In vitro and in vivo evaluation of chitosan–gelatin scaffolds for cartilage tissue engineering

Chitosan–gelatin polyelectrolyte complexes were fabricated and evaluated as tissue engineering scaffolds for cartilage regeneration in vitro and in vivo. The crosslinker for the gelatin component was selected among glutaraldehyde, bisepoxy, and a water-soluble carbodiimide (WSC) based upon the proliferation of chondrocytes on the crosslinked gelatin. WSC was found to be the most suitable crosslinker. Complex scaffolds made from chitosan and gelatin with a component ratio equal to one possessed the proper degradation rate and mechanical stability in vitro. Chondrocytes were able to proliferate well and secrete abundant extracellular matrix in the chitosan–gelatin (1:1) complex scaffolds crosslinked by WSC (C1G1_WSC) compared to the non-crosslinked scaffolds. Implantation of chondrocytes-seeded scaffolds in the defects of rabbit articular cartilage confirmed that C1G1_WSC promoted the cartilage regeneration. The neotissue formed the histological feature of tide line and lacunae in 6.5 months. The amount of glycosaminoglycans in C1G1_WSC constructs (0.187 ± 0.095 μg/mg tissue) harvested from the animals after 6.5 months was 14 wt.% of that in normal cartilage (1.329 ± 0.660 μg/mg tissue). The average compressive modulus of regenerated tissue at 6.5 months was about 0.539 MPa, which approached to that of normal cartilage (0.735 MPa), while that in the blank control (3.881 MPa) was much higher and typical for fibrous tissue. Type II collagen expression in C1G1_WSC constructs was similarly intense as that in the normal hyaline cartilage. According to the above results, the use of C1G1_WSC scaffolds may enhance the cartilage regeneration in vitro and in vivo.     

Xylan polysaccharides fabricated into nanofibrous substrate for myocardial infarction

Myocardial infarction, a main cause of heart failure, leads to loss of cardiac tissue impairment of left ventricular function. Repair of diseased myocardium with in vitro engineered cardiac muscle patch/injectable biopolymers with cells may become a viable option for myocardial infarction. We attempted to solve these problems by in vitro study by selecting a plant based polysaccharides beech wood Xylan for the normal functioning of infarcted myocardium. The present study fabricated Xylan based nanofibrous scaffolds cross-linked with glutaraldehyde (Glu) vapors for 24 h, 48 h and 1% Glu blended fibers for the culture of neonatal rat cardiac cells for myocardial infarction. These nanofibers were characterized by SEM, FT-IR, tensile testing and cell culture studies for the normal expression of cardiac proteins. The observed results showed that the Xylan/polyvinyl alcohol (PVA) 24 h Glu vapor cross-linked nanofibers (427 nm) having mechanical strength of 2.43 MPa and Young modulus of 3.74 MPa are suitable for the culture of cardiac cells. Cardiac cells proliferation increased only by 11% in Xylan/PVA 24 h Glu cross-linked nanofibers compared to control tissue culture plate (TCP). The normal cardiac cell morphology was observed in 24 h cross-linked Xylan/PVA nanofibers but 48 h cross-linked fibers cell morphology was changed to flattened and elongated on the fibrous surfaces. Confocal analysis for cardiac expression proteins actinin, connexin 43 was observed normally in 24 h Glu cross-linked nanofibers compared to all other nanofibrous scaffolds. The fabricated Xylan/PVA nanofibrous scaffold may have good potential for the normal functioning of infarcted myocardium.     

Preparation and characterization of bimodal porous poly(γ-benzyl-L-glutamate) scaffolds for bone tissue engineering

An ideal scaffold in bone tissue-engineering strategy should provide biomimetic extracellular matrix-like architecture and biological properties. Poly(γ-benzyl-L-glutamate) (PBLG) has been a popular model polypeptide for various potential biomedical applications due to its good biocompatibility and biodegradability. This study developed novel bimodal porous PBLG polypeptide scaffolds via a combination of biotemplating method and in situ ring-opening polymerization of γ-benzyl-L-gIutamate N-carboxyanhydride (BLG-NCA). The PBLG scaffolds were characterized by proton nuclear magnetic resonance spectroscopy, X-ray diffraction, differential scanning calorimetry, scanning electron microscope (SEM) and mechanical test. The results showed that the semi-crystalline PBLG scaffolds exhibited an anisotropic porous structure composed of honeycomb-like channels (100–200 μm in diameter) and micropores (5–20 μm), with a very high porosity of 97.4 ± 1.6%. The compressive modulus and glass transition temperature were 402.8 ± 20.6 kPa and 20.2 °C, respectively. The in vitro biocompatibility evaluation with MC3T3-E1 cells using SEM, fluorescent staining and MTT assay revealed that the PBLG scaffolds had good biocompatibility and favored cell attachment, spread and proliferation. Therefore, the bimodal porous polypeptide scaffolds are promising for bone tissue engineering.     

Biological evaluation of human hair keratin scaffolds for skin wound repair and regeneration

The cytocompatibility, in vivo biodegradation and wound healing of keratin biomaterials were investigated. For the purposes, three groups of keratin scaffolds were fabricated by freeze-drying reduced solutions at 2 wt.%, 4 wt.% and 8 wt.% keratins extracted from human hairs. These scaffolds exhibited evenly distributed high porous structures with pore size of 120–220 μm and the porosity > 90%. NIH3T3 cells proliferated well on these scaffolds in culture lasting up to 22 days. Confocal micrographs stained with AO visually revealed cell attachment and infiltration as well as scaffold architectural stability. In vivo animal experiments were conducted with 4 wt.% keratin scaffolds. Early degradation of subcutaneously implanted scaffolds occurred at 3 weeks in the outermost surface, in concomitant with inflammatory response. At 5 weeks, the overall porous structure of scaffolds severely deteriorated while the early inflammatory response in the outermost surface obviously subsided. A faster keratin biodegradation was observed in repairing full-thickness skin defects. Compared with the blank control, keratin scaffolds gave rise to more blood vessels at 2 weeks and better complete wound repair at 3 weeks with a thicker epidermis, less contraction and newly formed hair follicles. These preliminary results suggest that human hair keratin scaffolds are promising dermal substitutes for skin regeneration.     

Fabrication and evaluation of biomimetic scaffolds by using collagen–alginate fibrillar gels for potential tissue engineering applications

Pore architecture and its stable functionality under cell culturing of three dimensional (3D) scaffolds are of great importance for tissue engineering purposes. In this study, alginate was incorporated with collagen to fabricate collagen–alginate composite scaffolds with different collagen/alginate ratios by lyophilizing the respective composite gels formed via collagen fibrillogenesis in vitro and then chemically crosslinking. The effects of alginate amount and crosslinking treatment on pore architecture, swelling behavior, enzymatic degradation and tensile property of composite scaffolds were systematically investigated. The relevant results indicated that the present strategy was simple but efficient to fabricate highly interconnected strong biomimetic 3D scaffolds with nanofibrous surface. NIH3T3 cells were used as a model cell to evaluate the cytocompatibility, attachment to the nanofibrous surface and porous architectural stability in terms of cell proliferation and infiltration within the crosslinked scaffolds. Compared with the mechanically weakest crosslinked collagen sponges, the cell-cultured composite scaffolds presented a good porous architecture, thus permitting cell proliferation on the top surface as well as infiltration into the inner part of 3D composite scaffolds. These composite scaffolds with pore size ranging from 150 to 300 μm, over 90% porosity, tuned biodegradability and water-uptake capability are promising for tissue engineering applications.     

Thermal-crosslinked porous chitosan scaffolds for soft tissue engineering applications

The aim of this study was to demonstrate the feasibility of using a steam autoclave process for sterilization and simultaneously thermal-crosslinking of lyophilized chitosan scaffolds. This process is of great interest in biomaterial development due to its simplicity and low toxicity. The steam autoclave process had no significant effect on the average pore diameter (~ 70 μm) and overall porosity (> 80%) of the resultant chitosan scaffolds, while the sterilized scaffolds possessed more homogenous pore size distribution. The sterilized chitosan scaffolds exhibited an enhanced compressive modulus (109.8 kPa) and comparable equilibrium swelling ratio (23.3). The resultant chitosan scaffolds could be used directly for in vitro cell culture without extra sterilization. The data of in vitro studies demonstrated that the scaffolds facilitated cell attachment and proliferation, indicating great potential for soft tissue engineering applications.     

In vitro study on porous silver scaffolds prepared by electroplating method using cellular carbon skeleton as the substrate

Porous silver scaffolds, with the porosity ranging from 68% to 81% and the apparent density ranging from 0.4 to 1 g?cm^? 3 were prepared by electroplating method using cellular carbon skeleton as the substrate. The microstructure, mechanical property, cytotoxicity and antibacterial activity of the prepared porous silver scaffold were studied. The present porous silver scaffolds had a highly three-dimensional trabecular porous structure with the porosity and the apparent density close to that of the cancellous bone. Furthermore, the mechanical property such as elastic modulus and yield strength of the porous silver scaffolds were lower than that of commercial available porous Ti and porous Ti alloys but much closer to that of the cancellous bone and porous Ta. In addition, study of in vitro behavior showed that the porous silver scaffold possessed significant antibacterial capability of inhibition of bacterial proliferation and adherence against Staphylococcus aureus and Staphylococcus epidermidis, and little cytotoxicity to Mg-63 cell line and NIH-3T3 cell line. Consequently, the porous silver scaffolds prepared by electrodeposition possess a promising application for bone implants.     

Porous poly(vinyl alcohol)/sepiolite bone scaffolds: Preparation,structure and mechanical properties

Porous poly(vinyl alcohol) (PVA)/sepiolite nanocomposite scaffolds containing 0–10 wt.% sepiolite were prepared by freeze-drying and thermally crosslinked with poly(arylic acid). The microstructure of the obtained scaffolds was characterised by scanning electron microscopy and micro-computed tomography, which showed a ribbon and ladder like interconnected structure. The incorporation of sepiolite increased the mean pore size and porosity of the PVA scaffold as well as the degree of anisotropy due to its fibrous structure. The tensile strength, modulus and energy at break of the PVA solid material that constructed the scaffold were found to improve with additions of sepiolite by up to 104%, 331% and 22% for 6 wt.% clay. Such enhancements were attributed to the strong interactions between the PVA and sepiolite, the good dispersion of sepiolite nanofibres in the matrix and the intrinsic properties of the nanofibres. However, the tensile properties of the PVA scaffold deteriorated in the presence of sepiolite because of the higher porosity, pore size and degree of anisotropy. The PVA/sepiolite nanocomposite scaffold containing 6 wt.% sepiolite was characterised by an interconnected structure, a porosity of 89.5% and a mean pore size of 79 μm and exhibited a tensile strength of 0.44 MPa and modulus of 14.9 MPa, which demonstrates potential for this type of materials to be further developed as bone scaffolds.     

Crosslinked gelatin nanofibres: Preparation,characterisation and in vitro studies using glial-like cells

Gelatin (GL) nanofibrous matrices mimicking the complex biological structure of the natural extracellular matrix (ECM) were prepared from aqueous solutions by electrospinning technique. GL nanofibres with a diameter size of around 300 nm were obtained optimising the process and solution parameters. To increase the GL stability in aqueous environment γ-glycidoxypropyltrimethoxysilane (GPTMS) was used as GL crosslinker. GPTMS crosslinking did not modify the nanofibrous matrix morphology: fibre diameter and membrane pores size were 327 ± 45 nm and 1.64 ± 0.37 μm, respectively. The produced GPTMS crosslinked GL nanofibres (GL/GPTMS_NF) were found to support the in vitro adhesion, proliferation and survival of neonatal olfactory bulb ensheating cells (NOBECs).     

Modification of porous calcium phosphate surfaces with different geometries of bioactive glass nanoparticles

In this study, the effects of bioactive glass nanoparticles' (nBGs) size and shape incorporated into hydroxyapatite/β-tricalcium phosphate (BCP) scaffolds were investigated. We prepared a highly porous (> 85%) BCP scaffold and coated its surface with a nanocomposite layer consisted of polycaprolactone (PCL) and rod (~ 153 nm in height and ~ 29 nm in width) or spherical (~ 33 nm and 64 nm in diameter) nBGs. Osteogenic gene expression by primary human osteoblast-like cells (HOB) was investigated using quantitative real time polymerase chain reaction (q-RT-PCR). We demonstrated for the first time that in vitro osteogenesis is dramatically affected by the shape of the nBGs, whereby rod shaped nBGs showed the most significant osteogenic induction, compared to spherical particles (regardless of their size). Importantly, the good biological effect observed for the rod shaped nBGs was coupled by a marked increase in the modulus (~ 48 MPa), compressive strength (~ 1 MPa) and failure strain (~ 6%), compared to those for the BCP scaffolds (~ 4 MPa, ~ 1 MPa and ~ 0.5% respectively). The findings of this study demonstrated that the shape of the nBGs is of significant importance when considering bone regeneration.     

Preparation,characterization and biological test of 3D-scaffolds based on chitosan,fibroin and hydroxyapatite for bone tissue engineering

This work describes the preparation and characterization of porous 3D-scaffolds based on chitosan (CHI), chitosan/silk fibroin (CHI/SF) and chitosan/silk fibroin/hydroxyapatite (CHI/SF/HA) by freeze drying. The biomaterials were characterized by X-ray diffraction, attenuated total reflection Fourier transform infrared spectroscopy, thermogravimetric analysis, differential scanning calorimetry, scanning electron microscopy and energy dispersive spectroscopy. In addition, studies of porosity, pore size, contact angle and biological response of SaOs-2osteoblastic cells were performed. The CHI scaffolds have a porosity of 94.2 ± 0.9%, which is statistically higher than the one presented by CHI/SF/HA scaffolds, 89.7 ± 2.6%. Although all scaffolds were able to promote adhesion, growth and maintenance of osteogenic differentiation of SaOs-2 cells, the new 3D-scaffold based on CHI/SF/HA showed a significantly higher cell growth at 7 days and 21 days and the level of alkaline phosphatase at 14 and 21 days was statistically superior compared to other tested materials.     

Human-like collagen/nano-hydroxyapatite scaffolds for the culture of chondrocytes

Three dimensional (3D) biodegradable porous scaffolds play a key role in cartilage tissue repair. Freeze-drying and cross-linking techniques were used to fabricate a 3D composite scaffold that combined the excellent biological characteristics of human-like collagen (HLC) and the outstanding mechanical properties of nano-hydroxyapatite (nHA). The scaffolds were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and compression tests, using Relive® Artificial Bone (RAB) scaffolds as a control. HLC/nHA scaffolds displayed homogeneous interconnected macroporous structure and could withstand a compression stress of 2.67 ± 0.37 MPa, which was higher than that of the control group. Rabbit chondrocytes were seeded on the composite porous scaffolds and cultured for 21 days. Cell/scaffold constructs were examined using SEM, histological procedures, and biochemical assays for cell proliferation and the production of glycosaminoglycans (GAGs). The results indicated that HLC/nHA porous scaffolds were capable of encouraging cell adhesion, homogeneous distribution and abundant GAG synthesis, and maintaining natural chondrocyte morphology compared to RAB scaffolds. In conclusion, the presented data warrants the further exploration of HLC/nHA scaffolds as a potential biomimetic platform for chondrocytes in cartilage tissue engineering.     

Facile fabrication of the porous three-dimensional regenerated silk fibroin scaffolds

In the present work, we report a new facile method to fabricate porous three-dimensional regenerated silk fibroin (RSF) scaffolds through n-butanol- and freezing-induced conformation transition and phase separation. The effects of RSF concentration, freezing temperature and n-butanol addition on the microstructure, the secondary structures of silk fibroin and apparent mechanical properties of the RSF scaffolds were investigated by SEM, ¹³C CP-MAS NMR spectra and mechanical testing, respectively. By adjusting the RSF concentration and n-butanol addition, the pore size of the scaffold could be controlled in the range from of 10 μm to 350 μm with 84%–98% of porosity. The tensile strength of the wet scaffold reached the maximum of 755.2 ± 33.6 kPa when the concentration of RSF solution was increased to 15% w/w. Moreover, post-treatment with ethanol further induced conformation transition of RSF from random coil or helix to β-sheet. The porous scaffolds prepared by this facile and energy-saving method with good biocompatibility will have great potential for application in tissue engineering.     

Porous and strong bioactive glass (13–93) scaffolds fabricated by freeze extrusion technique

Scaffolds fabricated by current methods often lack the combination of high strength and high porosity for skeletal substitution of load-bearing bones. In this work, freeze extrusion fabrication (FEF), a solid freeform fabrication technique, was investigated for the creation of porous and strong bioactive glass (13–93) scaffolds for potential applications in the repair of loaded bone. The process parameters for forming three-dimensional (3D) scaffolds with a pre-designed, grid-like microstructure by FEF were determined. Following thermal treatment of the as-formed constructs at temperatures up to 700 °C, scaffolds consisting of dense glass struts and interconnecting pores (porosity ≈ 50%; pore width ≈ 300 μm) were obtained. These scaffolds showed an elastic mechanical response in compression, with a compressive strength of 140 ± 70 MPa and an elastic modulus of 5.5 ± 0.5 GPa, comparable to the values for human cortical bone. The scaffolds supported the proliferation of osteogenic cells in vitro, showing their biocompatibility. These results indicate that 13–93 bioactive glass scaffolds created by the FEF method could have potential application in the repair and regeneration of load-bearing bones.     

Stability and mechanical evaluation of bovine pericardium cross-linked with polyurethane prepolymer in aqueous medium

The present study investigates the potential use of non-catalyzed water-soluble blocked polyurethane prepolymer (PUP) as a bifunctional cross-linker for collagenous scaffolds. The effect of concentration (5, 10, 15 and 20%), time (4, 6, 12 and 24 h), medium volume (50, 100, 200 and 300%) and pH (7.4, 8.2, 9 and 10) over stability, microstructure and tensile mechanical behavior of acellular pericardial matrix was studied. The cross-linking index increased up to 81% while the denaturation temperature increased up to 12 °C after PUP crosslinking. PUP-treated scaffold resisted the collagenase degradation (0.167 ± 0.14 mmol/g of liberated amine groups vs. 598 ± 60 mmol/g for non-cross-linked matrix). The collagen fiber network was coated with PUP while viscoelastic properties were altered after cross-linking. The treatment of the pericardial scaffold with PUP allows (i) different densities of cross-linking depending of the process parameters and (ii) tensile properties similar to glutaraldehyde method.     

Development,characterization, and in vitro bioactivity studies of sol–gel bioactive glass/poly(l-lactide) nanocomposite scaffolds

Developing materials combining the advantages of synthetic polymers and bioactive glass nanoparticles can provide an efficient bone engineering scaffold. In this study, sol–gel bioactive glass (SG) nanoparticles were synthesized by quick alkali-mediation; sol–gel derived bioactive glass/poly(l-lactide) nanocomposite scaffolds were then developed. The influence of the glass content on the porosity of nanocomposite scaffolds was evaluated by SEM. The results showed that the neat polymer scaffold (PLA) has a highly interconnected porous structure with a maximum pore size of about 250 μm. For the composite scaffold containing 25 wt.% glass (SGP25), the decrease in the maximum pore size, (to about 200 μm) was not significant while for the SGP50 composite scaffold containing 50 wt.% glass it was a significant decrease (to about 100 μm). The apparent porosity of the scaffolds was 56.56% ± 7.15, 54.14% ± 3.84, and 53.11% ± 3.99 for PLA, SGP25, and, SGP50 respectively. FT-IR, TGA, and XRD results revealed some interaction of the glass filler with the polymeric matrix in the scaffolds. The degradation study showed that, by increasing the glass content in the scaffolds, the water absorption decreased, the weight loss increased, and the cumulative ion concentrations released from them also increased. This indicates the possibility of modulating the degradation rate by varying the glass/polymer ratio. At the end of the incubation period, the weight losses were around 5.44% ± 0.96, 32.50% ± 2.73, and 41.47% ± 3.02 for the PLA, SGP25, and SGP50, respectively. Moreover, the water uptake reached 119.65% ± 18.88 and 93.39% ± 13.01 for SGP25 and SGP50, respectively. The addition of the SG to the scaffolds was found to enhance their in vitro bioactivity. Therefore, these nanocomposite scaffolds have a potential to be applied in bone engineering. All data are expressed as mean ± standard deviation (n = 3).     

Radiation synthesis of gelatin/CM-chitosan/β-tricalcium phosphate composite scaffold for bone tissue engineering

A series of biodegradable composite scaffolds was fabricated from an aqueous solution of gelatin, carboxymethyl chitosan (CM-chitosan) and β-tricalcium phosphate (β-TCP) by radiation-induced crosslinking at ambient temperature. Ultrasonic treatment on the polymer solutions significantly influenced the distribution of β-TCP particles. An ultrasonic time of 20 min, followed by 30 kGy irradiation induced a crosslinked scaffold with homogeneous distribution of β-TCP particles, interconnected porous structure, sound swelling capacity and mechanical strength. Fourier Transform Infrared Spectroscopy and X-ray Diffraction analysis indicated that β-TCP successfully incorporated with the network of gelatin and CM-chitosan. In vivo implantation of the scaffold into the mandible of beagle dog revealed that the scaffolds had excellent biocompatibility and the presence of β-TCP can accelerate bone regeneration. The comprehensive results of this study paved way for the application of gelatin/CM-chitosan/β-TCP composite scaffolds as candidate of bone tissue engineering material.     

Nanoscale characterization of bone–implant interface and biomechanical modulation of bone ingrowth

Bone–implant interface is characterized by an array of cells and macromolecules. This study investigated the nanomechancial properties of bone–implant interface using atomic force microscopy in vitro, and the mechanical modulation of implant bone ingrowth in vivo using bone histomorphometry. Upon harvest of screw-type titanium implants placed in vivo in the rabbit maxilla and proximal femur for 4 weeks, nanoindentation was performed in the bone–implant interface at 60-μm intervals radially from the implant surface. The average Young's Moduli (E) of the maxillary bone–implant interface was 1.13 ± 0.27 MPa, lacking significant differences at all intervals. In contrast, an increasing gradient of E was observed radially from the femur bone–implant interface: 0.87 ± 0.25 MPa to 2.24 ± 0.69 MPa, representing significant differences among several 60-μm intervals. In a separate experiment, bone healing was allowed for 6 weeks for proximal femur implants. The right femoral implant received axial cyclic loading at 200 mN and 1 Hz for 10 min/d over 12 days, whereas the left femoral implant served as control. Cyclic loading induced significantly higher bone volume, osteoblast numbers per endocortical bone surface, mineral apposition rate, and bone formation rate than controls. These data demonstrate nanoscale and microscale characterizations of bone–implant interface, and mechanical modulation of bone ingrowth surrounding titanium implants.     

The effect of processing parameters and solid concentration on the mechanical and microstructural properties of freeze-casted macroporous hydroxyapatite scaffolds

The design and fabrication of macroporous hydroxyapatite scaffolds, which could overcome current bone tissue engineering limitations, have been considered in recent years. In the current study, controlled unidirectional freeze-casting at different cooling rates was investigated. In the first step, different slurries with initial hydroxyapatite concentrations of 7–37.5 vol.% were prepared. In the next step, different cooling rates from 2 to 14 °C/min were applied to synthesize the porous scaffold. Additionally, a sintering temperature of 1350 °C was chosen as an optimum temperature. Finally, the phase composition (by XRD), microstructure (by SEM), mechanical characteristics, and the porosity of sintered samples were assessed. The porosity of the sintered samples was in a range of 45–87% and the compressive strengths varied from 0.4 MPa to 60 MPa. The mechanical strength of the scaffolds increased as a function of initial concentration, cooling rate, and sintering temperature. With regards to mechanical strength and pore size, the samples with the initial concentration and the cooling rate of 15 vol.% and 5 °C/min, respectively, showed better results.     

Osteogenic effect of hyaluronic acid sodium salt in the pores of a hydroxyapatite scaffold

According to previous reports, a large volume of bone marrow cells (1 × 10⁷ cells/ml) is required for bone regeneration in the pores of a scaffold in vivo. We theorized that immersion of a porous hydroxyapatite (HA) scaffold in hyaluronic acid solution would facilitate bone formation in the scaffold at 1 × 10⁶ cells/ml density of bone marrow cells. The cells were respectively seeded into pores of the cylindrical HA scaffolds with a hollow center after immersion in hyaluronic acid solution or in culture medium. The scaffolds were implanted in the dorsal subcutis of rats for 4 weeks. Thereafter, serially sectioned paraffin specimens were made and observed histologically. Bone formation was observed in many pores of HA scaffold by immersion in hyaluronic acid solution. However, there were no or less pores with new bone formation in the scaffold by immersion in culture medium. The cells were cultured with and without hyaluronic acid in vitro. There was no significant difference in bone formation in vitro with and without hyaluronic acid. The results of this study suggest that hyaluronic acid binds to the cells on the wall of three-dimensional structure and effectively promotes new bone formation.