Hydroxyapatite Composite Scaffolds Research Articles

Magnetic responsive hydroxyapatite composite scaffolds construction for bone defect reparation

IntroductionIn recent years, interest in magnetic biomimetic scaffolds for tissue engineering has increased considerably. A type of magnetic scaffold composed of magnetic nanoparticles (MNPs) and hydroxyapatite (HA) for bone repair has been developed by our research group.Aim and methodsIn this study, to investigate the influence of the MNP content (in the scaffolds) on the cell behaviors and the interactions between the magnetic scaffold and the exterior magnetic field, a series of MNP-HA magnetic scaffolds with different MNP contents (from 0.2% to 2%) were fabricated by immersing HA scaffold into MNP colloid. ROS 17/2.8 and MC3T3-E1 cells were cultured on the scaffolds in vitro, with and without an exterior magnetic field, respectively. The cell adhesion, proliferation and differentiation were evaluated via scanning electron microscopy; confocal laser scanning microscopy; and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), alkaline phosphatase, and bone gla protein activity tests.ResultsThe results demonstrated the positive influence of the magnetic scaffolds on cell adhesion, proliferation, and differentiation. Further, a higher amount of MNPs on the magnetic scaffolds led to more significant stimulation.ConclusionThe magnetic scaffold can respond to the exterior magnetic field and engender some synergistic effect to intensify the stimulating effect of a magnetic field to the proliferation and differentiation of cells.

Synthesis, characterization and cytocompatibility studies of α-chitin hydrogel/nano hydroxyapatite composite scaffolds

α-chitin hydrogel/nano hydroxyapatite (nHAp) composite scaffold have been synthesized by freeze-drying approach with nHAp and α-chitin hydrogel. The prepared nHAp and nanocomposite scaffolds were characterized using DLS, SEM, FT-IR, XRD and TGA studies. The porosity, swelling, degradation, protein adsorption and biomineralization (calcification) of the prepared nanocomposite scaffolds were evaluated. Cell viability, attachment and proliferation were investigated using MG 63, Vero, NIH 3T3 and nHDF cells to confirm that the nanocomposite scaffolds were cytocompatible and cells were found to attach and spread on the scaffolds. All the results suggested that these scaffolds can be used for bone and wound tissue engineering.

β-Chitin hydrogel/nano hydroxyapatite composite scaffolds for tissue engineering applications

β-Chitin hydrogel/nano hydroxyapatite (nHAp) nanocomposite scaffolds were prepared by freeze-drying approach from the mixture of β-chitin hydrogel and nHAp in different concentrations such as 0.5 and 1%, respectively. The prepared nHAp and nanocomposite scaffolds were characterized using various modalities. Porosity, swelling ability, in vitro degradation, protein adsorption and biomineralization of the prepared composite scaffolds were evaluated. The composite scaffolds were found to have 70–80% porosity with well defined interconnected porous structure. The scaffolds also showed a swelling ratio of 15–20, controlled biodegradation of about 30–40% with enhanced protein adsorption. In addition, the cell viability, attachment and proliferation using MG 63, Vero, NIH3T3 and nHDF cells confirmed the cytocompatibility nature of the nanocomposite scaffolds with well improved cell attachment and proliferation. All these results essentially signify that this material can be a potential candidate for bone and wound tissue engineering applications.

Processing/structure/property relationship of multi‐scaled PCL and PCL–HA composite scaffolds prepared via gas foaming and NaCl reverse templating

In this study, we investigated the processing/structure/property relationship of multi-scaled porous biodegradable scaffolds prepared by combining the gas foaming and NaCl reverse templating techniques. Poly(ε-caprolactone) (PCL), hydroxyapatite (HA) nano-particles and NaCl micro-particles were melt-mixed by selecting different compositions and subsequently gas foamed by a pressure-quench method. The NaCl micro-particles were finally removed from the foamed systems in order to allow for the achievement of the multi-scaled scaffold pore structure. The control of the micro-structural properties of the scaffolds was obtained by the optimal combination of the NaCl templating concentration and the composition of the CO2-N2 mixture as the blowing agent. In particular, these parameters were accurately selected to allow for the fabrication of PCL and PCL-HA composite scaffolds with multi-scaled open pore structures. Finally, the biocompatibility of the scaffolds has been assessed by cultivating pre-osteoblast MG63 cells in vitro, thus demonstrating their potential applications for bone regeneration.

The influence of polymer concentrations on the structure and mechanical properties of porous polycaprolactone-coated hydroxyapatite scaffolds

Polycaprolactone (PCL)-coated porous hydroxyapatite (HA) composite scaffolds were prepared by combining polymer impregnating method with dip-coating method. Three different PCL solution concentrations were used in dip-coating process to improve the mechanical properties of porous HA scaffolds. The results indicated that as the concentration of PCL solution increases the compressive strength significantly increased from 0.09 MPa to 0.51 MPa while the porosity decreased from 90% to 75% for the composite scaffolds. An interlaced structure was found inside the pore wall for all composite scaffolds due to the penetration of PCL. The porous HA/PCL composite scaffolds dip-coated with 10% PCL exhibited optimal combination of mechanical properties and pore interconnectivity, and may be a potential bone candidate for the tissue engineering applications.

Preparation of Polyurethane / Hydroxyapatite Composite Scaffold for Tissue Regeneration

Preparation of Polyurethane / Hydroxyapatite Composite Scaffold for Tissue Regeneration

Preparation and characterization of chitosan–gelatin/nanohydroxyapatite composite scaffolds for tissue engineering applications

Chitosan is a novel biocompatible, biodegradable polymer for potential use in tissue engineering. In this work, chitosan–gelatin/nanophase hydroxyapatite composite scaffolds were prepared by blending chitosan and gelatin with nanophase hydroxyapatite (nHA). The prepared nHA was characterized using TEM, XRD and FT-IR. The prepared composite scaffolds were characterized using SEM, FT-IR and XRD studies. The composite scaffolds were highly porous with a pore size of 150–300 μm. In addition, density, swelling ratio, degradation, biomineralization, cytotoxicity and cell attachment of the composite scaffolds were studied. The scaffolds showed good swelling character, which could be modulated by varying ratio of chitosan and gelatin. Composite scaffolds in the presence of nHA showed a decreased degradation rate and increased mineralization in SBF. The biological response of MG-63 cells on nanocomposite scaffolds was superior in terms of improved cell attachment, higher proliferation, and spreading compared to chitosan–gelatin (CG) scaffold.

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

AbstractPoly(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

Preparation and characterization of aliphatic polyurethane and hydroxyapatite composite scaffold

AbstractNovel porous composite scaffolds for tissue engineering were prepared from aliphatic biodegradable polyurethane (PU) elastomer and hydroxyapatite (HA). It was found that the aliphatic PU was possible to load up to 50 wt % HA. The morphology and properties of the scaffolds were characterized by scanning electron microscope, X‐ray diffraction, infrared absorption spectra, mechanical testing, dynamic mechanical analysis, and in vitro degradation measurement. The results indicated that the HA/PU scaffolds had an interconnected porous structure with a pore size mainly ranging from 300 to 900 μm, and 50–200 μm micropores existed on the pores' walls. The average pore size of macropores and micropores are 510 and 100 μm, respectively. The compressive strength of the composite scaffolds showed higher enhancement with increasing HA content. In addition, the polymer matrix was completely composed of aliphatic component and exhibited progressive mass loss in vitro degradation, and the degradation rate depended on the HA content in PU matrix. The porous HA/PU composite may have a good prospect to be used as scaffold for tissue engineering. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009

Enhanced bone formation by marrow‐derived endothelial and osteogenic cell transplantation

Bone marrow-derived osteogenic cells can regenerate bone tissuesin vivo. The aim of the present study is to determine whether the cotransplantation of bone marrow-derived endothelial-like cells (BMECs) enhances bone regeneration by bone marrow-derived osteogenic cell (BMOC) transplantation in osseous defects. Canine bone marrow cells were differentiated separately into BMECs and BMOCs. Using apatite-coated poly(lactide-co-glycolide)/hydroxyapatite composite scaffolds as cell delivery vehicles, BMOCs were transplanted with or without BMECs into critical-sized calvarial defects in immunodeficient mice. Histological analyses, microcomputed tomography, and soft X-ray were performed to assess mineralized bone formation at 8 weeks. Cotransplantation of BMECs and BMOCs resulted in greater bone formation than transplantation of BMOCs alone. There was a significant (p < 0.05) increase in bone formation area following cotransplantation (30.8% +/- 2.5%), compared with transplantation of BMOCs alone (15.3% +/- 1.9%). These results demonstrate that the cotransplantation of BMECs enhances bone regeneration mediated by BMOC transplantation in osseous defects.

Effect of Saturation and Post Processing on 3D Printed Calcium Phosphate Scaffolds

Three dimensional printing was investigated for fabricating hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) composite scaffolds using calcium phosphate based ceramics and calcium phosphate cement chemistry. Scaffolds were formed by printing an aqueous sodium phosphate solution on the powder bed consisting of a mixture of dicalcium phosphate anhydrous (DCPA) and calcium hydroxide powders. The sodium phosphate solution was functioning as a binder material and also as the initiator of the wet chemical reaction. Compressive mechanical properties of printed samples were examined as a function of saturation level that was inversely proportional to the powder to liquid ratio. To increase mechanical properties and obtain hydroxyapatite and β-TCP composites, the printed samples were sintered. The effect of sintering parameters including dwell time and sintering temperature were also examined. X-ray diffraction (XRD) was used to examine material composition at different stages of the manufacturing process and to confirm the presence of HA and β-TCP in the final stage. The effect of sintering procedure on the surface topology of the samples was examined using scanning electron microscopy (SEM).

In Vivo Bone Formation Following Transplantation of Human Adipose–Derived Stromal Cells That Are Not Differentiated Osteogenically

A number of studies have shown in vivo bone regeneration by transplantation of osteogenic cells differentiated in vitro from adipose-derived stromal cells (ADSCs). However, the in vitro osteogenic differentiation process requires an additional culture period, and the dexamethasone that is generally used in the process may be cytotoxic. Here, we tested the hypothesis that ADSCs that are not differentiated osteogenically in vitro prior to transplantation would extensively regenerate bone in vivo when exogenous bone morphogenetic protein-2 (BMP-2) is delivered to the transplantation site. We fabricated a poly(dl-lactic-co-glycolic acid)/hydroxyapatite (PLGA/HA) composite scaffold with osteoactive HA that is highly exposed on the scaffold surface. This scaffold was able to release BMP-2 over a 4-week period in vitro. Human ADSCs cultured on BMP-2-loaded PLGA/HA scaffolds for 2 weeks differentiated toward osteogenic cells expressing alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN) mRNA, while cells on PLGA/HA scaffolds without BMP-2 expressed only ALP. To study in vivo bone formation, PLGA/HA scaffolds (group 1), BMP-2-loaded PLGA/HA scaffolds (group 2), undifferentiated ADSCs seeded on PLGA/HA scaffolds (group 3), and undifferentiated ADSCs seeded on BMP-2-loaded PLGA/HA scaffolds (group 4) were implanted into dorsal, subcutaneous spaces of athymic mice. Eight weeks after implantation, group 4 exhibited a 25-fold greater bone formation area and 5-fold higher calcium deposition than group 3. Bone regeneration by transplanted human ADSCs in group 4 was confirmed by expression of human-specific osteoblastic genes, ALP, collagen type I, OPN, OCN, and bone sialoprotein, while group 3 expressed much lower levels of collagen type I and OPN mRNA only. This study demonstrates the feasibility of extensive in vivo bone regeneration by transplantation of ADSCs without prior in vitro osteogenic differentiation, and that a PLGA/HA composite BMP-2 delivery system stimulates bone regeneration following transplantation of undifferentiated human ADSCs.

Development of specific collagen scaffolds to support the osteogenic and chondrogenic differentiation of human bone marrow stromal cells

Type I Collagen matrices of defined porosity, incorporating carbonate substituted hydroxyapatite (HA) crystals, were assessed for their ability to support osteo- and chondrogenic differentiation of human bone marrow stromal cells (HBMSCs). Collagen–HA composite scaffolds supported the osteogenic differentiation of HBMSCs both in vitro and in vivo as demonstrated by histological and micro-CT analyses indicating the extensive penetration of alkaline phosphatase expressing cells and new matrix synthesis with localised areas immunologically positive for osteocalcin. In vivo, extensive new osteoid formation of implant origin was observed in the areas of vasculature. Chondrogenic matrix synthesis was evidenced in the peripheral regions of pure collagen systems by an abundance of Sox9 expressing chondrocytes embedded within a proteoglycan and collagen II rich ECM. The introduction of microchannels to the scaffold architecture was seen to enhance chondrogenesis. Tissue specific gene expression and corresponding matrix synthesis indicate that collagen matrices support the growth and differentiation of HBMSCs and suggest the potential of this platform for understanding the ECM cues necessary for osteogenesis and chondrogenesis.

The impact of critical point drying with liquid carbon dioxide on collagen–hydroxyapatite composite scaffolds

Collagen–hydroxyapatite composites for bone tissue engineering are usually made by freezing an aqueous dispersion of these components and then freeze-drying. This method creates a foamed matrix which may not be optimum for growing cell colonies larger than a few hundred micrometres due to the limited diffusion of nutrients and oxygen, and the limited removal of waste metabolites. Incorporating a network of microchannels in the interior of the scaffold which may permit the flow of nutrient-rich media has been proposed as a method to overcome these diffusion constraints. A novel three-dimensional printing and critical point drying technique previously used to make collagen scaffolds has been modified to create collagen–hydroxyapatite scaffolds. This study investigates the properties of collagen and collagen–hydroxyapatite scaffolds and whether subjecting collagen and hydroxyapatite to critical point drying with liquid carbon dioxide results in any changes to the individual components. Specifically, the hydroxyapatite component was characterized before and after processing using wavelength-dispersive X-ray spectroscopy, X-ray diffraction and infrared spectroscopy. Critical point drying did not induce elemental, crystallographic or molecular changes in the hydroxyapatite. The quaternary structure of collagen was characterized using transmission electron microscopy and the quarter-staggered array characteristic of native collagen remained after processing. Microstructural characterization of the composites using scanning electron microscopy showed the hydroxyapatite particles were mechanically interlocked in the collagen matrix. The in vitro biological response of MG63 osteogenic cells to the composite scaffolds were characterized using the Alamar Blue™, PicoGreen™, alkaline phosphate and Live/Dead™ assays, and revealed that the critical point dried scaffolds were non-cytotoxic.

Synthesis and characterisation of gelatin–nano hydroxyapatite composite scaffolds for bone tissue engineering

Gelatin–hydroxyapatite (HAp) nanocomposites have been prepared by particulate leaching technique using glutaraldehyde (GTA) as cross-linking agent for polymer. The porosity in the scaffolds was controlled using sodium chloride as porogen agent. Microstructural investigation by scanning electron microscopy (SEM), revealed the formation of a well interconnected porous scaffold with pore size in the range of 100–200 μm. X-ray diffraction and Fourier transform infrared spectroscopy were used to confirm the formation of crystalline HAp as well the presence of both constituents in the composite samples. The bioactivity of the samples was evaluated by conducting MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and cell adhesion tests. The results suggest that the use of GTA in excess of 0˙25% can be detrimental to cell survival. Cell attachment on the nanocomposite scaffold was verified by microscopic analysis.

Preparation and Evaluation of Porous Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)— Hydroxyapatite Composite Scaffolds

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) and PHBHHx-hydroxyapatite (HAP) composite scaffolds have been prepared by phase separation and subsequent sublimation of the solvent for bone tissue engineering. Scanning electron microscopy (SEM), porosity measurement, mechanical tests, and thermogravimertric analysis (TGA) are used to analyze the physical properties of the scaffolds. The biocompatibility and osteoconductivity are assessed by examining the morphology, proliferation, and differentiation of MC3T3-E1 osteoprogenitor cells seeded on the scaffolds. The PHBHHx-HAP composite scaffolds show better mechanical properties, biocompatibility, and osteoconductivity than the PHBHHx scaffolds. The results suggest that PHBHHx-HAP composite scaffolds can be employed as a promising candidate for bone reconstruction.

In vivo bone formation from human embryonic stem cell-derived osteogenic cells in poly( d, l-lactic- co-glycolic acid)/hydroxyapatite composite scaffolds

We have previously reported the efficient osteogenic differentiation of human embryonic stem cells (hESCs) by co-culture with primary human bone-derived cells (hPBDs) without the use of exogenous factors. In the present study, we explored whether osteogenic cells derived from hESCs (OC-hESCs) using the previously reported method would be capable of regenerating bone tissue in vivo. A three-dimensional porous poly( d, l-lactic- co-glycolic acid)/hydroxyapatite composite scaffold was used as a cell delivery vehicle. In vivo implantation of OC-hESC-seeded scaffolds showed significant bone formation in the subcutaneous sites of immunodeficient mice at 4 and 8 weeks after implantation ( n=5 for each time point). Meanwhile, implantation of the control no cell-seeded scaffolds or human dermal fibroblast-seeded scaffolds did not show any new bone formation. In addition, the presence of BMP-2 (1 μg/scaffold) enhanced new bone tissue formation in terms of mineralization and the expression of bone-specific genetic markers. According to FISH analysis, implanted OC-hESCs remained in the regeneration sites, which suggested that the implanted cells participated in the formation of new bone. In conclusion, OC-hESCs successfully regenerated bone tissue upon in vivo implantation, and this regeneration can be further enhanced by the administration of BMP-2. These results suggest the clinical feasibility of OC-hESCs as a good alternative source of cells for bone regeneration.

Biodegradable Porous PCL/HA Scaffolds for Bone Tissue Engineering

Poly ε-caprolactone(PCL)/hydroxyapatite(HA) composite scaffolds were fabricated by particulate leaching and freeze drying routes with different HA content. Porosity was decreased with HA addition, while mean pore size was maintained at around porogen size regardless of HA content. Compressive modulus was increased with increasing HA content. In this study, the optimum content of HA was around 40% in weight against PCL to obtain the highest compressive modulus with keeping porosity above 85%. HA apparently enhanced proliferation of osteoblast-like MG63 cells in PCL/HA composite scaffolds. Typical adhesion, migration and aggregation procedure of MG63 cells were found on PCL, while spreading morphology only was found on HA even at the early stage of adhesion without migration or aggregation.

Amorphous hydroxyapatite‐sintered polymeric scaffolds for bone tissue regeneration: Physical characterization studies

Given the inherent shortcomings of autografts and allografts, donor-site morbidity and risk of disease transmission, respectively, alternatives to traditional bone grafting options are warranted. To this end, poly(lactide-co-glycolide) (PLAGA) and in situ-synthesized amorphous hydroxyapatite (HA) were used to construct three-dimensional microsphere-based composite scaffolds of varying HA content for bone regeneration. In the current study, the effect of adding amorphous HA to the PLAGA scaffolds on their physical characteristics and in vitro degradation mechanism was investigated. Porosimetry and uniaxial compression testing were used to analyze the internal structure and elastic modulus of the scaffolds, respectively. Additionally, gel permeation chromatography (GPC) was performed to assess the polymer molecular weight over the course of an 8-week degradation study. HA content (17% or 27%) of the composite scaffolds was found to increase scaffold pore volume from 33.86% for pure polymer scaffolds, to 40.49% or 46.29%, depending on the amount of incorporated HA. This increased pore volume provided the composite scaffolds with a greater surface area and a corresponding decrease in elastic modulus. Scaffold degradation studies conducted over 8 weeks showed PLAGA to degrade in a first-order mechanism, with the rate of polymer degradation for the 27% HA composite scaffold being significantly slower than that of the pure PLAGA scaffold (degradation constants of 0.0324 and 0.0232 week(-1), respectively). These results suggest that the addition of amorphous HA to PLAGA microspheres resulted in porous, bioactive scaffolds that offer potential as alternative bone grafting materials for the field of regenerative medicine.

Preparation and Evaluation of Porous Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)-Hydroxyapatite Composite Scaffolds

Preparation and Evaluation of Porous Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)-Hydroxyapatite Composite Scaffolds