Density–property relationships in mineralized collagen–glycosaminoglycan scaffolds

Mineralized collagen–glycosminoglycan scaffolds have previously been fabricated by freeze-drying a slurry containing a co-precipitate of calcium phosphate, collagen and glycosaminoglycan. The mechanical properties of the scaffold are low (e.g. the dry Young’s modulus for a 50 wt.% mineralized scaffold is roughly 780 kPa). Our previous attempt to increase the mechanical properties of the scaffold by increasing the mineralization (from 50 to 75 wt.%) was unsuccessful due to defects in the more mineralized scaffold. In this paper, we describe a new technique to improve the mechanical properties by increasing the relative density of the scaffolds. The volume fraction of solids in the slurry was increased by vacuum-filtration. The slurry was then freeze-dried in the conventional manner to produce scaffolds with relative densities between 0.045 and 0.187 and pore sizes of about 100–350 μm, values appropriate for bone growth. The uniaxial compressive stress–strain curves of the scaffolds indicated that the Young’s modulus in the dry state increased from 780 to 6500 kPa and that the crushing strength increased from 39 to 275 kPa with increasing relative density. In the hydrated state, the Young’s modulus increased from 6.44 to 34.8 kPa and the crushing strength increased from 0.55 to 2.12 kPa; the properties were further increased by cross-linking. The modulus and strength were well described by models for cellular solids.     

Silica–collagen bionanocomposites as three-dimensional scaffolds for fibroblast immobilization

Silica–collagen bionanocomposite hydrogels were obtained by addition of silica nanoparticles to a protein suspension followed by neutralization. Electron microscopy studies indicated that larger silica nanoparticles (80 nm) do not interact strongly with collagen, whereas smaller ones (12 nm) form rosaries along the protein fibers. However, the composite network structurally evolved with time due to the contraction of the cells and the dissolution of the silica nanoparticles. When compared to classical collagen hydrogels, these bionanocomposite materials showed lower surface contraction in the short term (1 week) and higher viability of entrapped cells in the long term (3 weeks). A low level of gelatinase MMP2 enzyme expression was also found after this period. Several proteins involved in the catabolic and anabolic activity of the cells could also be observed by immunodetection techniques. All these data suggest that the bionanocomposite matrices constitute a suitable environment for fibroblast adhesion, proliferation and biological activity and therefore constitute an original three-dimensional environment for in vitro cell culture and in vivo applications, in particular as biological dressings.     

Structure–property relationships of a biopolymer network: The eggshell membrane

The eggshell membrane (ESM) is a biopolymer network that may have potential applications in biomedicine, but it also may reveal important details regarding the behaviour of biopolymer networks. In this paper, we have studied the mechanical and morphological properties of the ESM in order to reveal important structure–property relationships. Light optical microscopy and atomic force microscopy were used to assess the morphology of the ESM. The mechanical properties of membranes and individual fibres were studied by means of tensile tests and nanoindentation tests, respectively. The mechanical behaviour of ESM networks in different environmental conditions showed a non-linear and a linear regime. As for elastomers and other biopolymer systems, the non-linear regime was modelled by the Mooney–Rivlin relation. The Young’s modulus in the linear regime of the network was related to the Young’s modulus of the individual fibres using Gibson and Ashby analysis for cellular solids. The results of morphological characterization were used to relate the properties of individual fibres to the properties of the whole networks. This enabled us to predict the macroscopical properties of the network based on the properties of the individual fibres. It was found that the ESM networks behaved as both Mooney–Rivlin and Hookean materials in different environmental conditions. This study helps elucidate the properties of the biopolymer networks found in nature and describes important mechanical properties for the use of the ESM as a biomaterial.     

Structure–property-processing correlations in freeze-cast composite scaffolds

Surprisingly few reports have been published, to date, on the structure–property-processing correlations observed in freeze-cast materials directionally solidified from polymer solutions, or ceramic or metal slurries. The studies that exist focus on properties of sintered ceramics, that is materials whose structure was altered by further processing. In this contribution, we report first results on correlations observed in alumina–chitosan–gelatin composites, which were chosen as a model system to test and compare the effect of particle size and processing parameters on their mechanical properties at a specific composition. Our study reveals that highly porous (>90%) hybrid materials can be manufactured by freeze casting, through the self-assembly of a polymer and a ceramic phase that occurs during directional solidification, without the need of additional processing steps such as sintering or infiltration. It further illustrates that the properties of freeze-cast hybrid materials can independently be tailored at two levels of their structural hierarchy, allowing for the simultaneous optimization of both mechanical and structural requirements. An increase in freezing rate resulted in decreases in lamellar spacing, cell wall thickness, pore aspect ratio and cross-sectional area, as well as increases in both Young’s modulus and compressive yield strength. The mechanical properties of the composite scaffolds increased with an increasing particle size. The results show that both structure and mechanical properties of the freeze-cast composites can be custom-designed and that they are thus ideally suited for a large variety of applications that require high porosity at low or medium load-bearing capacity.     

The delayed addition of human mesenchymal stem cells to pre-formed endothelial cell networks results in functional vascularization of a collagen–glycosaminoglycan scaffold in vivo

This paper demonstrates a method to engineer, in vitro, a nascent microvasculature within a collagen–glycosaminoglycan scaffold with a view to overcoming the major issue of graft failure due to avascular necrosis of tissue-engineered constructs. Human umbilical vein endothelial cells (ECs) were cultured alone and in various co-culture combinations with human mesenchymal stem cells (MSCs) to determine their vasculogenic abilities in vitro. Results demonstrated that the delayed addition of MSCs to pre-formed EC networks, whereby MSCs act as pericytes to the nascent vessels, resulted in the best developed vasculature. The results also demonstrate that the crosstalk between ECs and MSCs during microvessel formation occurs in a highly regulated, spatio-temporal fashion, whereby the initial seeding of ECs results in platelet derived growth factor (PDGF) release; the subsequent addition of MSCs 3 days later leads to a cessation in PDGF production, coinciding with increased vascular endothelial cell growth factor expression and enhanced vessel formation. Functional assessment of these pre-engineered constructs in a subcutaneous rat implant model demonstrated anastomosis between the in vitro engineered vessels and the host vasculature, with significantly increased vascularization occurring in the co-culture group. This study has thus provided new information on the process of in vitro vasculogenesis within a three-dimensional porous scaffold for tissue engineering and demonstrates the potential for using these vascularized scaffolds in the repair of critical sized bone defects.     

Fabrication and characterization of biomimetic collagen–apatite scaffolds with tunable structures for bone tissue engineering

The objective of the current study is to prepare a biomimetic collagen–apatite scaffold for improved bone repair and regeneration. A novel bottom–up approach has been developed, which combines a biomimetic self-assembly method with a controllable freeze-casting technology. In this study, the mineralized collagen fibers were generated using a simple one-step co-precipitation method which involved collagen self-assembly and in situ apatite precipitation in a collagen-containing modified simulated body fluid (m-SBF). The precipitates were then subjected to controllable freeze casting, forming scaffolds with either an isotropic equiaxed structure or a unidirectional lamellar structure. These scaffolds were comprised of collagen fibers and poorly crystalline bone-like carbonated apatite nanoparticles. The mineral content in the scaffold could be tailored in the range 0–54 wt.% by simply adjusting the collagen content in the m-SBF. Further, the mechanisms of the formation of both the equiaxed and the lamellar scaffolds were investigated, and freezing regimes for equiaxed and lamellar solidification were established. Finally, the bone-forming capability of such prepared scaffolds was evaluated in vivo in a mouse calvarial defect model. It was confirmed that the scaffolds well support new bone formation.     

Comparison of the properties of collagen–chitosan scaffolds after γ-ray irradiation and carbodiimide cross-linking

The property of collagen–chitosan porous scaffold varies according to cross-linking density and scaffold composition. This study was designed to compare the properties of collagen–chitosan porous scaffolds cross-linked with γ-irradiation and carbodiimide (CAR) for the first time. Eleven sets of collagen–chitosan scaffolds containing different concentrations of chitosan at a 5% increasing gradient were fabricated. Fourier transform infrared spectroscopy was performed to confirm the success of cross-linking in the scaffolds. The scaffold morphology was evaluated under scanning electron microscope (SEM). SEM revealed that chitosan was an indispensable material for the fabrication of γ-ray irradiation scaffold. The microstructure of γ-ray irradiation scaffold was less stable than those of alternative scaffolds. Based upon swelling ratio, porosity factor, and collagenase degradation, γ-ray irradiation scaffold was less stable than CAR and 25% proportion of chitosan scaffolds. Mechanical property determines the orientation in γ-irradiation and CAR scaffold. In vitro degradation test indicated that γ-irradiation and CAR cross-linking can elevate the scaffold biocompatibility. Compared with γ-ray irradiation, CAR cross-linked scaffold containing 25% chitosan can more significantly enhance the bio-stability and biocompatibility of collagen–chitosan scaffolds. CAR cross-linked scaffold may be the best choice for future tissue engineering.     

Structure–function relationships and source-to-ground distance in electrospun polycaprolactone

The strength of electrospun scaffolds has direct relevance to their function within tissue engineering. We characterized the effects of source-to-ground distance on the mechanical properties of electrospun poly(ε-caprolactone) (PCL). Source-to-ground distances of 10, 15 and 20 cm, solids concentrations of 12 and 18 wt.% and mandrel rotation surface speeds of 0–12 m s^?1 were utilized. Tensile tests evaluated elastic modulus, tensile strength and elongation at failure. Scanning electron microscopy provided morphology and quantified fiber alignment. Increased source-to-ground distance yielded a microstructure allowing greater fiber rearrangement under load, tripling the observed tensile strength. Increases in rotational speed generally increased fiber alignment and strength at high but not low to moderate speeds. As fiber is quickly pulled out of a comparatively gentle falling process, collision with neighboring fibers moving at different speeds and in different directions can occur. The source-to-ground distance influences these collisions and thus has critical implications for microstructure and biocompatibility. In larger diameter (18 wt.% PCL), heavily point-bonded fibers (produced using a shorter, 10 cm source-to-ground distance), elongation at failure in the aligned direction increases dramatically due to severe localized necking. These specimens show only half of the tensile strength (from 2.6 to 4.5 MPa) and a dramatic increase (from 94% to 503%) in elongation at failure vs. a longer 20 cm source-to-ground distance. Strains of several hundred per cent are accompanied by periodic necking of large-diameter fibers in which microstructural failure appears to occur in a sequential manner involving an equilibrium between localized strain in the tensile direction and anisotropic point bonding that locally resists strain.     

Improved performance of collagen scaffolds crosslinked by Traut’s reagent and Sulfo-SMCC

Collagen scaffolds are frequently employed for applications in regenerative medicine. In previous studies, we affirmed that Traut’s reagent (2-Iminothiolane hydrochloride) and Sulfo-SMCC (4-(N-Maleimidomethyl) cyclohexane-1-carboxylic acid 3-sulpho-N-hydroxysuccinimide ester sodium salt) could covalently bind growth factors on collagen scaffolds. We also observed that crosslinking formed within the collagen scaffolds with excess dosage of Sulfo-SMCC, which improved the biological performance of collagen scaffolds together with growth factors. In order to evaluate changes in capacity caused by crosslinking, Traut’s reagent and adjusted different concentrations of Sulfo-SMCC (0.263, 1.315, 2.63 and 5.26 mM) were used to construct collagen scaffolds with differing extents of crosslinking in this study. The results demonstrated that resistance of collagen scaffolds to enzymatic digestion, cellularization and vascularization in vivo were enhanced by the crosslinking procedure. The cell culture studies indicated that the crosslinking procedure did not influence biocompatibility. Moreover, there were no statistical differences in the degradation rate, cellularization or vascularization among 1.315, 2.63 and 5.26 mM crosslinked groups. These results demonstrated that crosslinking collagen scaffolds with an appropriate amount of Traut’s reagent and Sulfo-SMCC was an effective and safe method to modify naturally derived collagen scaffolds with notable potential uses in tissue regeneration.     

Nanostructuring of PEG–fibrinogen polymeric scaffolds

Recent studies have shown that nanostructuring of scaffolds for tissue engineering has a major impact on their interactions with cells. The current investigation focuses on nanostructuring of a biocompatible, biosynthetic polymeric hydrogel scaffold made from crosslinked poly(ethylene glycol)–fibrinogen conjugates. Nanostructuring was achieved by the addition of the block copolymer Pluronic^® F127, which self-assembles into nanometric micelles at certain concentrations and temperatures. Cryo-transmission electron microscopy experiments detected F127 micelles, both embedded within PEGylated fibrinogen hydrogels and in solution. The density of the F127 micelles, as well as their ordering, increased with increasing block copolymer concentration. The mechanical properties of the nanostructured hydrogels were investigated using stress-sweep rheological testing. These tests revealed a correlation between the block copolymer concentration and the storage modulus of the composite hydrogels. In vitro cellular assays confirmed that the increased modulus of the hydrogels did not limit the ability of the cells to form extensions and become spindled within the three-dimensional (3-D) hydrogel culture environment. Thus, altering the nanostructure of the hydrogel may be used as a strategy to control cellular behavior in 3-D through changes in mechanical properties of the environment.     

Incorporation of bioactive polyvinylpyrrolidone–iodine within bilayered collagen scaffolds enhances the differentiation and subchondral osteogenesis of mesenchymal stem cells

Polyvinylpyrrolidone–iodine (Povidone-iodine, PVP-I) is widely used as an antiseptic agent for lavation during joint surgery; however, the biological effects of PVP–I on cells from joint tissue are unknown. This study examined the biocompatibility and biological effects of PVP–I on cells from joint tissue, with the aim of optimizing cell-scaffold based joint repair. Cells from joint tissue, including cartilage derived progenitor cells (CPC), subchondral bone derived osteoblast and bone marrow derived mesenchymal stem cells (BM-MSC) were isolated. The concentration-dependent effects of PVP–I on cell proliferation, migration and differentiation were evaluated. Additionally, the efficacy and mechanism of a PVP–I loaded bilayer collagen scaffold for osteochondral defect repair was investigated in a rabbit model. A micromolar concentration of PVP–I was found not to affect cell proliferation, CPC migration or extracellular matrix production. Interestingly, micromolar concentrations of PVP–I promote osteogenic differentiation of BM-MSC, as evidenced by up-regulation of RUNX2 and Osteocalcin gene expression, as well as increased mineralization on the three-dimensional scaffold. PVP–I treatment of collagen scaffolds significantly increased fibronectin binding onto the scaffold surface and collagen type I protein synthesis of cultured BM-MSC. Implantation of PVP–I treated collagen scaffolds into rabbit osteochondral defect significantly enhanced subchondral bone regeneration at 6 weeks post-surgery compared with the scaffold alone (subchondral bone histological score of 8.80 ± 1.64 vs. 3.8 ± 2.19, p < 0.05). The biocompatibility and pro-osteogenic activity of PVP–I on the cells from joint tissue and the enhanced subchondral bone formation in PVP–I treated scaffolds would thus indicate the potential of PVP–I for osteochondral defect repair.     

The stimulation of healing within a rat calvarial defect by mPCL–TCP/collagen scaffolds loaded with rhBMP-2

Bone morphogenetic proteins (BMPs) have been widely investigated for their clinical use in bone repair and it is known that a suitable carrier matrix to deliver them is essential for optimal bone regeneration within a specific defect site. Fused deposited modeling (FDM) allows for the fabrication of medical grade poly ?-caprolactone/tricalcium phosphate (mPCL–TCP) scaffolds with high reproducibility and tailor designed dimensions. Here we loaded FDM fabricated mPCL–TCP/collagen scaffolds with 5 μg recombinant human (rh)BMP-2 and evaluated bone healing within a rat calvarial critical-sized defect. Using a comprehensive approach, this study assessed the newly regenerated bone employing micro-computed tomography (μCT), histology/histomorphometry, and mechanical assessments. By 15 weeks, mPCL–TCP/collagen/rhBMP-2 defects exhibited complete healing of the calvarium whereas the non-BMP-2-loaded scaffolds showed significant less bone ingrowth, as confirmed by μCT. Histomorphometry revealed significantly increased bone healing amongst the rhBMP-2 groups compared to non-treated scaffolds at 4 and 15 weeks, although the % BV/TV did not indicate complete mineralisation of the entire defect site. Hence, our study confirms that it is important to combine microCt and histomorphometry to be able to study bone regeneration comprehensively in 3D. A significant up-regulation of the osteogenic proteins, type I collagen and osteocalcin, was evident at both time points in rhBMP-2 groups. Although mineral apposition rates at 15 weeks were statistically equivalent amongst treatment groups, micro-compression and push-out strengths indicated superior bone quality at 15 weeks for defects treated with mPCL–TCP/collagen/rhBMP-2. Consistently over all modalities, the progression of healing was from empty defect < mPCL–TCP/collagen < mPCL–TCP/collagen/rhBMP-2, providing substantiating data to support the hypothesis that the release of rhBMP-2 from FDM-created mPCL–TCP/collagen scaffolds is a clinically relevant approach to repair and regenerate critically-sized craniofacial bone defects.     

Thermal dehydration treatment and glutaraldehyde cross-linking to increase the biostability of collagen–chitosan porous scaffolds used as dermal equivalent

A biodegradable scaffold for skin-tissue engineering was designed using collagen and chitosan, which are common materials for biomedical application. The scaffolds containing different amounts of chitosan were prepared by mixing the collagen and chitosan solutions followed by removal of the solvent using a freeze-drying method. The cross-linking treatment of these scaffolds was performed using the dehydrothermal treatment (DHT) method or glutaraldehyde (GA) to increase their biostability. The effect of the chitosan concentration and the cross-linking methods on the morphology of these scaffolds was studied by SEM. The water retention and the biodegradability in vitro of various collagen-chitosan scaffolds were investigated. Finally the biocompatibility of the collagen-chitosan (10 wt% chitosan) scaffold treated with different cross-linking methods was evaluated using a in vivo animal test. A mild inflammatory reaction could be detected in the early stages, and GA treatment can decrease the inflammatory reaction in a long-term implantation. After implantation for four weeks, all kinds of scaffolds, especially the GA-treated scaffolds (Col-GA) were filled with a large number of fibroblasts and were vascularized to a certain extent. These results suggest that the GA-treated scaffold has an increased biostability and excellent biocompatibility. It can be a potential candidate for skin-tissue engineering.     

The interplay between nanostructured carbon-grafted chitosan scaffolds and protein adsorption on the cellular response of osteoblasts: Structure–function property relationship

The rapid adsorption of proteins occurs during the early stages of biomedical device implantation into physiological systems. In this regard, the adsorption of proteins is a strong function of the nature of a biomedical device, which ultimately governs the biological functions. The objective of this study was to elucidate the interplay between nanostructured carbon-modified (graphene oxide and single-walled carbon nanohorn) chitosan scaffolds and consequent protein adsorption and biological function (osteoblast function). We compare and contrast the footprint of protein adsorption on unmodified chitosan and nanostructured carbon-modified chitosan. A comparative analysis of cell–substrate interactions using an osteoblast cell line (MC3T3-E1) implied that biological functions were significantly enhanced in the presence of nanostructured carbon, compared with unmodified chitosan. The difference in their respective behaviors is related to the degree and topography of protein adsorption on the scaffolds. Furthermore, there was a synergistic effect of nanostructured carbon and protein adsorption in terms of favorably modulating biological functions, including cell attachment, proliferation and viability, with the effect being greater on nanostructured carbon-modified scaffolds. The study also underscores that protein adsorption is favored in nanostructured carbon-modified scaffolds such that bioactivity and biological function are promoted.     

Collagen–hyaluronic acid scaffolds for adipose tissue engineering

Three-dimensional (3-D) in vitro models of the mammary gland require a scaffold matrix that supports the development of adipose stroma within a robust freely permeable matrix. 3-D porous collagen–hyaluronic acid (HA: 7.5% and 15%) scaffolds were produced by controlled freeze-drying technique and crosslinking with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. All scaffolds displayed uniform, interconnected pore structure (total porosity ～85%). Physical and chemical analysis showed no signs of collagen denaturation during the formation process. The values of thermal characteristics indicated that crosslinking occurred and that its efficiency was enhanced by the presence of HA. Although the crosslinking reduced the swelling of the strut material in water, the collagen–HA matrix as a whole tended to swell more and show higher dissolution resistance than pure collagen samples. The compressive modulus and elastic collapse stress were higher for collagen–HA composites. All the scaffolds were shown to support the proliferation and differentiation 3T3-L1 preadipocytes while collagen–HA samples maintained a significantly increased proportion of cycling cells (Ki-67+). Furthermore, collagen–HA composites displayed significantly raised Adipsin gene expression with adipogenic culture supplementation for 8 days vs. control conditions. These results indicate that collagen–HA scaffolds may offer robust, freely permeable 3-D matrices that enhance mammary stromal tissue development in vitro.     

Assessing both safe haven and secure base support in parent–child relationships

Parental sensitivity, a crucial element of attachment theory, refers to the ability to correctly interpret and respond appropriately to infants’ signals. The question of whether infants’ emotional expressions communicate discrete negative emotions has been widely debated in the literature on infant emotional development, but it has rarely been discussed in the parental sensitivity literature. This article explores how insights from the parental sensitivity literature and from evolutionary and dynamical systems perspectives on infant emotion expressions can be brought together to enhance our understanding of parental responsiveness to infant distress. The current research concludes that sensitivity does not rely on reading discrete negative emotions in infant signals, but rather on an integration of complex, graded distress expressions with contextual factors and iterative interaction processes.     

Microstructural and mechanical differences between digested collagen–fibrin co-gels and pure collagen and fibrin gels

Collagen and fibrin are important extracellular matrix (ECM) components in the body, providing structural integrity to various tissues. These biopolymers are also common scaffolds used in tissue engineering. This study investigated how co-gelation of collagen and fibrin affected the properties of each individual protein network. Collagen–fibrin co-gels were cast and subsequently digested using either plasmin or collagenase; the microstructure and mechanical behavior of the resulting networks were then compared with the respective pure collagen or fibrin gels of the same protein concentration. The morphologies of the collagen networks were further analyzed via three-dimensional network reconstruction from confocal image z-stacks. Both collagen and fibrin exhibited a decrease in mean fiber diameter when formed in co-gels compared with the pure gels. This microstructural change was accompanied by an increased failure strain and decreased tangent modulus for both collagen and fibrin following selective digestion of the co-gels. In addition, analysis of the reconstructed collagen networks indicated the presence of very long fibers and the clustering of fibrils, resulting in very high connectivities for collagen networks formed in co-gels.     

Vascularization of wide pore agarose–gelatin cryogel scaffolds implanted subcutaneously in diabetic and non-diabetic mice

Polymeric scaffolds have been reported to promote angiogenesis, facilitating oxygen delivery; however, little is known about the effect of diabetes on the neo-vascularization of implanted polymeric scaffolds at subcutaneous (SC) sites. In this study we compare the effect of diabetes on scaffold vascularization following SC implantation into diabetic and non-diabetic mice. Wide pore agarose cryogel scaffolds with grafted gelatin were prepared by a two-step freezing procedure and subsequent thawing. The scaffolds were implanted subcutaneously into streptozoticin-induced diabetic mice and control, non-diabetic mice. The vascularization process was estimated using histological sections, in which endothelial cells were identified by Von Willebrand factor (vWF) and CD31 antigen staining and the pericyte layer was confirmed by α-smooth muscle actin (α-SMA) visualization. Comparative analysis showed a similar thickness of fibrous capsules around the vascularized scaffolds in both diabetic and non-diabetic animals. Intensive staining for α-SMA indicated the formation of mature blood vessels in the surrounding fibrous capsule and tissue invading the scaffold area. No statistically significant differences in capillary density and area occupied by blood vessels were found between diabetic and non-diabetic mice. In conclusion, the present study shows no adverse effects of diabetes on new blood vessel formation in SC implanted agarose cryogel scaffolds with grafted gelatin.     

Biomimetic chitosan–nanohydroxyapatite composite scaffolds for bone tissue engineering

We describe a comparative assessment of the structure–property–process relationship of three-dimensional chitosan–nanohydroxyapatite (nHA) and pure chitosan scaffolds in conjunction with their respective biological response with the aim of advancing our insight into aspects that concern bone tissue engineering. High- and medium-molecular-weight (MW) chitosan scaffolds with 0.5, 1 and 2 wt.% fraction of nHA were fabricated by freezing and lyophilization. The nanocomposites were characterized by a highly porous structure and the pore size (～50 to 120 μm) was in a similar range for the scaffolds with different content of nHA. A combination of X-ray diffraction, Fourier transform infrared spectroscopy and electron microscopy indicated that nHA particles were uniformly dispersed in chitosan matrix and there was a chemical interaction between chitosan and nHA. The compression modulus of hydrated chitosan scaffolds was increased on the addition of 1 wt.% nHA from 6.0 to 9.2 kPa in high-MW scaffold. The water uptake ability of composites decreased with an increase in the amount of nHA, while the water retention ability was similar to pure chitosan scaffold. After 28 days in physiological condition, nanocomposites indicated about 10% lower degree of degradation in comparison to chitosan scaffold. The biological response of pre-osteoblasts (MC 3T3-E1) on nanocomposite scaffolds was superior in terms of improved cell attachment, higher proliferation, and well-spread morphology in relation to chitosan scaffold. In composite scaffolds, cell proliferation was about 1.5 times greater than pure chitosan after 7 days of culture and beyond, as implied by qualitative analysis via fluorescence microscopy and quantitative study through MTT assay. The observations related to well-developed structure morphology, physicochemical properties and superior cytocompatibility suggest that chitosan–nHA porous scaffolds are potential candidate materials for bone regeneration although it is necessary to further enhance the mechanical properties of the nanocomposite.