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.     

Fabrication and characterization of PLLA-chitosan hybrid scaffolds with improved cell compatibility

To combine the individual advantages of synthetic and natural polymers, poly(L-lactic acid) (PLLA)-chitosan hybrid scaffolds were fabricated. PLLA sponges were prepared by particulate-leaching, and then PLLA-chitosan hybrid scaffolds were obtained by dipping the PLLA sponges in chitosan solution and subsequently freeze-drying. Physicochemical properties of the scaffolds were characterized by scanning electron microscopy (SEM), water uptake test, and mechanical strength measurement. Moreover, cell adhesion, cell proliferation, and cell viability on the scaffolds were evaluated through osteoblast-like cell culture. The experimental results indicated that, PLLA sponges exhibited macroporous structure and the interconnected microporous structure of chitosan was formed within the macropores of PLLA sponges. The incorporation of chitosan reinforced PLLA sponges in dependence on chitosan content. The hybrid scaffolds had higher water uptake ability compared with PLLA sponges. Particularly, the hybrid scaffolds exhibited excellent cell attachment efficiency, cell proliferation, and cell viability. This study suggests that the hybrid scaffolds obtain good mechanical strength from PLLA and excellent cell compatibility from chitosan.     

Three-dimensional porous bioscaffolds for bone tissue regeneration: Fabrication via adaptive foam reticulation and freeze casting techniques, characterization, and cell study

Highly interconnected and 3D porous bioactive hydroxyapatite (HAP) and Bioglass scaffolds have been fabricated by an adaptive version of camphene based foam reticulation (ARM) and camphene freeze casting (CFC) methods. Controlled sublimation of camphene during freeze casting at -78°C produced process optimized bioscaffolds with open, uniform, and interconnected porous structures. HAP and Bioglass scaffolds with desired porosity, pore size, and microtopography were successfully fabricated using polyurethane foam templates of appropriate structures. Macropores of 50-1100 μm with microporosity of 1-10 μm, known to facilitate cell adhesion and proliferation, were obtained. Compressive yield strength of 0.8 MPa close to the upper range of cancellous bone was achieved. The mean compressive strength of HAP scaffolds compared favorably with the theoretical model of porosity variation with strength and was higher than reported values. The nature of pore development, morphology, porosity, crystal structure, chemical composition, and thermal behavior were characterized using scanning electron and optical microscopy, X-ray diffraction, thermal analysis, and mercury porosimetry. These scaffolds are suited for nonstructural graft and were not cytotoxic in vitro when osteoblast-like MG63 cells were cultured with the HAP constructs. The cells attached indicated by cell metabolic activity by resazurin assay and spread well when cultured on the surface of the materials. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 100A:2948-2959, 2012.     

Preparation of Interconnected Porous Chitosan Scaffolds by Sodium Acetate Particulate Leaching

For tissue-engineering applications, a 3D porous chitosan scaffold was simply prepared from a mixture of acidic chitosan solution and sodium acetate particles as the porogen by a salt-leaching method. Differences in the porous structure in terms of pore morphology and interconnectivity between the salt-leached chitosan scaffold and phase-separated scaffold as the control were examined by using scanning electron microscopy, protein release and enzymatic degradation tests. A fibroblast (NIH-3T3) cell culture was performed for cell affinity evaluation. The chitosan scaffold prepared by salt-leaching showed good interconnectivity and improved mechanical properties. Furthermore, the chitosan scaffolds showed a high initial cell adhesion after 4 h cell culture and increased cell proliferation than the control. Thus, salt-leached chitosan scaffolds can be used for various tissue-engineering applications.     

Fabrication and characterization of gelatin-based biocompatible porous composite scaffold for bone tissue engineering

In this study, composite scaffolds were prepared with polyethylene oxide (PEO)-linked gelatin and tricalcium phosphate (TCP). Chitosan, a positively charged polysaccharide, was introduced into the scaffolds to improve the properties of the artificial bone matrix. The chemical and thermal properties of composite scaffolds were investigated by Fourier transform infrared spectroscopy, thermogravimetric analyzer, differential thermal analyzer. In vitro cytotoxicity of the composite scaffold was also evaluated and the sample showed no cytotoxic effect. The morphology was studied by SEM and light microscopy. It was observed that the prepared scaffold had an open interconnected porous structure with pore size of 230-354 μm, which is suitable for osteoblast cell proliferation. The mechanical properties were assessed and it was found that the composite had compressive modulus of 1200 MPa with a strength of 5.2 MPa and bending modulus of 250 MPa having strength of 12.3 MPa. The porosity and apparent density were calculated and it was found that the incorporation of TCP can reduce the porosity and water absorption. It was revealed from the study that the composite had a 3D porous microstructure and TCP particles were dispersed evenly among the crosslinked gelatin/chitosan scaffold. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 100A:3020-3028, 2012.     

Engineering new bone tissue in vitro on highly porous poly(alpha-hydroxyl acids)/hydroxyapatite composite scaffolds

Engineering new bone tissue with cells and a synthetic extracellular matrix (scaffolding) represents a new approach for the regeneration of mineralized tissues compared with the transplantation of bone (autografts or allografts). In the present work, highly porous poly(L-lactic acid) (PLLA) and PLLA/hydroxyapatite (HAP) composite scaffolds were prepared with a thermally induced phase separation technique. The scaffolds were seeded with osteoblastic cells and cultured in vitro. In the pure PLLA scaffolds, the osteoblasts attached primarily on the outer surface of the polymer. In contrast, the osteoblasts penetrated deep into the PLLA/HAP scaffolds and were uniformly distributed. The osteoblast survival percentage in the PLLA/HAP scaffolds was superior to that in the PLLA scaffolds. The osteoblasts proliferated in both types of the scaffolds, but the cell number was always higher in the PLLA/HAP composite scaffolds during 6 weeks of in vitro cultivation. Bone-specific markers (mRNAs encoding bone sialoprotein and osteocalcin) were expressed more abundantly in the PLLA/HAP composite scaffolds than in the PLLA scaffolds. The new tissue increased continuously in the PLLA/HAP composite scaffolds, whereas new tissue formed only near the surface of pure PLLA scaffolds. These results demonstrate that HAP imparts osteoconductivity and the highly porous PLLA/HAP composite scaffolds are superior to pure PLLA scaffolds for bone tissue engineering.     

Uniform deposition of protein incorporated mineral layer on three-dimensional porous polymer scaffolds

Inorganic-organic hybrid materials designed to facilitate bone tissue regeneration use a calcium phosphate mineral layer to encourage cell adhesion, proliferation, and osteogenic differentiation. Mineral formed on porous materials is often discontinuous through the thickness of the scaffold. This study aimed to uniformly coat the pores of three-dimensional (3D) porous, polymer scaffolds with a bone-like mineral layer in addition to uniformly incorporating a model protein within this mineral layer. A filtration system designed to induce simulated body fluid flow through the interstices of 3D polylactic-co-glycolic acid scaffolds (10-mm diameter x 2-mm thickness) illustrated that a uniform, continuous mineral layer can be precipitated on the pore surfaces of a 3D porous structure within 5 days. MicroCT analysis showed increased mineral volume percent (MV%) (7.86 +/- 3.25 MV%, p = 0.029) and continuous mineralization of filtered scaffolds compared with two static control groups (floating, 0.16 +/- 0.26 MV% and submerged, 0.20 +/- 0.01 MV%). Furthermore, the system was effective in coprecipitating a model protein, bone sialoprotein (BSA), within the mineral layer. A 10-fold increase in BSA incorporation was seen when coprecipitated filtered scaffolds (1308 +/- 464 microg) were compared to a submerged static control group (139 +/- 45 microg), p < 0.001. Confocal microscopy visually confirmed uniform coprecipitation of BSA throughout the thickness of the filtration scaffolds. The designed system enables 3D mineralization through the thickness of porous materials, and provides the option of including coprecipitated biomolecular cues within the mineral layer. This approach of providing a 3D conductive and osteoinductive environment could be conducive to bone tissue regeneration.     

Novel chitosan-based hyaluronan hybrid polymer fibers as a scaffold in ligament tissue engineering

To clarify the feasibility of using novel chitosan-based hyaluronan hybrid polymer fibers as a scaffold in ligament tissue engineering, their mechanical properties and ability to promote cellular adhesion, proliferation, and extracellular matrix production were studied in vitro. Chitosan fibers and chitosan-based 0.05% and 0.1% hyaluronan hybrid fibers were developed by the wet spinning method. Hyaluronan coating significantly increased mechanical properties, compared to the chitosan fibers. Rabbit fibroblasts adhesion onto hybrid fibers was significantly greater than for the control and chitosan fibers. For analysis of cell proliferation and extracellular matrix production, a three-dimensional scaffold was created by simply piling up each fiber. At 1 day after cultivation, the DNA content in the hybrid scaffolds was higher than that in the chitosan scaffold. Scanning electron microscopy showed that the fibroblasts had produced collagen fibers after 14 days of culture. Immunostaining for type I collagen was clearly predominant in the hybrid scaffolds, and the mRNA level of type I collagen in the hybrid scaffolds were significantly greater than that in the chitosan scaffold. The present study revealed that hyaluronan hybridization with chitosan fibers enhanced fiber mechanical properties and in vitro biological effects on the cultured fibroblasts.     

Porous alginate/hydroxyapatite composite scaffolds for bone tissue engineering: preparation, characterization, and in vitro studies

In this study a series of alginate/hydroxyapatite (HAP) composite scaffolds was prepared by phase separation. HAP was incorporated into the alginate gel solution to improve both the mechanical and cell-attachment properties of the scaffolds. These scaffolds had a well-interconnected porous structure with an average pore size of 150 microm and over 82% porosity. The alginate/HAP scaffold prepared at -40 degrees C with a 50% HAP content showed the best mechanical properties. The morphology of scaffolds could be manipulated by tuning the quenching temperature during the preparation. The dissolution of alginate/HAP composite scaffolds could be slowed by the pretreating them by immersion in 1.0 M CaCl(2) solution. The rat osteosarcoma UMR106 cells, an osteoblastic cell line, seeded in the scaffolds, displayed better cell attachment to the 75/25 and 50/50 alginate/HAP composite scaffolds than to the pure alginate scaffold. The natural polymeric sponges that fabricated in this study may be a promising approach for tissue-engineering applications.     

Chitosan-alginate as scaffolding material for cartilage tissue engineering

Tissue compatibility of chitosan-alginate scaffolds was studied in vitro in terms of cell morphology, proliferation, and functionality using HTB-94 cells. The scaffold has an interconnected 3D porous structure, and was fabricated by thermally induced phase separation followed by freeze drying. Cell proliferation on the chitosan-alginate scaffold was found to be faster than on a pure chitosan scaffold. After cell culture for 2 weeks in vitro, the cells on the chitosan scaffold gradually assumed a fibroblast-like morphology while the cells on the chitosan-alginate scaffold retained their spherical morphology throughout the period of study. SDS-PAGE electrophoresis and Western blot assays for proteins extracted from cells grown on scaffolds indicated that production of cartilage-specific collagen type II, a marker for chondrocytic phenotype, increased from week 2 to week 3 on the chitosan-alginate scaffold but decreased on the chitosan scaffold. This study suggested that chitosan-alginate scaffolds promote cell proliferation, enhance phenotype expression of HTB-94 chondrocytes, and may potentially serve as an improved alternative to chitosan scaffolds for cartilage tissue engineering.     

The precision structural regulation of PLLA porous scaffold and its influence on the proliferation and differentiation of MC3T3-E1 cells

A thermal-induced phase separation combined sugar template method was used to fabricate the Poly (L-lactide) acid (PLLA) scaffolds with precisely regulated porous structure. The effect of tuned porous structure of scaffolds on osteoblasts proliferation and differentiation was investigated. The results showed that the pore diameters (200–300, 300–400, 400–500 μm), porosity and interconnectivity of PLLA scaffolds can be accurately controlled indicated by scanning electron microscope. The results of cell experiments showed that the porous structure including the pore size and interconnectivity of scaffolds dramatically influence the cell proliferation and differentiation. The scaffold with pore diameter of 400–500 μm exhibited the highest cell viability and alkaline phosphatase activity among all the scaffolds for the MC3T3-E1 cells. The higher cell proliferation and biocompatibility observed in the 400–500 μm scaffold indicated the high selectivity for MC3T3-E1cells on the pore size of scaffold in tissue engineering. The precise control of the porous structure of scaffold may better guide the cell–matrix interaction in the future research.     

Mesenchymal stem cell proliferation and differentiation on load-bearing trabecular Nitinol scaffolds

Bone tissue regeneration in load-bearing regions of the body requires high-strength porous scaffolds capable of supporting angiogenesis and osteogenesis. 70% porous Nitinol (NiTi) scaffolds with a regular 3-D architecture resembling trabecular bone were produced from Ni foams using an original reactive vapor infiltration technique. The “trabecular Nitinol” scaffolds possessed a high compressive strength of 79 MPa and high permeability of 6.9 × 10^?6 cm². The scaffolds were further modified to produce a near Ni-free surface layer and evaluated in terms of Ni ion release and human mesenchymal stem cell (hMSC) proliferation (AlamarBlue), differentiation (alkaline phosphatase activity, ALP) and mineralization (Alizarin Red S staining). Scanning electron microscopy was employed to qualitatively corroborate the results. hMSCs were able to adhere and proliferate on both as-produced and surface-modified trabecular NiTi scaffolds, to acquire an osteoblastic phenotype and produce a mineralized extracellular matrix. Both ALP activity and mineralization were increased on porous scaffolds compared to control polystyrene plates. Experiments in a model coculture system of microvascular endothelial cells and hMSCs demonstrated the formation of prevascular structures in trabecular NiTi scaffolds. These data suggest that load-bearing trabecular Nitinol scaffolds could be effective in regenerating damaged or lost bone tissue.     

Natural origin scaffolds with in situ pore forming capability for bone tissue engineering applications

This work describes the development of a biodegradable matrix, based on chitosan and starch, with the ability to form a porous structure in situ due to the attack by specific enzymes present in the human body (alpha-amylase and lysozyme). Scaffolds with three different compositions were developed: chitosan (C100) and chitosan/starch (CS80-20, CS60-40). Compressive test results showed that these materials exhibit very promising mechanical properties, namely a high modulus in both the dry and wet states. The compressive modulus in the dry state for C100 was 580+/-33MPa, CS80-20 (402+/-62MPa) and CS60-40 (337+/-78MPa). Degradation studies were performed using alpha-amylase and/or lysozyme at concentrations similar to those found in human serum, at 37 degrees C for up to 90 days. Scanning electron micrographs showed that enzymatic degradation caused a porous structure to be formed, indicating the potential of this methodology to obtain in situ forming scaffolds. In order to evaluate the biocompatibility of the scaffolds, extracts and direct contact tests were performed. Results with the MTT test showed that the extracts of the materials were clearly non-toxic to L929 fibroblast cells. Analysis of cell adhesion and morphology of seeded osteoblastic-like cells in direct contact tests showed that at day 7 the number of cells on CS80-20 and CS60-40 was noticeably higher than that on C100, which suggests that starch containing materials may promote cell adhesion and proliferation. This combination of properties seems to be a very promising approach to obtain scaffolds with gradual in vivo pore forming capability for bone tissue engineering applications.     

Effects of in situ and physical mixing on mechanical and bioactive behaviors of nano hydroxyapatite-chitosan scaffolds

Nano hydroxyapatite (HAP) was employed to intensify chitosan (CS) scaffolds by two methods. The first one is nano HAP crystallized in situ from the CS matrix by a biomimetic method (in situ scaffold). In the second method the sol-gel nano HAP powder was added directly to the CS solution (physical mixing scaffold). The distribution status of nano HAP was examined by scanning electron microscopy. The compressive performance was measured by a universal material testing machine. The in vitro study in stimulated body fluid was performed to evaluate the biological properties of both scaffolds. MTT testing and alkaline phosphatase activity from human bone mesenchymal stem cell culture showed differences in biocompatibility and bioactivity between the scaffolds. The results indicated that the in situ scaffold possessed more excellent mechanical and bioactive behaviors than that of the physical mixing scaffold.     

Preparation and in vitro characterization of electrospun PVA scaffolds coated with bioactive glass for bone regeneration

An important objective in bone tissue engineering is to fabricate biomimetic three-dimensional scaffolds that stimulate mineralization for rapid regeneration of bone. In this work, scaffolds of electrospun poly(vinyl alcohol) (PVA) fibers (diameter = 286 ± 14 nm) were coated with a sol-gel derived bioactive glass (BG) and evaluated in vitro for potential applications in bone repair. Structural and chemical analyses showed that the BG coating was homogeneously deposited on the PVA fibers. In vitro cell culture studies showed that the BG-coated PVA scaffold had a greater capacity to support proliferation of osteogenic MC3T3-E1 cells, alkaline phosphatase activity, and mineralization than the uncoated PVA scaffold. The BG coating improved the tensile strength of the PVA scaffold from 18 ± 2 MPa to 21 ± 2 MPa, but reduced the elongation to failure from 94 ± 4% to 64 ± 5%. However, immersion of the BG-coated PVA scaffolds in a simulated body fluid for 5 days resulted in an increase in the tensile strength (24 ± 2 MPa) and elongation to failure (159 ± 4%). Together, the results show that these BG-coated PVA scaffolds could be considered as candidate materials for bone tissue engineering applications.     

Balancing mechanical strength with bioactivity in chitosan–calcium phosphate 3D microsphere scaffolds for bone tissue engineering: air- vs. freeze-drying processes

The objective of this study was to evaluate the potential benefit of 3D composite scaffolds composed of chitosan and calcium phosphate for bone tissue engineering. Additionally, incorporation of mechanically weak lyophilized microspheres within those air-dried (AD) was considered for enhanced bioactivity. AD microsphere, alone, and air- and freeze-dried microsphere (FDAD) 3D scaffolds were evaluated in vitro using a 28-day osteogenic culture model with the Saos-2 cell line. Mechanical testing, quantitative microscopy, and lysozyme-driven enzymatic degradation of the scaffolds were also studied. FDAD scaffold showed a higher concentration (p?<?0.01) in cells per scaffold mass vs. AD constructs. Collagen was ～31% greater (p?<?0.01) on FDAD compared to AD scaffolds not evident in microscopy of microsphere surfaces. Alternatively, AD scaffolds demonstrated a superior threefold increase in compressive strength over FDAD (12 vs. 4?MPa) with minimal degradation. Inclusion of FD spheres within the FDAD scaffolds allowed increased cellular activity through improved seeding, proliferation, and extracellular matrix production (as collagen), although mechanical strength was sacrificed through introduction of the less stiff, porous FD spheres.     

Characterization and cytocompatibility of biphasic calcium phosphate/polyamide 6 scaffolds for bone regeneration

Porous scaffolds of biphasic calcium phosphate (BCP)/polyamide 6 (PA6) with weight ratios of 30/70, 45/55, and 55/45 have been fabricated through a modified thermally induced phase separation technique. The chemical structure properties, macrostructure, and mechanical strength of the scaffolds were characterized by Fourier transform infrared spectroscopy, X-ray diffraction, thermogravimetric analysis, scanning electron microscopy, and mechanical testing. The results indicated that the BCP/PA6 scaffolds had an interconnected porous structure with a pore size mainly ranging from 100 to 900 μm and many micropores on the rough pore walls. The mechanical property of the scaffold was significantly enhanced by the addition of BCP inorganic fillers. The 55/45 BCP/PA6 composite scaffold with 76.5% ± 2.1% porosity attained a compressive strength of 1.86 ± 0.14 MPa. Moreover, the BCP/PA6 porous scaffold was cultured with rat calvarial osteoblasts to investigate the cell proliferation, viability, and differentiation function (alkaline phosphatase). The type I collagen expression was also used to characterize the differentiation of rat calvarial osteoblasts on BCP/PA6 composite scaffold by immunocytochemistry. The in vitro cytocompatibility evaluation demonstrated that the BCP/PA6 scaffold acted as a good template for the cells adhesion, spreading, growth, and differentiation. These results suggest that the BCP/PA6 porous composite could be a candidate as an excellent substitute for damaged or defect bone.     

Synergistic Stimuli by Hydrodynamic Pressure and Hydrophilic Coating on PLGA Scaffolds for Extracellular Matrix Synthesis of Engineered Cartilage

Abstract

Scaffold surface properties and mechanical stimuli have been known to have a critical influence on cell proliferation and extracellular matrix synthesis in cultured cells for tissue engineering. Hydrophilic surface and hydrodynamic pressure (HP) stimulation have been shown to have a beneficial effect on chondrocyte activity. The aim of this study was, thus, to assess the synergic influences of HP and hydrophilic coating on cell activity using primary porcine chondrocytes inoculated in hydrophilic-coated poly(lactide-co-glycolide) (PLGA) sponge scaffolds. The natural materials hyaluronic acid (HA), chitosan and HA/chitosan were cross-linked on porous PLGA as a hydrophilic surface modification. HP was applied to scaffolds at an amplitude of 2.24 MPa and a frequency of 0.1 Hz for 30 min per day, twice a week, over a period of 28 days. Cell activities were determined by the MTS assay and the dimethylmethylene blue assay for glycosaminoglycan (GAG) quantification. Our results displayed that PLGA coated with both HA and chitosan had the best hydrophilicity (contact angle 49.46°) and initial compressive modulus (1.10 ± 0.13 MPa) among the tested scaffold groups. Additionally, HP stimulation enhanced cell proliferation as well as GAG production (up to 3-fold in culture medium and 15-fold in scaffolds at 28 days compared to static culture of PLGA alone in the scaffold group) in the hydrophilic-coated scaffold groups. The synergistic benefit from hydrophilic coating and HP stimulation may be imperative in regenerating engineered cartilage in the long-term.     

Organic/inorganic hybrid network structure nanocomposite scaffolds based on grafted chitosan for tissue engineering

We describe the first study of structure-processing-property relationship in organic/inorganic hybrid network structure nanocomposite scaffolds based on grafted chitosan for bone tissue engineering. Chitosan was first grafted with propylene oxide to form hydroxypropylated chitosan, which was subsequently linked with ethylene glycol functionalized nanohydroxyapatite to form an organic/inorganic network structure. The resulting scaffold was characterized by a highly porous structure and significantly superior physico-chemical, mechanical and biological properties compared to pure chitosan. The scaffolds exhibited high modulus, controlled swelling behavior and reduced water uptake, but the water retention ability was similar to pure chitosan scaffold. MTT assay studies confirmed the non-cytotoxic nature of the scaffolds and enabled degradation products to be analyzed. The nanocomposite scaffolds were biocompatible and supported adhesion, spreading, proliferation and viability of osteoblasts cells. Furthermore, the cells were able to infiltrate and colonize into the pores of the scaffolds and establish cell-cell interactions. The study suggests that hydroxypropylation of chitosan and forming a network structure with a nano-inorganic constituent is a promising approach for enhancing physico-chemical, functional and biological properties for utilization in bone tissue engineering applications.     

Effects of In Situ and Physical Mixing on Mechanical and Bioactive Behaviors of Nano Hydroxyapatite–Chitosan Scaffolds

Nano hydroxyapatite (HAP) was employed to intensify chitosan (CS) scaffolds by two methods. The first one is nano HAP crystallized in situ from the CS matrix by a biomimetic method (in situ scaffold). In the second method the sol–gel nano HAP powder was added directly to the CS solution (physical mixing scaffold). The distribution status of nano HAP was examined by scanning electron microscopy. The compressive performance was measured by a universal material testing machine. The in vitro study in stimulated body fluid was performed to evaluate the biological properties of both scaffolds. MTT testing and alkaline phosphatase activity from human bone mesenchymal stem cell culture showed differences in biocompatibility and bioactivity between the scaffolds. The results indicated that the in situ scaffold possessed more excellent mechanical and bioactive behaviors than that of the physical mixing scaffold.