Investigation of Magnesium Incorporation within Gelatin/Calcium Phosphate Nanocomposite Scaffold for Bone Tissue Engineering

In this study, diffusional method was used to prepare a calcium phosphate/gelatin nanocomposite as a scaffold for bone tissue repair. Incorporation of magnesium (Mg) into mineral phase of the scaffold was also investigated. Addition of Mg ions to the synthesis process caused formation of magnesium phosphate (MgP) and hydroxyapatite (HAp). However, analyses data for the sample lacking Mg showed that the mineral formed within GEL had a low crystalline nature, consisting of HAp and octacalcium phosphate (OCP). With addition of Mg within the structure of precipitated minerals, morphology of minerals was dramatically changed toward being irregular and less ordered.     

Direct ink writing of vancomycin-loaded polycaprolactone/ polyethylene oxide/ hydroxyapatite 3D scaffolds

Novel inks were formulated by dissolving polycaprolactone (PCL), a hydrophobic polymer, in organic solvent systems; polyethylene oxide (PEO) was incorporated to extend the range of hydrophilicity of the system. Hydroxyapatite (HAp) with a weight ratio of 55–85% was added to the polymer-based solution to mimic the material composition of natural bone tissue. The direct ink writing (DIW) technique was applied to extrude the formulated inks to fabricate the predesigned tissue scaffold structures; the influence of HAp concentration was investigated. The results indicate that in comparison to other inks containing HAp (55%, 75%, and 85%w/w), the ink containing 65% w/w HAp had faster ink recovery behavior; the fabricated scaffold had a rougher surface as well as better mechanical properties and wettability. It is noted that the 65% w/w HAp concentration is similar to the inorganic composition of natural bone tissue. The elastic modulus values of PCL/PEO/HAp scaffolds were in the range of 4–12 MPa; the values were dependent on the HAp concentration. Furthermore, vancomycin as a model drug was successfully encapsulated in the PCL/PEO/HAp composite scaffold for drug release applications. This paper presents novel drug-loaded PCL/PEO/HAp inks for 3D scaffold fabrication using the DIW printing technique for potential bone scaffold applications.     

Fabrication of Hydroxyapatite with Bioglass Nanocomposite for Human Wharton’s-Jelly-Derived Mesenchymal Stem Cell Growing Substrate

Recently, composite scaffolding has found many applications in hard tissue engineering due to a number of desirable features. In this present study, hydroxyapatite/bioglass (HAp/BG) nanocomposite scaffolds were prepared in different ratios using a hydrothermal approach. The aim of this research was to evaluate the adhesion, growth, viability, and osteoblast differentiation behavior of human Wharton’s-jelly-derived mesenchymal stem cells (hWJMSCs) on HAp/BG in vitro as a scaffold for application in bone tissue engineering. Particle size and morphology were investigated by TEM and bioactivity was assessed and proven using SEM analysis with hWJMSCs in contact with the HAp/BG nanocomposite. Viability was evaluated using PrestoBlue^TM assay and early osteoblast differentiation and mineralization behaviors were investigated by ALP activity and EDX analysis simultaneously. TEM results showed that the prepared HAp/BG nanocomposite had dimensions of less than 40 nm. The morphology of hWJMSCs showed a fibroblast-like shape, with a clear filopodia structure. The viability of hWJMSCs was highest for the HAp/BG nanocomposite with a 70:30 ratio of HAp to BG (HAp70/BG30). The in vitro biological results confirmed that HAp/BG composite was not cytotoxic. It was also observed that the biological performance of HAp70/BG30 was higher than HAp scaffold alone. In summary, HAp/BG scaffold combined with mesenchymal stem cells showed significant potential for bone repair applications in tissue engineering.     

Preparation,characterization and mechanical properties of controlled porous gelatin/hydroxyapatite nanocomposite through layer solvent casting combined with freeze-drying and lamination techniques

In this present study, to mimic the mineral and organic component of natural bone, hydroxyapatite (HA) and gelatin (GEL) nanocomposite was prepared via layer solvent casting combined with freeze-drying and lamination techniques. Glutaraldehyde (GA) was used as cross-linking agent. The synthesized nanocrystalline hydroxyapatite and nanocomposite samples were characterized by the commonly used bulk techniques. The results showed that GEL/HA nanocomposite were porous with 3-dimension interconnected microstructure, pore sizes were 100 μm to 1 mm, porosity were 75% to 93% and HA particles are dispersed evenly among gelatin fibers. It was also found that increasing initial GEL concentration and HA content enhance the elastic modulus (E) and reduce toughness and affect pore size and morphology. Finally, the stress–strain behavior in compression was very similar to natural spongy bone where the compressive modulus obtained was about 180 MPa.     

Preparation and characterization of gelatin/hydroxyapatite nanocomposite for bone tissue engineering

Homogeneous gelatin/hydroxyapatite (GEL/HA) nano‐composites were synthesized by a novel in situ precipitation method, and its corresponding characterizations, including composition, morphology, pore structure, thermal stability, mechanical strength, and biocompatibility, were carried out. High‐magnified scanning electron microscope (SEM) images indicated that nano‐HA with particle size ranging from 20 to 50 nm were uniformly distributed in GEL matrix and tightly integrated with organic phase. Wide angle X‐ray diffraction (XRD) analysis and transmission electron microscope (TEM) images showed that, during the process of mineralization, there existed preferred oriented growth of HA crystals along (002) and (211) crystal planes. Thermogravimetric analysis (TGA) and differential thermal analysis (DTA) indicated that, the thermal stability of GEL molecules enhanced by hybridizing with HA nanocrystals. Interconnected porous GEL/HA nanocomposites with pore size ranging between 50 and 350 μm were prepared by a freeze‐drying method. This pore size was adequate for bone tissue engineering (BTE) applications. In addition, in vitro MG63 osteoblast‐like cell culture illuminated that GEL/HA nanocomposites had excellent cytocompatibility and could promote proliferation of cells. These results suggested that GEL/HA nanocomposite might be an ideal bone substitute. POLYM. COMPOS., 38:1579–1590, 2017. © 2015 Society of Plastics Engineers     

Development of bone scaffold using Puntius conchonius fish scale derived hydroxyapatite: Physico-mechanical and bioactivity evaluations

Naturally derived Hydroxyapatite (HAp) from fish scale is finding wide applications in the development of bone scaffold to promote bone regeneration. But porous HAp scaffold is fragile in nature making it unsuitable for bone repair or replacement applications. Thus, it is essential to improve the mechanical property of HAp scaffolds while retaining the interconnected porous structure for tissue ingrowth in vivo. In this study solvent casting particulate leaching technique is used to develop novel Puntius conchonius fish scale derived HAp bone scaffold by varying the wt.% of the HAp from 60 to 80% in PMMA matrix. Physico-chemical, mechanical, structural and bioactive properties of the developed scaffolds are investigated. The obtained results indicate that HAp-PMMA scaffold at 70?wt % HAp loading shows optimal properties with 7.26?±?0.45?MPa compressive strength, 75?±?0.8% porosity, 8.0?±?0.68% degradation and 190?±?11% water absorption. The obtained results of the scaffold can meet the physiological demands to guide bone regeneration. Moreover, in vitro bioactivity analysis also confirms the formation of bone like apatite in the scaffold surface after 28 days of SBF immersion. Thus, the developed scaffold has the potential to be effectively used in bone tissue engineering applications.     

Effect of ZnO reinforcement on the compressive properties,in vitro bioactivity,biodegradability and cytocompatibility of bone scaffold developed from bovine bone-derived HAp and PMMA

In this study, the effect of zinc oxide (ZnO) incorporation on the properties of Hydroxyapatite (HAp)/Poly(methyl methacrylate) (PMMA)/ZnO based composite bone scaffold is investigated. HAp is derived from calcination of bovine bone bio-waste and ZnO is synthesized by direct precipitation technique. Porous scaffolds are developed by gas foaming process using ammonium bicarbonate as the foaming agent and adding ZnO nanoparticles (NPs) at 2.5, 5, 7.5 and 10% (w/w) respectively. Incorporation of ZnO up to 5% (w/w) is found to significantly enhance the porosity, compressive strength, thermal stability and swelling properties of the developed scaffolds. In-vitro bioactivity and biodegradability assessment using simulated body fluid (SBF) show improved results of 5% ZnO loaded scaffolds. Furthermore, the composite scaffold show enhanced cytocompatibility during the in vitro cytotoxicity test performed using XTT assay. A comprehensive study on the scaffold properties shows that 5% ZnO composite scaffold exhibits the best-optimized properties suitable for bone tissue engineering applications.     

In situ synthesized chitosan–gelatin/ZnO nanocomposite scaffold with drug delivery properties: Higher antibacterial and lower cytotoxicity effects

In this work, chitosan–gelatin/zinc oxide nanocomposite hydrogel scaffolds (CS–GEL/nZnO) were prepared via in situ synthesis of ZnO nanoparticles (nZnO) to reach a scaffold with both inherent antibacterial and drug delivery properties. The prepared nanocomposite hydrogel scaffolds were characterized using scanning electron microscopy, transmission electron microscopy, atomic absorption spectrometer, Fourier transform infrared spectroscopy, and X-ray diffraction. In addition, swelling, biodegradation, antibacterial, cytocompatibility, and cell attachment of the scaffolds were evaluated. The results showed that the prepared scaffolds had high porosity with a pore size of 50–400 μm and nZnO were well distributed without any agglomeration on the CS–GEL matrix. In addition, the nanocomposite scaffolds showed enhanced swelling, biodegradation, and antibacterial properties. Moreover, the drug delivery studies using naproxen showed that nZnO could control naproxen release. Cytocompatibility of the samples was proved using normal human dermal fibroblast cells (HFF2). In comparison to the previous reports which nZnO were simply added to the matrix of the scaffold, in situ synthesis of nZnO was led to higher antibacterial and lower cytotoxicity effects as a result of well distribution of nZnO in this method. According to the findings, the in situ synthesized CS–GEL/nZnO is strongly recommended for biomedical applications especially skin tissue engineering. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 136, 47590.     

Synthesis of intercalated lamellar hydroxyapatite/gelatin nanocomposite for bone substitute application

Hydroxyapatite (HAp)/polymer composites have been widely investigated for bone substitute applications in recent years. Inspired by the arrangement of ordered organic and inorganic layers in natural bones and seashells, for the first time a novel intercalated nanocomposite of gelatin and lamellar HAp was prepared via solution intercalation process. X‐ray diffraction (XRD) results showed that the basal spacing of HAp lamellas enlarged by 3.0 nm from 3.1 nm to 6.1 nm, indicating that the gelatin molecules had been intercalated into the gallery of lamellar HAp. The microstructures of pure lamellar HAp and intercalated gelatin/HAp nanocomposite were observed by transmission electron microscopy (TEM) analysis. Fourier transform infrared spectroscopy (FT‐IR) analysis revealed that there were chemical interactions between gelatin molecules and HAp. Thermogravimetric analysis (TGA) results confirmed that thermal stability of the composites was enhanced. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

An investigation into the influence of electrospinning parameters on the diameter and alignment of poly(hydroxybutyrate‐co‐hydroxyvalerate) fibers

Electrospinning is an effective technology for the fabrication of ultrafine fibers, which can be the basic component of a tissue engineering scaffold. In tissue engineering, because cells seeded on fibrous scaffolds with varying fiber diameters and morphologies exhibit different responses, it is critical to control these characteristics of electrospun fibers. The diameter and morphology of electrospun fibers can be influenced by many processing parameters (e.g., electrospinning voltage, needle inner diameter, solution feeding rate, rotational speed of the fiber‐collecting cylinder, and working distance) and solution properties (polymer solution concentration and conductivity). In this study, a factorial design approach was used to systematically investigate the degree of influence of each of these parameters on fiber diameter, degree of fiber alignment, and their possible synergetic effects, using a natural biodegradable polymer, poly(hydroxybutyrate‐co‐hydroxyvalerate), for the electrospinning experiments. It was found that the solution concentration invoked the highest main effect on fiber diameter, whereas both rotational speed of the fiber‐collecting cylinder and addition of a conductivity‐enhancing salt could significantly affect the degree of fiber alignment. By carefully controlling the electrospinning parameters and solution properties, fibrous scaffolds of desired characteristics could be made to meet the requirements of different tissue engineering applications. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Extrusion-based 3D printing of gelatin methacryloyl with nanocrystalline hydroxyapatite

The particle shape and size distribution of inorganic fillers play a crucial role in the scaffold buildability when those are incorporated in the viscoelastic polymers. In order to address this issue, the phase pure rod-shaped nanocrystalline hydroxyapatite (HAp) powders with varying particle sizes and shapes were synthesized by a one-pot hydrothermal method without any regulatory surfactant at an initial solution pH of 9. As-synthesized nanocrystalline HAp particles (0–5 wt%) were incorporated into 15 wt% pre-cross-linked gelatin methacryloyl (GelMA) hydrogel matrix to fabricate a predesigned scaffold architecture using a custom-made 3D bioprinter. The printing parameters (nozzle diameter, extrusion pressure, and printing speed) were optimized for each composition. The biophysical properties (uniaxial compression behavior, swelling ratio, and in vitro degradation) of the composite hydrogel scaffolds were critically analyzed to unravel the role of nano-sized HAp addition. The compression strength and modulus were substantially improved, while the rate of water uptake and bio-enzymatic degradation significantly reduced with HAp content. We propose that the inorganic–organic nanocomposite hydrogel could be efficiently assembled to formulate a potential bioink for 3D bioprinting applications toward tissue regeneration.     

One‐Pot Fabrication of Poly(ε‐Caprolactone)‐Incorporated Bovine Serum Albumin/Calcium Alginate/Hydroxyapatite Nanocomposite Scaffolds by High Internal Phase Emulsion Templates

In this work, the authors report an effective one‐pot method to prepare poly(ε‐caprolactone) (PCL)‐incorporated bovine serum albumin (BSA)/calcium alginate/hydroxyapatite (HAp) nanocomposite (NC) scaffolds by templating oil‐in‐water high internal phase emulsion (HIPE), which includes alginate, BSA, and HAp in water phase and PCL in oil phase. The water phase of HIPEs is solidified to form hydrogels containing emulsion droplets via gelation of alginate induced by Ca²⁺ ions released from HAp. And the prepared hydrogels are freeze‐dried to obtain PCL‐incorporated porous scaffolds. The obtained scaffolds possess interconnected pore structures. Increasing PCL concentration clearly enhances the compressive property and BSA stability, decreases the swelling ratio of scaffolds, which assists in improving the scaffold stability. The anti‐inflammatory drug ibuprofen can be highly efficiently loaded into scaffolds and released in a sustained rate. Furthermore, mouse bone mesenchymal stem cells can successfully proliferate on the scaffolds, proving the biocompatibility of scaffolds. All results show that the PCL‐incorporated NC scaffolds possess promising potentials in tissue engineering application.

     

Biomimetic biphasic 3‐D nanocomposite scaffold for osteochondral regeneration

Scaffold‐based interfacial tissue engineering aims to not only provide the structural and mechanical framework for cellular growth and tissue regeneration, but also direct cell behavior. Due to the disparity in composition of the osteochondral (cartilage and bone) interface, this work has developed a novel biomimetic biphasic nanocomposite scaffold integrating two biocompatible polymers containing tissue‐specific growth factor‐encapsulated core–shell nanospheres. Specifically, a poly(caprolactone) (PCL)‐based bone layer was successfully integrated with a poly(ethylene glycol) (PEG) hydrogel cartilage layer. In addition, a novel nanosphere fabrication technique for efficient growth factor encapsulation and sustained delivery via a wet coaxial electrospray technique was developed. Human bone marrow mesenchymal stem cell (hMSC) adhesion, osteogenic, and chondrogenic differentiation were evaluated. Our in vitro results showed significantly improved hMSC adhesion and differentiation in bone and cartilage layers, respectively. Studies have demonstrated promising results with novel biphasic nanocomposite scaffold for osteochondral tissue regeneration, thus, warranting further studies. © 2013 American Institute of Chemical Engineers AIChE J 60: 432–442, 2014     

Electrospun poly(L‐lactic acid)/hydroxyapatite composite fibrous scaffolds for bone tissue engineering

Poly(L ‐lactic acid) (PLLA) is one of the most studied synthetic biodegradable polymeric materials as a bone graft substitute. Taking into account the osteoconductive property of hydroxyapatite (HAp), we prepared fibrous matrices of PLLA without and with HAp particles in amounts of 0.25 or 0.50% (w/v, based on the volume of the base 15% w/v PLLA solution in 70:30 v/v dichloromethane/tetrahydrofuran). These fibrous matrices were assessed for their potential as substrates for bone cell culture. The presence of HAp in the composite fibre mats was confirmed using energy dispersive X‐ray spectroscopy mapping. The average diameters of both neat PLLA and PLLA/HAp fibres, as determined using scanning electron microscopy, ranged between 2.3 and 3.5 µm, with the average spacing between adjacent fibres ranging between 5.7 and 8.5 µm. The porosity of these fibrous membranes was high (ca 97–98%). A direct cytotoxicity evaluation with L929 mouse fibroblasts indicated that the neat PLLA fibre mats released no substance at a level that was toxic to the cells. The presence of HAp particles at 0.50% w/v in the PLLA fibrous scaffolds not only promoted the attachment and the proliferation of MC3T3‐E1 mouse pre‐osteoblastic cells, but also increased the expression of osteocalcin mRNA and the extent of mineralization after the cells had been cultured on the scaffolds for 14 and 21 days, respectively. The results obtained suggested that the PLLA/HAp fibre mats could be materials of choice for bone tissue engineering. Copyright © 2009 Society of Chemical Industry     

Comparative study of hydroxyapatite/gelatin composites reinforced with bio-inert ceramic particles

Recently, nano bio-composites have emerged as an efficient strategy to upgrade the structural and functional properties of synthetic bone grafts. Bioinert ceramics have attracted wide attention because of their biocompatibility. Novel composites of nano-hydroxyapatite/GEL with incorporation of bioinert ceramics like Al₂O₃, TiO₂ and ZrO₂ for different composites as a reinforcing phase to increase its mechanical properties was prepared. The nHAp with the size of 10–50 nm in diameter and 50–100 nm in length was uniformly distributed into GEL matrix to form the composite. It was found that the composite with a high ceramic content has good homogeneity and mechanical strength, which are close to the cancellous bone. An interconnected porous material with porosity of at least 74% was achieved by phase inversion method. The formation reaction of the nHAp/GEL/bioinert ceramic nanocomposite was then investigated via FT-IR, XRD, TG/DTA and SEM. The organic–inorganic interaction between HAp nano crystallites and GEL molecules were confirmed from FT-IR and TG/DTA. The compressive strength of bioinert ceramic reinforced nanocomposites scaffolds could high up to 13.15 MPa while those of nHAp/GEL were 4.87 MPa. The nano indentation technique was used to find nano hardness and fracture toughness was evaluated by Vickers indentation.     

Separation of gibberellic acid (GA3) by macroporous adsorption resins

Adsorption rates of gibberellic acid (GA3) on S‐8 and X‐5 resins were measured with the results indicating that the adsorption attained equilibrium for adsorption times longer than 4 h. The adsorption isotherms of GA3 on AB‐8, X‐5, S‐8, D4020, D3520, D4006 and NKA‐9 were determined at 20 °C. S‐8 resin had the largest solid/liquid distribution coefficient of 10.5 and was selected as the adsorbent for separation of GA3 from the aqueous solution. The breakthrough curves of GA3 from the aqueous solution on S‐8 resin were determined at different flow rates. The dynamic adsorption capacity of GA3 was measured as 4.56 mgg⁻¹ wet resin at a flow rate of 0.5 cm³min⁻¹. GA3 adsorbed on S‐8 resin was eluted with several eluents with 80% (v%) acetone aqueous solution having the best desorption performance. When GA3 was adsorbed by S‐8 resin from the fermentation liquor, the dynamic adsorption capacity of GA3 was increased to 12.02 mgg⁻¹ wet resin in virtue of the salting‐out effect of the ammonium sulfate in the fermentation liquor. The yield of GA3 was above 90% after GA3 was adsorbed by S‐8 resin from the fermentation liquor at pH 2 and eluted by 80% (v%) acetone aqueous solution. The GA3 concentration was increased seven‐fold from the fermentation liquor after an adsorption and desorption cycle. © 2000 Society of Chemical Industry     

Synthesis of hydroxyapatite/poly(vinyl alcohol phosphate) nanocomposite and its characterization

Hydroxyapatite (HAp)/poly(vinyl alcohol phosphate) (PVAP) nanocomposite has been prepared using a solution‐based method varying HAp from 10 to 60% (w/w). X‐ray diffraction, Fourier transform infrared absorption spectra (FTIR), and thermal analysis have indicated the presence of bonding between HAp particles and PVAP matrix. Transmission electron microscope analysis shows the needle‐like crystals of HAp powder having a diameter of 6–10 nm and a length of 26–38 nm. The surface roughness and the homogeneous dispersion of HAp particles in the polymer matrix have been observed by scanning electron microscopy. Particle size distribution analysis shows the narrow distribution of hydrodynamic particles in the polymer matrix. The tensile stress–strain curves show the improvement in mechanical properties of the composites with increase in amount of HAp particles loading. The composites along with polymer are highly hemocompatible. The use of PVAP promotes the homogeneous distribution of particles on the polymer matrix along with strong particle–polymer interfacial bonding, which has supported the improvement in mechanical properties of the composites. The prepared HAp/PVAP composite with uniform microstructure would be effective to act as a potential biomaterial. POLYM. COMPOS., 2008. © 2008 Society of Plastics Engineers     

Physical and mechanical properties of a poly-3-hydroxybutyrate-coated nanocrystalline hydroxyapatite scaffold for bone tissue engineering

A major challenge for tissue engineers is the design of scaffolds with appropriate physical and mechanical properties. The present research discusses the formation of ceramic scaffolding in tissue engineering. Hydroxyapatite (HAp) powder was made from bovine bone by thermal treatment at 900?°C; 40, 50 and 60%wt porous HAp was then produced using the polyurethane sponge replication method. Scaffolds were coated with poly-3-hydroxybutyrate (P3HB) for 30?s and 1?min in order to increase the scaffold??s mechanical properties. XRD, SEM and FT-IR were used to study phase structure, morphology and agent groups, respectively. In XRD and FT-IR data, established hydrogen bands between polymer and ceramic matrix confirm that the scaffold is formed as a composite. The scaffold obtained with 50%wt HAp and a 30?s coating was 90% porous, with an average diameter of 100?C400???m, and demonstrated a compressive strength and modulus of 1.46 and 21.27?MPa, respectively. Based on these results, this scaffold is optimised for the aforementioned properties and can be utilised in bone tissue engineering.     

The influence of the preparation conditions and filler content on thermal properties of poly‐l‐lactide and hydroxyapatite/poly‐l‐lactide nanocomposite

Poly‐l ‐lactide (PLLA) and hydroxyapatite/poly‐l ‐lactide (HAp/PLLA) are two widely used biomaterials for three‐dimensional scaffolds, drug release matrices and implantable medical devices for reparation of bone tissue; diversity in the initial preparation and filler content has a significant influence on different properties such as morphology and crystallinity, thus playing a considerable role in most of these applications. For this reason, PLLA and HAp/PLLA samples with a large difference in crystallinity (from below 20% to over 70%) and filler content (up to 86 wt% of HAp nanoparticles with an average diameter of 80 nm) were prepared and consequent dissimilarities in morphology, crystallinity and thermal properties were investigated by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), wide angle X‐ray diffraction (WAXD) measurements and Fourier transform infrared (FTIR) spectroscopy. Special attention was devoted to analyzing data obtained from thermal measurements. A three‐phase model was employed in order to describe the heat capacity step decline in the nanocomposite; the evolution in different polymer fractions, the crystalline fraction and the mobile and rigid amorphous fractions, with filler content was determined. © 2017 Society of Chemical Industry     

Chitosan/gelatin porous bone scaffolds made by crosslinking treatment and freeze‐drying technology: Effects of crosslinking durations on the porous structure,compressive strength,and in vitro cytotoxicity

In this study, freezing was used to separate a solute (polymer) and solvent (deionized water). The polymer in the ice crystals was then crosslinked with solvents, and this diminished the linear pores to form a porous structure. Gelatin and chitosan were blended and frozen, after which crosslinking agents were added, and the whole was frozen again and then freeze‐dried to form chitosan/gelatin porous bone scaffolds. Stereomicroscopy, scanning electron microscopy, compressive strength testing, porosity testing, in vitro biocompatibility, and cytotoxicity were used to evaluate the properties of the bone scaffolds. The test results show that both crosslinking agents, glutaraldehyde (GA) and 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide, were able to form a porous structure. In addition, the compressive strength increased as a result of the increased crosslinking time. However, the porosity and cell viability were not correlated with the crosslinking times. The optimal porous and interconnected pore structure occurred when the bone scaffolds were crosslinked with GA for 20 min. It was proven that crosslinking the frozen polymers successfully resulted in a division of the linear pores, and this resulted in interconnected multiple pores and a compressively strong structure. The 48‐h cytotoxicity did not affect the cell viability. This study successfully produced chitosan/gelatin porous materials for biomaterials application. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41851.