In Vitro Anti-Inflammation and Chondrogenic Differentiation Effects of Inclusion Nanocomplexes of Hyaluronic Acid-Beta Cyclodextrin and Simvastatin.

The aim of this study was to prepare inclusion nanocomplexes of hyaluronic acid-ß-cyclodextrin and simvastatin (HA-ß-CD/SIM) and evaluate in vitro anti-inflammation effects on lipopolysaccharide (LPS)-activated synoviocytes and chondrogenic differentiation effects on rat adipose-derived stem cells (rADSCs). The ß-CD moieties in HA-ß-CD could incorporate SIM to form HA-ß-CD/SIM nanocomplexes with diameters of 297-350 nm. HA-ß-CD/SIM resulted in long-term release of SIM from the nanocomplexes for up to 63 days in a sustained manner. In vitro studies revealed that HA-ß-CD/SIM nanocomplexes were able to effectively and dose-dependently suppress the mRNA expression levels of pro-inflammatory markers such as matrix metallopeptidase-3 (MMP-3), MMP-13, cyclooxygenase-2 (COX-2), a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5), interleukin-6 (IL-6), and tumor necrosis factor (TNF-α) in LPS-stimulated synoviocytes. HA-ß-CD/SIM-treated rADSCs significantly and dose-dependently enhanced mRNA expressions of aggrecan, collagen type II (COL2A1), and collagen type X (COL10A1), implying that HA-ß-CD/SIM greatly induced the chondrogenic differentiation of rADSCs. Conclusively, HA-ß-CD/SIM nanocomplexes will be a promising therapeutic material to alleviate inflammation as well as promote chondrogenesis.

Biphasic Calcium Phosphate (BCP)-Immobilized Porous Poly (d,l-Lactic-co-Glycolic Acid) Microspheres Enhance Osteogenic Activities of Osteoblasts.

The purpose of this study was to evaluate the potential of porous poly (d,l-lactic-co-glycolic acid) (PLGA) microspheres (PMSs) immobilized on biphasic calcium phosphate nanoparticles (BCP NPs) (BCP-IM-PMSs) to enhance osteogenic activity. PMSs were fabricated using a fluidic device, and their surfaces were modified with l-lysine (aminated-PMSs), whereas the BCP NPs were modified with heparin⁻dopamine (Hep-DOPA) to obtain heparinized⁻BCP (Hep-BCP) NPs. BCP-IM-PMSs were fabricated via electrostatic interactions between the Hep-BCP NPs and aminated-PMSs. The fabricated BCP-IM-PMSs showed an interconnected pore structure. In vitro studies showed that MG-63 cells cultured on BCP-IM-PMSs had increased alkaline phosphatase activity, calcium content, and mRNA expression of osteocalcin (OCN) and osteopontin (OPN) compared with cells cultured on PMSs. These data suggest that BCP NP-immobilized PMSs have the potential to enhance osteogenic activity.

3D printed alendronate-releasing poly(caprolactone) porous scaffolds enhance osteogenic differentiation and bone formation in rat tibial defects.

The aim of this study was to evaluate the in vitro osteogenic effects and in vivo new bone formation of three-dimensional (3D) printed alendronate (Aln)-releasing poly(caprolactone) (PCL) (Aln/PCL) scaffolds in rat tibial defect models. 3D printed Aln/PCL scaffolds were fabricated via layer-by-layer deposition. The fabricated Aln/PCL scaffolds had high porosity and an interconnected pore structure and showed sustained Aln release. In vitro studies showed that MG-63 cells seeded on the Aln/PCL scaffolds displayed increased alkaline phosphatase (ALP) activity and calcium content in a dose-dependent manner when compared with cell cultures in PCL scaffolds. In addition, in vivo animal studies and histologic evaluation showed that Aln/PCL scaffolds implanted in a rat tibial defect model markedly increased new bone formation and mineralized bone tissues in a dose-dependent manner compared to PCL-only scaffolds. Our results show that 3D printed Aln/PCL scaffolds are promising templates for bone tissue engineering applications.

Ibuprofen-loaded porous microspheres suppressed the progression of monosodium iodoacetate-induced osteoarthritis in a rat model.

The objectives of this study were (1) to fabricate ibuprofen-loaded porous microspheres (IBU/PMSs), (2) to evaluate the in vitro anti-inflammatory effects of the microspheres using LPS-induced inflammation in cultured synoviocytes, and (3) to evaluate the in vivo effect of the IBU/PMSs on the progression of monosodium iodoacetate (MIA)-induced osteoarthritis (OA) in a rat model. A dose-dependent in vitro anti-inflammatory effect on pro-inflammatory cytokine markers (matrix metallopeptidase-3 (MMP-3), matrix metallopeptidase-13 (MMP-13), cyclooxygenase-2 (COX-2), a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5)), interleukin-6 (IL-6), and tumor necrosis factor (TNF-α) was observed by confirming with real-time PCR analyses. In vivo, treatment with IBU/PMSs reduced MIA-stimulated mRNA expression of MMP-3, MMP-13, COX-2, ADAMTS-5, IL-6, and TNF-α in rat synoviocytes. In addition, we demonstrated that intra-articular IBU/PMSs suppressed the progression of MIA-induced OA in the rat model via anti-inflammatory mechanisms. In conclusion, IBU/PMSs are a promising therapeutic material to control the pain and progression of OA.

Heparin-immobilized hydroxyapatite nanoparticles as a lactoferrin delivery system for improving osteogenic differentiation of adipose-derived stem cells.

The aim of this study is to fabricate lactoferrin (LF)-carrying hydroxyapatite nanoparticles (HAp NPs) to enhance osteogenic differentiation of rabbit adipose-derived stem cells (rADSCs). HAp NPs were modified with heparin-dopamine (Hep-DOPA) (Hep-HAp) and further immobilized with LF (LF/Hep-HAp). Heparin immobilization on HAp NPs prevented aggregation of HAp NPs in aqueous solution and prolonged the release of LF from LF/Hep-HAp NPs. In vitro studies of rADSCs have demonstrated that LF-Hep/HAp NPs significantly increase alkaline phosphatase (ALP) activity, calcium deposition, and both mRNA expression of osteocalcin (OCN) and osteopontin (OPN) in comparison with HAp and Hep-HAp NPs. These results suggest that LF/Hep-HAp NPs can effectively induce osteogenic differentiation of rADSCs.

The Effect of Alendronate Loaded Biphasic Calcium Phosphate Scaffolds on Bone Regeneration in a Rat Tibial Defect Model.

This study investigated the effect of alendronate (Aln) released from biphasic calcium phosphate (BCP) scaffolds. We evaluated the in vitro osteogenic differentiation of Aln/BCP scaffolds using MG-63 cells and the in vivo bone regenerative capability of Aln/BCP scaffolds using a rat tibial defect model with radiography, micro-computed tomography (CT), and histological examination. In vitro studies included the surface morphology of BCP and Aln-loaded BCP scaffolds visualized using field-emission scanning electron microscope, release kinetics of Aln from BCP scaffolds, alkaline phosphatase (ALP) activity, calcium deposition, and gene expression. The in vitro studies showed that sustained release of Aln from the BCP scaffolds consisted of porous microstructures, and revealed that MG-63 cells cultured on Aln-loaded BCP scaffolds showed significantly increased ALP activity, calcium deposition, and gene expression compared to cells cultured on BCP scaffolds. The in vivo studies using radiograph and histology examination revealed abundant callus formation and bone maturation at the site in the Aln/BCP groups compared to the control group. However, solid bony bridge formation was not observed at plain radiographs until 8 weeks. Micro-CT analysis revealed that bone mineral density and bone formation volume were increased over time in an Aln concentration-dependent manner. These results suggested that Aln/BCP scaffolds have the potential for controlling the release of Aln and enhance bone formation and mineralization.

Fabrication of a BMP-2-immobilized porous microsphere modified by heparin for bone tissue engineering.

The purpose of this study was to fabricate BMP-2-immobilized porous poly(lactide-co-glycolide) (PLGA) microspheres (PMS) modified with heparin for bone regeneration. A fluidic device was used to fabricate PMS and the fabricated PMS was modified with heparin-dopamine (Hep-DOPA). Bone morphogenic protein-2 (BMP-2) was immobilized on the heparinized PMS (Hep-PMS) via electrostatic interactions. Both PMS and modified PMS were characterized using scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). MG-63 cell activity on PMS and modified PMS were assessed via alkaline phosphatase (ALP) activity, calcium deposition, and osteocalcin and osteopontin mRNA expression. Immobilized Hep-DOPA and BMP-2 on PMS were demonstrated by XPS analysis. BMP-2-immobilized Hep-PMS provided significantly higher ALP activity, calcium deposition, and osteocalcin and osteopontin mRNA expression compared to PMS alone. These results suggest that BMP-2-immobilized Hep-PMS effectively improves MG-63 cell activity. In conclusion, BMP-2-immobilized Hep-PMS can be used to effectively regenerate bone defects.

Alendronate-Eluting Biphasic Calcium Phosphate (BCP) Scaffolds Stimulate Osteogenic Differentiation.

Biphasic calcium phosphate (BCP) scaffolds have been widely used in orthopedic and dental fields as osteoconductive bone substitutes. However, BCP scaffolds are not satisfactory for the stimulation of osteogenic differentiation and maturation. To enhance osteogenic differentiation, we prepared alendronate- (ALN-) eluting BCP scaffolds. The coating of ALN on BCP scaffolds was confirmed by scanning electron microscopy (FE-SEM), energy-dispersive X-ray spectroscopy (EDS), and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). An in vitro release study showed that release of ALN from ALN-eluting BCP scaffolds was sustained for up to 28 days. In vitro results revealed that MG-63 cells grown on ALN-eluting BCP scaffolds exhibited increased ALP activity and calcium deposition and upregulated gene expression of Runx2, ALP, OCN, and OPN compared with the BCP scaffold alone. Therefore, this study suggests that ALN-eluting BCP scaffolds have the potential to effectively stimulate osteogenic differentiation.

Improving Osteogenesis Activity on BMP-2-Immobilized PCL Fibers Modified by the γ-Ray Irradiation Technique.

The purpose of this study was to demonstrate the ability of BMP-2-immobilized polycaprolactone (PCL) fibers modified using the γ-ray irradiation technique to induce the osteogenic differentiation of MG-63 cells. Poly acrylic acid (AAc) was grafted onto the PCL fibers by the γ-ray irradiation technique. BMP-2 was then subsequently immobilized onto the AAc-PCL fibers (BMP-2/AAc-PCL). PCL and surface-modified PCL fibers was characterized by evaluation with a scanning electron microscope (SEM), X-ray photoelectron spectroscopy (XPS), and contact angle. The biological activity of the PCL and surface-modified PCL fibers were characterized by alkaline phosphatase (ALP) activity, calcium deposition, and the mRNA expression of osteocalcin and osteopontin in MG-63 cells. Successfully grafted AAc and PCL fibers with immobilized BMP-2 were confirmed by XPS results. The results of the contact angle showed that BMP-2/AAc-PCL fibers have more hydrophilic properties in comparison to PCL fibers. The ALP activity, calcium deposition, and gene expressions of MG-63 cells grown on BMP-2/AAc-PCL fibers showed greatly induced osteogenic differentiation in comparison to the PCL fibers. In conclusion, these results demonstrated that BMP-2/AAc-PCL fibers have the potential to effectively induce the osteogenic differentiation of MG-63 cells.

Co-delivery of platelet-derived growth factor (PDGF-BB) and bone morphogenic protein (BMP-2) coated onto heparinized titanium for improving osteoblast function and osteointegration.

The aim of this study was to improve osteoblast function by delivering two growth factors, PDGF-BB and BMP-2, incorporated onto heparinized titanium (Hep-Ti) substrate. To achieve co-delivery of PDGF-BB and BMP-2, the surface of anodized Ti was immobilized with heparin, and then the two growth factors were coated onto the Hep-Ti surface. Incorporation of the two growth factors onto Hep-Ti was evaluated by SEM and XPS. Incorporated PDGF-BB and BMP-2 were released from the Hep-Ti substrate in a sustained manner. In vitro studies revealed that osteoblasts grown on PDGF-BB- and BMP-2-immobilized Hep-Ti increased ALP activity, calcium deposition, osteocalcin and osteopontin levels as compared to those grown on PDGF-BB alone- or BMP-2 alone-immobilized Hep-Ti. These results suggested that co-delivery of PDGF-BB and BMP-2 using Hep-Ti substrate will be a promising material for the enhancement of osteoblast function and osteointegration.

Osteoblast activity of MG-63 cells is enhanced by growth on a lactoferrin-immobilized titanium substrate.

The aim of this study was to develop a lactoferrin (LF)-immobilized titanium (Ti) substrate to enhance the osteoblast activity of MG-63 cells. Ti substrates were first modified through heparin-dopamine (Hep-DOPA) anchorage. Then, LF was immobilized on the Hep-Ti substrates via electrostatic interactions. Hep-Ti substrates, with or without LF, were evaluated by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and contact angle measurements. Sustained release of LF on the Ti substrates was observed over a 28-day period. In vitro studies of osteoblast activity showed increased alkaline phosphatase activity and calcium deposition by MG-63 cells cultured on LF-immobilized Ti substrates as compared to those cultured on pristine Ti substrates, indicating that LF-immobilized Ti substrates were effective at enhancing osteoblast activity.

Improving osteoblast functions and bone formation upon BMP-2 immobilization on titanium modified with heparin.

The aim of this study was to develop bone morphogenetic protein-2 (BMP-2) immobilization on titanium (Ti) modified by heparin for improving osteoblast function in vitro and bone formation in vivo. The Ti surface was first modified with heparin and then immobilized with BMP-2 (BMP-2/Hep-Ti). The Ti and modified Ti were characterized by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and contact angle. In vitro studies demonstrated that osteoblasts cultured on BMP-2/Hep-Ti substrate increased ALP activity, calcium deposition, osteocalcin (OCN) and osteopontin (OPN) levels as compared to those cultured on Ti or BMP-2/Ti. In addition, intra-thread bone density and bone to implant contact ratio of BMP-2/Hep-Ti were significantly greater than those of Ti in the in vivo study. In conclusion, BMP-2 immobilized Ti modified heparin implants seemed to be a suitable delivery system for BMP-2 to improve osteoblast functions and new bone formation at the defect area around an implant.

Effect of lactoferrin-impregnated porous poly(lactide-co-glycolide) (PLGA) microspheres on osteogenic differentiation of rabbit adipose-derived stem cells (rADSCs).

The aim of this study was to develop lactoferrin (LF)-impregnated porous poly(lactide-co-glycolide) (PLGA) microspheres (PMs) to induce osteogenic differentiation of rabbit adipose-derived stem cells (rADSCs). Porous PLGA PMs were fabricated by a fluidic device and their surfaces were modified with heparin-dopamine (Hep-DOPA). Then, LF (100µg, 500µg, and 1000µg) was impregnated on the surface of heparinized PMs (Hep-PMs) via electrostatic interactions to yield LF-impregnated PMs. PMs and modified PMs were characterized by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Osteogenic differentiation of rADSCs on PMs and modified PMs was demonstrated by alkaline phosphatase (ALP) activity, calcium deposition, and mRNA expression of osteocalcin and osteopontin. Successful immobilization of Hep-DOPA and LF on the surface of PMs was confirmed by XPS analysis. LF-impregnated PMs generated significantly greater ALP activity, calcium deposition, and mRNA expression of osteocalcin and osteopontin compared with PMs. These results suggested that LF-impregnated PMs effectively induced osteogenic differentiation of rADSCs.

Bone formation in a rat tibial defect model using carboxymethyl cellulose/BioC/bone morphogenic protein-2 hybrid materials.

The objective of this study was to assess whether carboxymethyl cellulose- (CMC-) based hydrogel containing BioC (biphasic calcium phosphate (BCP); tricalcium phosphate (TCP) : hydroxyapatite (Hap) = 70 : 30) and bone morphogenic protein-2 (BMP-2) led to greater bone formation than CMC-based hydrogel containing BioC without BMP-2. In order to demonstrate bone formation at 4 and 8 weeks, plain radiographs, microcomputed tomography (micro-CT) evaluation, and histological studies were performed after implantation of all hybrid materials on an 8 mm defect of the right tibia in rats. The plain radiographs and micro-CT analyses revealed that CMC/BioC/BMP-2 (0.5 mg) led to much greater mineralization at 4 and 8 weeks than did CMC/BioC or CMC/Bio/BMP-2 (0.1 mg). Likewise, bone formation and bone remodeling studies revealed that CMC/BioC/BMP-2 (0.5 mg) led to a significantly greater amount of bone formation and bone remodeling at 4 and 8 weeks than did CMC/BioC or CMC/BioC/BMP-2 (0.1 mg). Histological studies revealed that mineralized bone tissue was present around the whole circumference of the defect site with CMC/BioC/BMP-2 (0.5 mg) but not with CMC/BioC or CMC/BioC/BMP-2 (0.1 mg) at 4 and 8 weeks. These results suggest that CMC/BioC/BMP-2 hybrid materials induced greater bone formation than CMC/BioC hybrid materials. Thus, CMC/BioC/BMP-2 hybrid materials may be used as an injectable substrate to regenerate bone defects.

The effect of alendronate-loaded polycarprolactone nanofibrous scaffolds on osteogenic differentiation of adipose-derived stem cells in bone tissue regeneration.

The osteogenic effect of culturing adipose-derived stem cells (ADSCs) on alendronate (Aln)-loaded polycarprolactone (PCL) nanofibrous scaffolds was evaluated by examining alkaline phosphatase (ALP) activity, calcium content, and expression of osteogenic differentiation genes in vitro. The 10% Aln/PCL nanofibrous scaffolds showed more ALP activity, mineralization, and osteocalcin and osteopontin mRNA than the 1% or 5% Aln/PCL nanofibrous scaffolds. The capacity of Aln/PCL nanofibrous scaffolds to regenerate new bone was studied in a rat calvarial defect model. New bone formation in vivo was evaluated by radiography, micro-computed tomography, and histological analysis. At 8 weeks after implantation, Aln/PCL scaffolds had a positive effect on bone regeneration and matrix formation. These results suggested that Aln/PCL nanofibrous scaffolds enhanced the osteogenic differentiation of ADSCs in vitro and bone formation in vivo.

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

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

Osteogenesis induction of periodontal ligament cells onto bone morphogenic protein-2 immobilized PCL fibers.

The purpose of this study was to develop bone morphogenic protein-2 (BMP-2) immobilized PCL fibers to induce osteogenic differentiation of periodontal ligament cells (PDLCs). The PCL fiber surface was modified with heparin-dopamine (Hep-DOPA) (Hep-PCL) and further immobilized with BMP-2 (BMP-2/Hep-PCL). PCL fibers and surface-modified PCL fibers (Hep-PCL and BMP-2/Hep-PCL) were characterized by X-ray photoelectron spectroscopy (XPS) and contact angle. Osteogenic differentiation of PDLCs was demonstrated by alkaline phosphatase (ALP) activity, calcium deposition, and gene expression. The results of XPS and contact angle revealed that Hep-DOPA and BMP-2 were successfully immobilized onto the PCL surface and that the BMP-2/Hep-PCL fibers have more hydrophilic surface properties than PCL fibers alone. ALP activity, calcium deposition, and gene expression on BMP-2/Hep-PCL fibers showed significantly induced osteogenic differentiation relative to PCL fibers. Therefore, we suggest that BMP-2/Hep-PCL fibers have the potential to effectively induce osteogenic differentiation of PDLCs.

The effect of titanium with heparin/BMP-2 complex for improving osteoblast activity.

The objective of this study was to investigate the enhanced osteoblast activity of MG-63 cells cultured on titanium (Ti) with a heparin/BMP-2 (Hep/BMP-2) complex. The Ti substrates were initially modified by chemical grafting poly-L-lysine (PLL) using condensing agent, followed by immobilizing the heparin/BMP-2 complex to the PLL-grafted Ti substrate via electrostatic interactions. The surface modification of Ti substrates with PLL and/or Hep/BMP-2 complex were confirmed with scanning electron microscopy, contact angle measurements, and X-ray photoelectron spectroscopy. Immobilized BMP-2 was released from the Hep/BMP-2/Ti substrate in a sustained manner. In vitro studies revealed that osteoblasts grown on Hep/BMP-2/Ti substrate increased ALP activity, calcium deposition, ALP and osteocalcin levels as compared to those grown on pristine Ti or PLL-Ti. These results indicated that heparin/BMP-2 complex immobilized Ti substrate can be useful to effectively improve osteoblast activity.

Bone formation of middle ear cavity using biphasic calcium phosphate lyophilized with Escherichia coli-derived recombinant human bone morphogenetic protein 2 using animal model.

OBJECTIVE: The aim of the study was to analyze the value of Escherichia coli-derived recombinant human bone morphogenetic protein-2 (ErhBMP-2) coated biphasic calcium phosphate (BCP) for the obliteration of middle ear bone defect after mastoid surgery. METHODS: Twenty-four specific pathogen-free Sprague-Dawley rats were randomly assigned to the BCP group (n=12) and BCP-ErhBMP-2 group (n=12; in which BCP scaffold of the granular type was coated with ErhBMP-2). In both groups, BCP scaffold was used to surgically fill the middle ear bulla. New bone formation was evaluated by measuring bone density (%) after 4 and 8 weeks in all rats in both groups. RESULTS: At 4 weeks, new bone was visible at the periphery and center of the middle ear cavity in both groups. In the BCP group, a moderate amount of fibrous tissue had infiltrated into the interspace of the scaffolds. New bone almost totally filled the interspace in the BCP-ErhBMP-2 group. At 8 weeks, copious new bone formation had occurred. Histometric measurements showed that bone density in the BCP group was smaller than in the BCP-ErhBMP-2 group at 4 weeks (25.10% and 38.43%, respectively; p<0.05) and 8 weeks (25.54% and 34.18%, respectively; p<0.05). CONCLUSIONS: New bone formation was greater in the presence of BCP-ErhBMP-2 scaffolds. ErhBMP-2 coated BCP scaffolds is a potentially useful material for middle ear obliteration after mastoidectomy.

Local delivery of alendronate eluting chitosan scaffold can effectively increase osteoblast functions and inhibit osteoclast differentiation.

The aim of this study was to investigate the effect of alendronate released from chitosan scaffolds on enhancement of osteoblast functions and inhibition of osteoclast differentiation in vitro. The surface and cell morphologies of chitosan scaffolds and alendronate-loaded chitosan scaffolds were characterized by variable pressure field emission scanning electron microscope (VP-FE-SEM). Alendronate was released in a sustained manner. For evaluating osteoblast functions in MG-63 cells, we investigated cell proliferation, alkaline phosphatase (ALP) activity, and calcium deposition. Furthermore, for evaluating inhibition of osteoclast differentiation in RAW 264.7 cells, we investigated tartrate-resistant acid phosphatase (TRAP) activity, TRAP staining, and gene expressions. The in vitro studies revealed that osteoblasts grown on alendronate-loaded chitosan scaffold showed a significant increment in cell proliferation, ALP activity, and calcium deposition as compared to those grown on chitosan scaffolds. In addition, the in vitro study showed that osteoclast differentiation in RAW 264.7 cells cultured on alendronate-loaded chitosan scaffolds was greatly inhibited as compared to those cultured on chitosan scaffolds by the results of TRAP activity, TRAP staining, and gene expressions. Taken together, alendronate-loaded chitosan scaffolds could achieve the dual functions of improvement in osteoblast functions and inhibition of osteoclast differentiation. Thus, alendronate-eluting chitosan substrates are promising materials for enhancing osteoblast functions and inhibiting osteoclast differentiation in orthopedic and dental fields.