances. The report also lengthy discusses the clinical applicability of magnetic nanoparticles for drug and gene delivery systems, targeted cancer therapy as well bone tissue engineering regulting their intrinsic superparamagnetism properites, biocompatibility with other therapies etc. We present detailed review of these technologies and their potential applications in clinical settings.

One-step synthesis of a piezoelectric hybrid BNNT/BaTiO3 composite and its application in bone tissue engineering

Nanomaterials with piezoelectric properties can significantly improve the applicability of polymers used in tissue engineering applications. In this study, we report the one-step synthesis of a novel hybrid piezoelectric composite comprising barium titanates and boron nitride nanotubes. This composite is distinguished by its unique microstructures, including nanoflakes, triangular boron nitride structures, and fiber-like boron nitride nanotube configurations, which contribute to its enhanced piezoelectric properties. The composite was incorporated into a chitosan-based tissue scaffold and evaluated in vitro. Electric-responsive Human Osteoblast cells cultured on the scaffolds are exposed to low-frequency ultrasound stimulation during cell growth. The biocompatibility, cell adhesion, alkaline phosphatase activities, and mineralization of osteoblast cells on the piezo-composite scaffolds were evaluated. The results show that the hybrid piezoelectric composite significantly enhances the properties of chitosan-based scaffold.

Facile synthesis of in situ bismuth-doped calcium phosphate nanocomposite integrated injectable biopolymer hydrogel slurry for bone regeneration

The bionics of natural bone structures plays an essential role in the selection of materials for bone tissue engineering. Although the content of trace metallic elements in bone is low, they have significant effects on the process of bone growth and metabolism. Up to now, the applications of “green” heavy metals in bone regeneration are limited. Herein, in this study, we present a straightforward one-pot strategy for the synthesis of in situ bismuth-doped amorphous calcium phosphate nanocomposites (RBCP), effectively integrating the beneficial properties of each component. The characterization of these products can be readily optimized by adjusting reaction parameters. Our in vitro studies show that under coordination of each component, the RBCP biomaterial demonstrates distinguished biocompatibility and significantly accelerates vascular pattern formation within just 4 h by stimulating the expression of angiogenesis-related genes in human umbilical vein endothelial cells (HUVECs). In vivo experiments indicate that the incorporation of bismuth effectively enhances bone regeneration and osseointegration in a rat femur defect model. In conclusion, the as-prepared RBCP biomaterials hold promising prospects for treating segmental bone defects, owing to the facile, cost-effective, and eco-friendly preparation process, along with their remarkable capabilities.

Knowledge mapping of induced membrane technique: a scientometric study from 2004 to 2023

BackgroundThe induced membrane technique (IMT) is a two-step procedure used for reconstructing segmental bone defects in the limbs. The osteogenic mechanism after bone grafting using IMT remains unclear, and efforts to modify the original techniques are limited to the investigative phase. Therefore, reviewing existing knowledge and identifying hotspots and new trends in IMT is critical.MethodsWe retrieved reviews and articles associated with IMT published between 2004 and 2023 from the Web of Science Core Collection (WoSCC). The keywords included induced membrane technique, guided bone regeneration, bone defect reconstruction, bone graft, stem cells, Masquelet technique, management of bone defects, and scaffold. HistCite, VOSviewer, CiteSpace, and R-bibliometrics were used for scientometric analysis.ResultsA total of 1019 publications from 374 academic journals with 33,995 co-cited references by 2,331 institutions from 65 countries or regions were included. China (n = 235) and the United States (n = 215) were the most productive countries, with Shanghai Jiao Tong University producing the most number of publications (n = 18). Journal Injury [co-citations = 1774; impact factor (IF) 2022 = 2.5] published the most manuscripts, while Masquelet AC and Giannoudis PV published literature with a significant influence on IMT, showing more co-citations (n = 727; n = 355). Two preface hotspots of IMT focused on investigating the microscopic mechanism (such as the membrane supporting graft-to-bone union and the role of inflammatory cells) and developing new techniques to improve IMT (such as bone tissue engineering and new drugs).ConclusionThis study comprehensively reviewed the literature about IMT published in the last 20 years using qualitative and quantitative methods, providing valuable information for researchers investigating IMT.

Zinc and chitosan-enhanced β-tricalcium phosphate from calcined fetal bovine bone for mandible reconstruction.

Mandibular defects pose significant challenges in reconstructive surgery, and scaffold materials are increasingly recognized for their potential to address these challenges. Among various scaffold materials, Beta-tricalcium phosphate (β-TCP) is noted for its exceptional osteogenic properties. However, improvements in its biodegradation rate and mechanical strength are essential for optimal performance. In this study, we developed a novel β-TCP-based scaffold, CFBB, by calcining fetal bovine cancellous bone. To enhance its properties, we modified CFBB with Chitosan (CS) and Zinc (Zn), creating three additional scaffold materials: CFBB/CS, CFBB/Zn2+, and CFBB/Zn2+/CS. We conducted comprehensive assessments of their physicochemical and morphological properties, degradation rates, biocompatibility, osteogenic ability, new bone formation, and neovascularization both in vitro and in vivo. Our findings revealed that all four materials were biocompatible and safe for use. The modifications with CS and Zn2+ significantly improved the mechanical strength, osteogenic, and angiogenic properties of CFBB, while concurrently decelerating its resorption rate. Among the tested materials, CFBB/Zn2+/CS demonstrated superior performance in promoting bone regeneration and vascularization, making it a particularly promising candidate for mandibular reconstruction. The CFBB/Zn2+/CS scaffold material, with its enhanced mechanical, osteogenic, and angiogenic properties, and a controlled resorption rate, emerges as a highly effective alternative for the repair of oral mandible defects. This study underscores the potential of combining multiple bioactive agents in scaffold materials to improve their functionality for specific clinical applications in bone tissue engineering.

Leveraging Machine Learning for Optimized Mechanical Properties and 3D Printing of PLA/cHAP for Bone Implant.

This study explores the fabrication and characterisation of 3D-printed polylactic acid (PLA) scaffolds reinforced with calcium hydroxyapatite (cHAP) for bone tissue engineering applications. By varying the cHAP content, we aimed to enhance PLA scaffolds' mechanical and thermal properties, making them suitable for load-bearing biomedical applications. The results indicate that increasing cHAP content improves the tensile and compressive strength of the scaffolds, although it also increases brittleness. Notably, incorporating cHAP at 7.5% and 10% significantly enhances thermal stability and mechanical performance, with properties comparable to or exceeding those of human cancellous bone. Furthermore, this study integrates machine learning techniques to predict the mechanical properties of these composites, employing algorithms such as XGBoost and AdaBoost. The models demonstrated high predictive accuracy, with R2 scores of 0.9173 and 0.8772 for compressive and tensile strength, respectively. These findings highlight the potential of using data-driven approaches to optimise material properties autonomously, offering significant implications for developing custom-tailored scaffolds in bone tissue engineering and regenerative medicine. The study underscores the promise of PLA/cHAP composites as viable candidates for advanced biomedical applications, particularly in creating patient-specific implants with improved mechanical and thermal characteristics.

Ex Ovo Chorioallantoic Membrane Assay as a Model of Bone Formation by Biomaterials.

Biomaterials play an increasingly critical role in bone tissue engineering. However, achieving effective clinical translation requires a careful choice of biomimetic materials and thorough assessment of their efficacy and safety. Existing in vitro and in vivo models have drawbacks including time and cost constraints, invasive procedures, and discordance between animal models and clinical outcomes. Therefore, there is a demand for an alternative model. We hypothesized that the chick embryo chorioallantoic membrane can serve as a bioreactor to evaluate the initial sign of bone formation on scaffolds. In parallel, we investigated the osteogenic potential of a previously fabricated fibrin-alginate-calcium phosphate biomaterial (FACaP). Blood vessels were observed to infiltrate the scaffolds with early signs of bone formation, confirmed via RUNX-2 and alpha smooth muscle actin markers. The scaffolds' chemical composition was evaluated by Fourier-transform infrared spectroscopy, and ion chromatography was used to assess calcium ion release. Finally, the topography was examined by atomic force microscopy. In conclusion, this system offers simple refinement for in vivo models in bone tissue engineering and highlights the great potential of FACaP as an angiogenic and osteogenic biomaterial for non-load-bearing applications.

Recent Advances in the Design and Structural/Functional Regulations of Biomolecule‐Reinforced Graphene Materials for Bone Tissue Engineering Applications

Recent Advances in the Design and Structural/Functional Regulations of Biomolecule‐Reinforced Graphene Materials for Bone Tissue Engineering Applications

DDIT3 switches osteogenic potential of BMP9 to lipogenic by attenuating Wnt/β-catenin signaling via up-regulating DKK1 in mesenchymal stem cells.

Bone morphogenetic protein 9 (BMP9) functions as a potent inducer of osteogenic differentiation in mesenchymal stem cells (MSCs), holding promise for bone tissue engineering. However, BMP9 also concurrently triggers lipogenic differentiation in MSCs, potentially compromising its osteogenic potential. In this study, we explored the role of DNA damage inducible transcript 3 (DDIT3) in regulating the balance between BMP9-induced osteogenic and lipogenic differentiation in MSCs. Utilizing techniques such as PCR, Western blot, histochemical staining, and in vivo experiments, we analyzed the osteogenic and lipogenic markers induced by BMP9 and delved into the underlying molecular mechanism. We found a significant upregulation of DDIT3 in C3H10T1/2 cells treated with BMP9. This upregulation led to a reduction in BMP9-induced osteogenic markers but an enhancement in lipogenic markers. Conversely, knocking down DDIT3 produced the opposite effects. Furthermore, BMP9-induced bone formation was decreased in the presence of DDIT3, but adipocyte formation was increased. Further investigations demonstrated that BMP9 increased the phosphorylation level of GSK-3β and promoted nuclear translocation of β-catenin, both of which were suppressed by DDIT3. Moreover, DDIT3 decreased the total β-catenin protein level while BMP9 increased the DKK1 protein level, which was further enhanced by DDIT3. Notably, knocking down DKK1 partially reversed the effect of DDIT3 on reducing BMP9-induced osteogenic markers and increasing lipogenic markers. Our findings indicated that DDIT3 enhances lipogenic differentiation by diminishing BMP9's osteogenic potential, possibly through inhibiting Wnt/β-catenin signaling via DKK1 upregulation in MSCs.

Effect of bioceramic inclusions on gel-cast aliphatic polymer membranes for bone tissue engineering applications: An in vitro study.

Polylactic acid (PLA) has been extensively used in tissue engineering. However, poor mechanical properties and low cell affinity have limited its pertinence in load bearing bone tissue regeneration (BTR) devices. Augmenting PLA with β-Tricalcium Phosphate (β-TCP), a calcium phosphate-based ceramic, could potentially improve its mechanical properties and enhance its osteogenic potential. Gels of PLA and β-TCP were prepared of different % w/w ratios through polymer dissolution in acetone, after which polymer-ceramic membranes were synthesized using the gel casting workflow and subjected to characterization. Gel-cast polymer-ceramic constructs were associated with significantly higher osteogenic capacity and calcium deposition in differentiated osteoblasts compared to pure polymer counterparts. Immunocytochemistry revealed cell spreading over the gel-cast membrane surfaces, characterized by trapezoidal morphology, distinct rounded nuclei, and well-aligned actin filaments. However, groups with higher ceramic loading expressed significantly higher levels of osteogenic markers relative to pure PLA membranes. Rule of mixtures and finite element models indicated an increase in theoretical mechanical strength with an increase in β-TCP concentration. This study potentiates the use of PLA/β-TCP composites in load bearing BTR applications and the ability to be used as customized patient-specific shape memory membranes in guided bone regeneration.

Bone Tissue Engineering Scaffold Film with Controlled Release of Tea Polyphenol-Magnesium Nanoformulations to Prevent Bacterial Infection

Bone Tissue Engineering Scaffold Film with Controlled Release of Tea Polyphenol-Magnesium Nanoformulations to Prevent Bacterial Infection

Fabrication of 3D microfibrous composite polycaprolactone/hydroxyapatite scaffolds loaded with piezoelectric poly (lactic acid) nanofibers by sequential near-field and conventional electrospinning for bone tissue engineering

Near-field electrospinning (NFES) has recently gained considerable interest in fabricating tissue engineering scaffolds. This technique combines the advantages of both 3D printing and electrospinning. It allows for the production of fibers with smaller resolution and the ability to make regular structures with suitable pores. In this study, a microfibrous composite scaffold of polycaprolactone (PCL)/hydroxyapatite (HA) was prepared by NFES in the first step. The microfibrous scaffold had a fiber spacing of 414.674 ± 24.9 μm with an average fiber diameter of 94.695 ± 16.149 μm. However, due to the large fiber spacing, the surface area was insufficient for cell adhesion. Therefore, the hybrid scaffold was prepared by adding aligned and random electrospun poly (L-lactic acid) (PLLA) nanofibers to the microfibrous scaffold. Cellular studies showed that cell adhesion to the hybrid scaffold increased by 334 % compared to the microfibrous scaffold. These nanofibers also exhibited piezoelectric properties, which helped stimulate bone regeneration. Aligned nanofibers in the hybrid scaffold enhanced alkaline phosphatase activity and the intensity of alizarin red staining 1.5 and 1.6 times, respectively, compared to the microfibrous scaffold. Furthermore, the elastic modulus and ultimate tensile strength increased by 268 % and 130 %, respectively, by adding aligned nanofibers to the microfibrous scaffold. Therefore, the hybrid microfibrous composite scaffold of PCL/HA containing aligned electrospun PLLA nanofibers with improved properties showed the potential for bone regeneration.

Synthesis of N, N,O and O-carboxymethyl chitosan derivatives of controllable substitution degrees and their utilization as electrospun scaffolds for bone tissue engineering

Most chitosan (CS) carboxymethylation approaches are weighed down by insufficient description protocols regarding the reaction specificity and the degree of substitution (DS). Here, we provide three carboxymethylation protocols of enhanced specificity towards the amine (N-), amine/hydroxy (N,O-) and hydroxy (O-) groups of CS. The DS for all samples was found to be ∼70 %, as confirmed by NMR analysis. To illustrate the modified materials' potential in bone tissue regeneration, each derivative was blended with poly(vinyl alcohol) to prepare scaffolds via electrospinning. Both materials and electrospun membranes were characterized in terms of their physicochemical properties by Fourier transform infrared spectroscopy, thermogravimetric analysis, differential scanning calorimetry and water contact angle measurements. The electrospun membranes' swelling ratio, degradation, tensile strength, as well as morphology were also examined through scanning electron microscopy before and after heat treatment at 120 °C, which was used as a method of physical stabilization, leading to significantly enhanced degradation rate and mechanical strength. The three electrospun scaffold types were loaded with MC3T3-E1 pre-osteoblastic cells and their cell adhesion, viability and growth rate were assessed. Finally, osteogenic differentiation was examined by means of alkaline phosphatase activity measurement, calcium mineralization and formation of extracellular matrix markers, with all materials showing promising prospects.

Enzyme-delivery Metal-organic Framework Composite Coatings for Restoration of Hyperglycemia-damaged Osteoblast Differentiation

There is a significant clinical need to develop effective treatments for bone defects in patients with diabetes mellitus (DM), as they are at higher risk of fractures and impaired healing. Guided bone tissue engineering using biocompatible and biodegradable polymers is a promising approach. However, current diabetic bone regenerative therapies often fail due to the accumulation of advanced glycation products, which can affect the integration of traditional tissue engineering scaffolds with native bone. Therefore, novel approaches are needed to improve the efficacy of diabetic bone regeneration. This study presents a proof-of-concept development of a multifunctional polymer composite coating tailored towards restoring diabetes-related damage in osteoblast differentiation. Our composite system involves 3D-printed poly(caprolactone fumarate) (PCLF) and poly(caprolactone) (PCL) blend scaffolds coated with multifunctional chitosan methacrylate (chiMA). The chiMA coating is embedded with a sustained-release formulation of glucose oxidase (GOx) from MIL-127 metal-organic frameworks making the coating a stimuli-responsive biomolecule delivery system. The multifunctional coating is designed for the sustained release of GOx and sodium pyruvate for in vitro glucose modulation and oxidative stress reduction, respectively. We propose that sustained release of GOx from MIL-127 embedded chiMA coatings can modulate the high glucose (HG) cellular milieu towards normal glucose (NG), enhancing osteoblast (OB) differentiation via downstream effects. Our results show successful synthesis of MIL-127, encapsulation of GOx, and fabrication of composite coating on the PCLF/PCL scaffolds with effective enzyme activity measured as a function of lowering glucose concentration in HG media for 144 h to normal levels. In vitro evaluation of OB viability, attachment, proliferation, and differentiation showed an overall decrease in cellular activity in HG conditions, which was restored through the glucose-modulating functionality of the GOx-releasing MIL-127 coatings. Our results also presented preliminary evidence of a statistical correlation between DM-related gene markers and osteogenic markers in vitro that requires further exploration. Although this proof-of-concept study holds promise for advancing precision biomaterials development for diabetic tissue engineering and meeting the unmet clinical need for effective treatments and warrants future in vivo evaluation of the composite coating and molecular biology understanding of correlations between DM and osteogenic markers.

Osteogenic and osteoclastic differentiation by control release of adenosine within PEI/HA/Collagen composite coatings

In virtue of the rapid metabolism and short half-life of adenosine (AD) in the human body, it is highly desired to explore new adenosine delivery system with controlled and steady release capacity. In this study, composite coatings consisting of polyethyleneimine (PEI), hyaluronic acid (HA) and collagen type I (COL-1) on titanium substrate surface are developed by layer-by-layer self-assembly technique, in which different concentrations of AD are encapsulated. In-vitro release study demonstrated a steady and sustained release of AD from the composite coating system. Adenosine within composite coatings depicted a concentration-dependent regulatory effect on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and osteoclastic differentiation of mouse leukemia cells of monocyte macrophage (RAW264.7) cells. All of these coatings exhibited good cell viability and promoted osteoblast cell proliferation. Among various composite coatings, the optimized coating specimen with loading of 65 μg AD (AD2) exhibits the best osteoconductive and osteoinductive activities with significantly enhanced osteoblast-associated gene expressions, and increased alkaline phosphatase (ALP) activity and calcium deposits. On the other hand, AD2 specimen shows the activity of inhibiting osteoclast differentiation and osteobclast-associated gene expressions as well as suppressing tartrate-resistant acid phosphatase (TRAP) activity. The superior performance of AD/PEI/HA/COL composite coatings indicated that this coating system could be useful for bone tissue engineering.