Candidate For Bone Tissue Engineering Research Articles

Amino acid surface modified bioglass: A candidate biomaterial for bone tissue engineering1.

Bioglasses are solid materials consisted of sodium oxide, calcium oxide, silicon dioxide and phosphorus in various proportions and have used in bone tissue engineering. There have been ongoing efforts to improve the surface properties of bioglasses to increase biocompatibility and performance. The aim of the present study is to modify the bioglass surface with an amino acid mixture consisting of arginine, aspartic acid, phenylalanine, cysteine, histidine and lysine, to characterize the surface, and to evaluate the performance and biocompatibility in vitro and in vivo. The untreated bioglass, bioglass kept in simulated body fluid (SBF), and modified bioglass were used in further evaluation. After confirmation of the surface modification with FT-IR analyses and SEM analyses, MC3T3-E1 preosteoblasts adhesion on the surface was also revealed by SEM. The modified bioglass had significantly higher ALP activity in colorimetric measurement, rate of calcium accumulations in Alizarin red s staining, lower rate of cell death in Annexin-V/PI staining to determine apoptosis and necrosis. Having higher cell viability rate in MTT test and absence of genotoxicity in micronucleus test (OECD 487), the modified bioglass was further confirmed for biocompatibility in vitro. The results of the rat tibial defect model revealed that the all bioglass treatments had a significantly better bone healing score compared to the untreated negative control. However, the modified bioglass exhibited significantly better bone healing efforts especially during the first and the second months compared to the other bioglass treatment treatments. As a result, the amino acid surface modification of bioglasses improves the surface biocompatibility and osteogenic performance that makes the amino acid modified bioglass a better candidate for bone tissue engineering. RESEARCH HIGHLIGHTS: Bioglass surface modification with amino acids contributes to bioglass-tissue interaction with an improved cell attachment. Modified bioglass increases in vitro Alp activity and calcium accumulation, and also positively affects cell behavior by supporting cell adaptation. Bioglass exerts osteogenic potential in vivo especially during early bone healing.

In Vitro Evaluation of Cellular Interactions with Nanostructured Spheres of Alginate and Zinc-Substituted Carbonated Hydroxyapatite.

The increasing demand for effective bone regeneration materials drives the exploration of biomaterials with enhanced bioactivity and biocompatibility, such as zinc-substituted compounds. This study investigates the in vitro cellular interactions with nanostructured spheres composed of alginate/carbonated hydroxyapatite (CHA), compared to zinc-substituted CHA (ZnCHA). This work aimed to compare the physicochemical properties and biological effects of ZnCHA and CHA on osteoblasts. ZnCHA was synthesized using a wet chemical method, followed by characterization through X-ray diffraction, Fourier transform infrared spectroscopy, total organic carbon analysis, Wavelength-dispersive X-ray spectroscopy, and BET surface area analysis to assess ion release and structural changes. Biological evaluation was conducted using cell viability, proliferation, and biomineralization assays on osteoblasts. Results showed successful incorporation of zinc and carbonate, leading to reduced crystallinity and increased surface area. Cell viability and proliferation assays indicated ZnCHA's cytocompatibility and enhanced osteoblastic activity, with increased mineralization nodules compared to CHA samples. The study concludes that ZnCHA composites are promising candidates for bone tissue engineering, demonstrating improved cytocompatibility and potential for further preclinical evaluations.

Mussel shell-derived biogenic hydroxyapatite as reinforcement on chitosan-loaded gentamicin composite for antibacterial activity and bone regeneration

In this study, hydroxyapatite (HAp) was synthesized from natural biowaste materials, specifically mussel shells, and combined with chitosan (CS) and gentamicin sulfate antibiotic (GA) using an in-situ method. The resulting composite material, designated HAp/CS-GA, has its physicochemical and structural properties characterized by Fourier transform infrared spectroscopy (FTIR) analysis. The structure was confirmed by X-ray diffraction (XRD) analysis. Additionally, field emission scanning electron microscopy (FE-SEM) equipped with the energy dispersive X-ray spectroscopic (EDX) technique was used to determine the surface topography and main components. The composite of HAp/CS-GA was analyzed using a drug release profile by UV–visible spectroscopy (UV–Vis). The fabricated composites antimicrobial behavior was examined against bone infection-causing Gram-positive and Gram-negative bacteria, showing potential activity against Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus compared to Escherichia coli, respectively. Simultaneously, the cytotoxicity of the composite was evaluated by MTT assay using an MG-63 osteoblast-like cell line that exhibited no toxicity in the prepared composite. After a 24 h incubation period, the MG-63 cells on the HAp/CS-GA composite showed good proliferation, according to Hoechst 33258 fluorescence staining results. The results suggested that the composite had excellent biocompatibility and antibacterial activity and enhanced the osteoblast cell proliferation. Therefore, the designed HAp/CS-GA composite would be a promising candidate for bone tissue engineering.

Bredigite Bioceramic: A Promising Candidate for Bone Tissue Engineering

Bredigite Bioceramic: A Promising Candidate for Bone Tissue Engineering

Additive manufacturing of polylactic acid scaffolds dip-coated with polycaprolactone for bone tissue engineering

Bone tissue engineering aims to create scaffolds that support bone regeneration, addressing the needs of approximately 1.71 billion people with bone structure problems worldwide. This study explores the fabrication and characterization of 3D-printed polylactic acid (PLA) scaffolds dip-coated with polycaprolactone (PCL), producing complex geometries with interconnected pores, specifically RoundBar Sphere and RoundBar Cube. The scaffolds were then coated with PCL solutions of 0.5 % and 1 % concentrations, applying up to three layers. Surface topology analysis indicated that PCL coating slightly reduced pore size (1150 µm to 900) while improving coverage and integrity. After coating, Fourier Transform Infrared Spectroscopy (FTIR) confirmed the presence of PCL on scaffold surfaces (characteristic bands at 1726, 1175 and 728 cm−1), whose a better coverage was obtained with more layers and higher concentrations of PCL. Coated scaffolds showed not significant change in compressive strengths (2–12 MPa), remaining suitable for trabecular bone applications. Hemolysis assays of the 3D scaffolds promoted non-hemolytic properties (0 % hemolysis), ensuring their blood compatibility. Metabolic activity (>70 %) and live/dead cell assays in human dermal fibroblasts (HDF) exhibited biocompatibilities for all samples, with coated scaffolds promoting enhanced cell proliferation compared to uncoated ones. Additionally, osteoblast metabolic activity (>90 %) and osteoblasts scratch assay demonstrated coated scaffolds promoted an area reduction of 1.36 and 1.53-fold higher than the control group and uncoated scaffold, respectively. In short, these coated scaffolds are promising candidates for bone tissue engineering and bone repair applications.

Exosome loaded 3D printed magnetic PLA constructs: a candidate for bone tissue engineering

Exosome loaded 3D printed magnetic PLA constructs: a candidate for bone tissue engineering

Transforming Natural Eggshell and Diatomite into Bioactive Calcium Silicate Material for Bone Regeneration.

Calcium silicate (CS), a new and important bioceramic bone graft material, is prepared by using eggshells, which have a porous structure and are rich in calcium ions. Furthermore, the preparation of new CS materials using eggshells and diatomaceous earth minimizes their negative impact on the environment. In this study, we prepared CS materials using a high-temperature calcination method. The composition of the material was demonstrated by X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) analysis. Scanning electron microscopy (SEM) analysis confirmed the porous structure of the CS material. We also introduced ZnO to prepare ZnO-CS with antibacterial properties and showed that ZnO-CS exhibits excellent antibacterial effects through in vitro antibacterial experiments. Subsequent in vitro mineralization experiments demonstrated that ZnO-CS promoted the formation of a hydroxyapatite layer. Furthermore, in vitro cytotoxicity experiments demonstrated that ZnO-CS had very good biosafety and promoted cell proliferation. These findings were confirmed through subsequent cell proliferation experiments. Our results indicate that the novel ZnO-CS is a promising candidate for bone tissue engineering.

3D bioprinting of mouse pre-osteoblasts and human MSCs using bioinks consisting of gelatin and decellularized bone particles

One of the key challenges in biofabrication applications is to obtain bioinks that provide a balance between printability, shape fidelity, cell viability, and tissue maturation. Decellularization methods allow the extraction of natural extracellular matrix, preserving tissue-specific matrix proteins. However, the critical challenge in bone decellularization is to preserve both organic (collagen, proteoglycans) and inorganic components (hydroxyapatite) to maintain the natural composition and functionality of bone. Besides, there is a need to investigate the effects of decellularized bone (DB) particles as a tissue-based additive in bioink formulation to develop functional bioinks. Here we evaluated the effect of incorporating DB particles of different sizes (≤45 and ≤100 μm) and concentrations (1%, 5%, 10% (wt %)) into bioink formulations containing gelatin (GEL) and pre-osteoblasts (MC3T3-E1) or human mesenchymal stem cells (hTERT-MSCs). In addition, we propose a minimalistic bioink formulation using GEL, DB particles and cells with an easy preparation process resulting in a high cell viability. The printability properties of the inks were evaluated. Additionally, rheological properties were determined with shear thinning and thixotropy tests. The bioprinted constructs were cultured for 28 days. The viability, proliferation, and osteogenic differentiation capacity of cells were evaluated using biochemical assays and fluorescence microscopy. The incorporation of DB particles enhanced cell proliferation and osteogenic differentiation capacity which might be due to the natural collagen and hydroxyapatite content of DB particles. Alkaline phosphatase activity is increased significantly by using DB particles, notably, without an osteogenic induction of the cells. Moreover, fluorescence images display pronounced cell-material interaction and cell attachment inside the constructs. With these promising results, the present minimalistic bioink formulation is envisioned as a potential candidate for bone tissue engineering as a clinically translatable material with straightforward preparation and high cell activity.

Inhibition of lipopolysaccharide-induced NF-κB maintains osteogenesis of dental pulp and periodontal ligament stem cells.

Dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) can differentiate into osteoblasts, indicating that both are potential candidates for bone tissue engineering. Osteogenesis is influenced by many environmental factors, one of which is lipopolysaccharide (LPS). LPS-induced NF-κB activity affects the osteogenic potencies of different types of MSCs differently. This study evaluated the effect of LPS-induced NF-κB activity and its inhibition in DPSCs and PDLSCs. DPSCs and PDLSCs were cultured in an osteogenic medium, pretreated with/without NF-κB inhibitor Bay 11-7082, and treated with/without LPS. Alizarin red staining was performed to assess bone nodule formation, which was observed under an inverted light microscope. NF-κB and alkaline phosphatase (ALP) activities were measured to examine the effect of Bay 11-7082 pretreatment and LPS supplementation on osteogenic differentiation of DPSCs and PDLSCs. LPS significantly induced NF-κB activity (p = 0.000) and reduced ALP activity (p = 0.000), which inhibited bone nodule formation in DPSCs and PDLSCs. Bay 11-7082 inhibited LPS-induced NF-κB activity, and partially maintained ALP activity and osteogenic potency of LPS-supplemented DPSCs and PDLSCs. Thus, inhibition of LPS-induced NF-κB activity can maintain the osteogenic potency of DPSCs and PDLSCs.

An integrated design strategy coupling additive manufacturing and matrix-assisted pulsed laser evaporation (MAPLE) towards the development of a new concept 3D scaffold with improved properties for tissue regeneration.

Bioinspired strategies for scaffold design and optimization were improved by the introduction of Additive Manufacturing (AM), thus allowing for replicating and reproducing complex shapes and structures in a reliable manner, adopting different kinds of polymeric and nanocomposite materials properly combined according to the features of the natural host tissues. Benefiting from recent findings in AM, a Matrix-Assisted Pulsed Laser Evaporation (MAPLE) technique was employed for obtaining graphene-like material (GL) uniform coatings on 3D scaffolds for tissue repair strategies, towards the development of a new concept 3D scaffold with controlled morphological/architectural and surface features and mechanical and biological properties. The effect of the material-design combination through an integrated technological approach (i.e., MAPLE deposition of GL on 3D AM PCL scaffolds) was assessed through scanning electron microscopy, atomic force microscopy, contact angle measurements, mechanical measurements and biological analyses (cell viability assay and alkaline phosphatase activity) in conjunction with confocal laser scanning microscopy. The differentiation of hMSCs towards the osteoblast phenotype was also investigated analysing the gene expression profile. The obtained findings provided a further insight into the development of improved strategies for the functionalization or combination of GL with other materials and 3D structures in a hybrid fashion for ensuring a tighter adhesion onto the substrates, improving cell fate over time, without negatively altering the mechanical properties and behaviour of the neat constructs. In particular, the results provided interesting information, making 3D AM GL-coated scaffolds potential candidates for bone tissue engineering.

The potential of the mineralized bone allograft block as an appropriate candidate for bone tissue engineering in periodontology

The potential of the mineralized bone allograft block as an appropriate candidate for bone tissue engineering in periodontology

Bio composite of porous hydroxyapatite and collagen extracted from eggshell membrane and Oreochromis niloticus fish skin for bone tissue applications

AbstractPorous hydroxyapatite (HA) was prepared by a chemical reaction between eggshell and phosphoric acid mixed with a foaming agent. Hydroxyapatite was composited with collagen prepared from a fermentation process of Acetobacter xylinum with eggshell membranes, extraction process of Oreochromis niloticus fish skin, and commercial‐grade collagen. Results of physical‐mechanical‐thermal properties of the bio composite materials yielded them as potential candidates for bone tissue engineering, drug delivery through the body, as well as tissue regeneration. The porous hydroxyapatite added foaming agent namely 64HA‐1NaHCO3‐500 and 64HA‐1NaHCO3‐600 having pore size and pore volume suitable for good adhering of collagen fiber types prepared from the fermentation process of Acetobacter xylinum of eggshell membrane and freeze‐drying process of Oreochromis niloticus. Because after reinforcing with collagen gave the highest compressive strength and three‐point bending strength values higher than HA samples without collagen three to five magnitudes. Furthermore, both bio composite samples served as positive thermal shock absorbers with significantly strong scanning electron microscope adhesion microstructures of collagen fibers within the porous HA matrices.

Feather keratin-montmorillonite nanocomposite hydrogel promotes bone regeneration by stimulating the osteogenic differentiation of endogenous stem cells

Bone defects caused by bone trauma, infection, surgery, or other systemic diseases remain a severe challenge for the medical field. To address this clinical problem, different hydrogels were exploited to promote bone tissue regrowth and regeneration. Keratins are natural fibrous proteins found in wool, hair, horns, nails, and feather. Due to their unique characteristics of outstanding biocompatibility, great biodegradability, and hydrophilic, keratins have been widely applicated in different fields. In our study, the feather keratin-montmorillonite nanocomposite hydrogels that consist of keratin hydrogels serving as the scaffold support to accommodate endogenous stem cells and montmorillonite is synthesized. The introduction of montmorillonite greatly improves the osteogenic effect of the keratin hydrogels via bone morphogenetic protein 2 (BMP-2)/phosphorylated small mothers against decapentaplegic homolog 1/5/8 (p-SMAD 1/5/8)/runt-related transcription factor 2 (RUNX2) expression. Moreover, the incorporation of montmorillonite into hydrogels can improve the mechanical properties and bioactivity of the hydrogels. The morphology of feather keratin-montmorillonite nanocomposite hydrogels was shown by scanning electron microscopy (SEM) to have an interconnected porous structure. The incorporation of montmorillonite into the keratin hydrogels was confirmed by the energy dispersive spectrum (EDS). We prove that the feather keratin-montmorillonite nanocomposite hydrogels enhance the osteogenic differentiation of BMSCs. Furthermore, micro-CT and histological analysis of rat cranial bone defect demonstrated that feather keratin-montmorillonite nanocomposite hydrogels dramatically stimulated bone regeneration in vivo. Collectively, feather keratin-montmorillonite nanocomposite hydrogels can regulate BMP/SMAD signaling pathway to stimulate osteogenic differentiation of endogenous stem cells and promote bone defect healing, indicating their promising candidate in bone tissue engineering.

Advanced injectable hydrogels for bone tissue regeneration.

Diseases or defects of the skeleton are hazardous because of their specificity and intricacy. Bone tissue engineering has become an important area of research that offers promising new tools for making biomimetic hydrogels that can be used to treat bone diseases. New hydrogels with a distinctive 3D network structure, high water content, and functional capabilities are ranked among the most promising candidates for bone tissue engineering. This makes them helpful in treating cartilage injury, skull deformity, and arthritis. This review will briefly introduce the variety of biocompatible functional hydrogels used in cell culture and bone tissue regeneration. Many gel design concepts, such as crosslinking procedures, controlled release properties, and alternative bionic methodology, were stressed regarding injectable hydrogels to form bone tissue. Hydrogels manufactured from biocompatible materials are a promising option for minimally invasive surgery because of their adaptable physicochemical qualities, ability to fill irregularly shaped defect sites, and ability to grow hormones or release drugs in response to external stimuli. Also included in this overview is a quick rundown of the more practical designs employed in treating bone disorders. Essential details on injectable hydrogel scaffolds for bone tissue regeneration are described in this article.

Phosphoserine-loaded chitosan membranes promote bone regeneration by activating endogenous stem cells.

Bone defects that result from trauma, infection, surgery, or congenital malformation can severely affect the quality of life. To address this clinical problem, a phosphoserine-loaded chitosan membrane that consists of chitosan membranes serving as the scaffold support to accommodate endogenous stem cells and phosphoserine is synthesized. The introduction of phosphoserine greatly improves the osteogenic effect of the chitosan membranes via mutual crosslinking using a crosslinker (EDC, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide). The morphology of PS-CS membranes was shown by scanning electron microscopy (SEM) to have an interconnected porous structure. The incorporation of phosphoserine into chitosan membranes was confirmed by energy dispersive spectrum (EDS), Fourier Transforms Infrared (FTIR), and X-ray diffraction (XRD) spectrum. The CCK8 assay and Live/Dead staining, Hemolysis analysis, and cell adhesion assay demonstrated that PS-CS membranes had good biocompatibility. The osteogenesis-related gene expression of BMSCs was higher in PS-CS membranes than in CS membranes, which was verified by alkaline phosphatase (ALP) activity, immunofluorescence staining, and real-time quantitative PCR (RT-qPCR). Furthermore, micro-CT and histological analysis of rat cranial bone defect demonstrated that PS-CS membranes dramatically stimulated bone regeneration in vivo. Moreover, H&E staining of the main organs (heart, liver, spleen, lung, or kidney) showed no obvious histological abnormalities, revealing that PS-CS membranes were no additional systemic toxicity in vivo. Collectively, PS-CS membranes may be a promising candidate for bone tissue engineering.

The effects of BaTiO3 on the handleability and mechanical strength of the prepared piezoelectric calcium phosphate silicate for bone tissue engineering

Natural bone is a piezoelectric material that can generate electrical signals when subjected to an external force. Although many studies have attempted to develop piezoelectric biomaterials for bone regeneration, post-treatment steps, such as sintering, are always needed. In this study, we prepared an injectable and piezoelectric bone substitute based on nanosized BaTiO3 (nBT)-added calcium phosphate silicate (CPS). The impacts of nBT on the CPS handleability and mechanical strength were characterized, and show that adding nBT could improve the CPS handleability but affect the CPS mechanical strength in a concentration-dependent manner (from 25.3 ± 1.0 MPa for 10BC to 13.5 ± 1.0 MPa for 40BC). In addition, our approach could fabricate a piezoelectric bone substitute with comparable piezoelectricity to the native bone without any post-treatment. The in vitro analyses demonstrated that nBT/CPS was biocompatible and could promote osteoblast differentiation. In conclusion, our results strongly indicate that the injectable formulation based on nBT/CPS can be a promising candidate in bone tissue engineering, and further research is needed to investigate the biomaterial's performance in bone defect animal models.

Cell Adhesion and Initial Bone Matrix Deposition on Titanium-Based Implants with Chitosan–Collagen Coatings: An In Vitro Study

In orthopedics, titanium (Ti)-alloy implants, are often considered as the first-choice candidates for bone tissue engineering. An appropriate implant coating enhances bone matrix ingrowth and biocompatibility, improving osseointegration. Collagen I (COLL) and chitosan (CS) are largely employed in several different medical applications, for their antibacterial and osteogenic properties. This is the first in vitro study that provides a preliminary comparison between two combinations of COLL/CS coverings for Ti-alloy implants, in terms of cell adhesion, viability, and bone matrix production for probable future use as a bone implant. Through an innovative spraying technique, COLL-CS-COLL and CS-COLL-CS coverings were applied over Ti-alloy (Ti-POR) cylinders. After cytotoxicity evaluations, human bone marrow mesenchymal stem cells (hBMSCs) were seeded onto specimens for 28 days. Cell viability, gene expression, histology, and scanning electron microscopy evaluations were performed. No cytotoxic effects were observed. All cylinders were biocompatible, thus permitting hBMSCs' proliferation. Furthermore, an initial bone matrix deposition was observed, especially in the presence of the two coatings. Neither of the coatings used interferes with the osteogenic differentiation process of hBMSCs, or with an initial deposition of new bone matrix. This study sets the stage for future, more complex, ex vivo or in vivo studies.

Biomimetic Growth of Hydroxyapatite in Hybrid Polycaprolactone/Graphene Oxide Ultra-Porous Scaffolds.

This paper reports the preparation and characterization of hybrid scaffolds composed of polycaprolactone (PCL) and different graphene oxide (GO) amounts, intending to incorporate the intrinsic characteristics of their constituents, such as bioactivity and biocidal effect. These materials were fabricated by a solvent-casting/particulate leaching technique showing a bimodal porosity (macro and micro) that was around 90%. The highly interconnected scaffolds were immersed in a simulated body fluid, promoting the growth of a hydroxyapatite (HAp) layer, making them ideal candidates for bone tissue engineering. The growth kinetics of the HAp layer was influenced by the GO content, a remarkable result. Furthermore, as expected, the addition of GO neither significantly improves nor reduces the compressive modulus of PCL scaffolds. The thermal behavior of composites was investigated by differential scanning calorimetry, showing an increase in crystallinity as the addition of GO raised, which implies that GO nanosheets can act as seeds to induce the crystallization of PCL. The improved bioactivity was demonstrated by the deposition of an HAp layer on the surface of the scaffold with GO, especially with a 0.1% GO content.

Investigating of physical, mechanical, and biological properties of polyhydroxybutyrate-keratin/alumina electrospun scaffold utilized in bone tissue engineering

Electrospun nanofiber scaffolds have a similar structure to extracellular matrix, leading to increased cell adhesion, proliferation, and migration. In this study, 1–5wt % of alumina nanowires were added to polyhydroxybutyrate-keratin (PHB–K) solution, and the morphology of the electrospun scaffolds in terms of fiber diameter, porosity percentage, and the uniformity of the alumina nanowires distribution was evaluated by SEM. FTIR and raman tests were also used to evaluate the chemical bonds of nanofibers and the presence of alumina and keratin in electrospun scaffolds. The crystallinity of the scaffolds was also measured using DSC and confirmed by XRD. The increase of alumina augmented the crystallinity of scaffolds because alumina is a nucleating agent. The tensile strength of PHB-K scaffolds increased up to 3 fold in the presence of 3 wt % of alumina.The MG63 cells survived, and the secretion of alkaline phosphatase and mineralization due to the presence of alumina was significantly higher than PHB and PHB-K scaffolds. Based on the results, the electrospun PHB-K/AL2O3 scaffolds are potential candidates for bone tissue engineering.

Preparation and characterization of 3D hydroxyapatite/collagen scaffolds and its application in bone regeneration with bone morphogenetic protein-2.

Desirable bone engineering materials should have a conducive three-dimensional (3D) structure and bioactive mediators for guided bone regeneration. In the present study, hydroxyapatite (HA)/collagen (Col) scaffolds were prepared by an optimized freeze-drying process. The porosity, moisture content, and mechanical properties of the composite have been investigated. The micro-morphology and structure were analyzed with scanning electron microscopy (SEM) and transmission electron microscopy (TEM), confirmed that self-cross-linked HA/Col was evenly distributed and formed a 3D porous scaffold. The physicochemical/mechanical characterization was carried out by Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD). Morphological observation and CCK-8 assay of co-culture cells indicated that HA/Col scaffolds were biocompatible. Then HA/Col scaffolds coupled with recombinant human bone morphogenetic proteins 2 (rhBMP-2) were implanted in the mandibular critical size defect in rats, and histological staining was used to evaluate the bone reconstruction. The result showed that HA/Col coupled with rhBMP-2 could significantly improve the formation of new bone and angiogenesis within the scaffolds as well as the proliferation and differentiation of osteoblasts. Thanks to the encouraging osteogenesis effects, the well-defined 3D scaffolds (HA/Col) cooperating with bioactive agents (rhBMP-2) are expected to be a promising candidate for bone tissue engineering applied to regenerative medicine.