Biocomposite Scaffolds Research Articles

Scaffolds made of nanostructured phosphates, collagen and chitosan for cell culture

Abstract In the present study a new strategy of synthesis was developed for nanostructured calcium phosphates, such as hydroxyapatite (HAP) or silicon, magnesium and zinc modified hydroxyapatites of controlled characteristics (such as the shape and size of nanoparticles, porosity and crystallinity) by precipitation reactions. These inorganic powders were jointly used with chitosan (CHI) and collagen type I (COL) to manufacture fibrous biocomposite scaffolds made of self-assembled layers by using layer by layer technique for cell cultures. The resulting materials were characterized by using X-ray diffraction, SEM, TEM and AFM. This work also reports the structural data obtained on the surface of the newly prepared nanostructured porous scaffolds made of self-assembled nanoHAP–Si(0.2%)–Mg(0.6%)–Zn(0.2%)/CHI/COL layers at 7 days in human osteoblastic cell culture. A new porous structure was found by SEM imaging on the surface of these scaffolds due to the cells and scaffold interactions. This structure is apparently similar to the bone tissue. Our data have indicated that the incorporation of small amounts of silicon, magnesium and zinc within the nanoHAP lattice improved the biological activity of human osteoblasts on these scaffolds, in vitro. The data have revealed the high importance of HAP nanostructure and its composition in the preparation of fibrous scaffolds with potential applications in particle technology, bone tissue engineering and nano medicine.

Fabrication and characterization of poly(vinyl alcohol)/graphene oxide nanofibrous biocomposite scaffolds

AbstractNanofibrous biocomposite scaffolds of poly(vinyl alcohol) (PVA) and graphene oxide (GO) were prepared by using electrospinning method. The microstructure, crystallinity, and morphology of the scaffolds were characterized through X‐ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). The mechanical properties were investigated by tensile testing. Moreover, Mouse Osteoblastic Cells (MC3T3‐E1) attachment and proliferation on the nanofibrous scaffolds were investigated by MTT [3‐(4,5‐dimeth‐ylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide] assay, SEM observation and fluorescence staining. XRD and FTIR results verify the presence of GO in the scaffolds. SEM images show the three‐dimensional porous fibrous morphology, and the average diameter of the composite fibers decreases with increasing the content of GO. The mechanical properties of the scaffolds are altered by changing the content of GO as well. The tensile strength and elasticity modulus increase when the content of GO is lower than 1 wt %, but decrease when GO is up to 3 and 5 wt %. MC3T3‐E1 cells attach and grow on the surfaces of the scaffolds, and the adding of GO do not affect the cells' viability. Also, MC3T3‐E1 cells are likely to spread on the PVA/GO composite scaffolds. Above all, these unique features of the PVA/GO nanofibrous scaffolds prepared by electrospinning would open up a wide variety of future applications in bone tissue engineering and drug delivery systems. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013

Biocomposites based on collagen and phosphorylated dextran for bone regeneration

Abstract

Fabrication and properties of porous scaffold of zein/PCL biocomposite for bone tissue engineering

The aim of this study was to fabricate porous scaffolds of zein/poly(ε-caprolactone) (PCL) biocomposite by solvent casting–particulate leaching method using sodium chloride particles as the porogen. Porous biocomposite scaffolds with porosity around 70% and well-interconnected network were obtained. The incorporation of zein into PCL led to the improvement of hydrophilicity as indicated by the results of water contact angle measurement. After immersion in phosphate buffered saline (PBS) in vitro for 28days, it was observed that the degradation rate of the zein/PCL biocomposite scaffold was faster than the PCL scaffold and that the rate could be tailored by adjusting the amount of zein in the composite. The results demonstrate the potential of the zein/PCL biocomposite scaffolds to be used in tissue engineering strategies to regenerate bone defects.

Preparation and characterization of polycaprolactone/forsterite nanocomposite porous scaffolds designed for bone tissue regeneration

Biocomposite scaffolds made from polymers and bioceramics can provide the mechanical structure necessary for osteoinductivity in the growth of new bone. The aim of this research was to investigate the properties of a novel nanocomposite scaffold made from a combination of polycaprolactone (PCL) and forsterite nanopowder which could find use in bone tissue engineering applications. The scaffold itself was fabricated by a method of solvent casting and particle leaching. The effect of forsterite content on the mechanical properties, bioactivity, biodegradability, and cytotoxicity of the scaffolds was investigated. Significant improvement in the mechanical properties was observed in the nanocomposite scaffolds as compared to that seen in the pure PCL scaffolds. Bioactivity was also observed in the nanocomposite scaffolds, a trait which was not present in the pure PCL scaffolds. Biodegradation assay indicated that the addition of forsterite nanopowder could modulate the degradation rate of PCL. In vitro tests of cytotoxicity and osteoblast proliferation showed that the nanocomposite scaffolds were non-cytotoxic, thereby allowing cells to adhere, grow, and proliferate on the surface of these scaffolds. The results obtained in this experiment suggest that the combination of PCL with forsterite nanopowder can be used to form scaffolds suitable for use in bone tissue engineering. The exact material behavior required can be adjusted through variation of the ratio between PCL and forsterite nanopowder used to form the scaffold.

Regeneration of mastoid air cells using polycaprolactone/β‐tricalcium phosphate biocomposites: An experimental study

The study aimed to regenerate pneumatic air cells in guinea pig bulla using three-dimenisonal (3D) biocomposite scaffolds consisting of polycaprolactone/β-tricalcium phosphate (PCL/β-TCP). Prospective controlled study in experimental animals. PCL/β-TCP composites were implanted into the bulla with mucosa preservation in group A (n = 10). PCL/β-TCP composites coated with collagen were implanted in group B (n = 10). After 12 weeks, the bullae were extracted and evaluated by micro-computed tomography (micro-CT) and were processed for histological analyses. In group A, micro-CT showed a well-maintained honeycomb appearance of micropores without obstruction. Regeneration of the mucosa was noted inside the pores of the 3D scaffold. However, partial obstruction of the micropores with new bone formation was evident in group B. Group A showed more satisfactory mucosal regeneration into the micropores. Our results indicate that the 3D scaffold may be amenable for use during mastoidectomy. Further studies for gas exchange in the regenerated mucosa are necessary.

Bio-composite scaffolds containing chitosan/nano-hydroxyapatite/nano-copper–zinc for bone tissue engineering

The current study involves fabrication and characterization of bio-composite scaffolds containing chitosan (CS), nano-hydroxyapatite (nHAp) and Cu–Zn alloy nanoparticles (nCu–Zn) by freeze drying technique. The fabricated composite scaffolds (CS/nHAp and CS/nHAp/nCu–Zn) were characterized by SEM, EDX, XRD and FT-IR studies. The addition of nCu–Zn in the CS/nHAp scaffolds significantly increased swelling, decreased degradation, increased protein adsorption, and increased antibacterial activity. The CS/nHAp/nCu–Zn scaffolds had no toxicity towards rat osteoprogenitor cells. So the developed CS/nHAp/nCu–Zn scaffolds have advantageous and potential applications over the CS–nHAp scaffolds for bone tissue engineering.

Injectable polyurethane composite scaffolds delay wound contraction and support cellular infiltration and remodeling in rat excisional wounds

Injectable scaffolds present compelling opportunities for wound repair and regeneration because of their ability to fill irregularly shaped defects and deliver biologics such as growth factors. In this study, we investigated the properties of injectable polyurethane (PUR) biocomposite scaffolds and their application in cutaneous wound repair using a rat excisional model. The scaffolds have a minimal reaction exotherm and clinically relevant working and setting times. Moreover, the biocomposites have mechanical and thermal properties consistent with rubbery elastomers. In the rat excisional wound model, injection of settable biocomposite scaffolds stented the wounds at early time points, resulting in a regenerative rather than a scarring phenotype at later time points. Measurements of wound length and thickness revealed that the treated wounds were less contracted at day 7 compared to blank wounds. Analysis of cell proliferation and apoptosis showed that the scaffolds were biocompatible and supported tissue ingrowth. Myofibroblast formation and collagen fiber organization provided evidence that the scaffolds have a positive effect on extracellular matrix remodeling by disrupting the formation of an aligned matrix under elevated tension. In summary, we have developed an injectable biodegradable PUR biocomposite scaffold that enhances cutaneous wound healing in a rat model.

Fabrication and biocompatibility of electrospun silkbiocomposites.

Silk fibroin has attracted great interest in tissue engineering because of its outstanding biocompatibility, biodegradability and minimal inflammatory reaction. In this study, two kinds of biocomposites based on regenerated silk fibroin are fabricated by electrospinning and post-treatment processes, respectively. Firstly, regenerated silk fibroin/tetramethoxysilane (TMOS) hybrid nanofibers with high hydrophilicity are prepared, which is superior for fibroblast attachment. The electrospinning process causes adjacent fibers to ‘weld’ at contact points, which can be proved by scanning electron microscope (SEM). The water contact angle of silk/tetramethoxysilane (TMOS) composites shows a sharper decrease than pure regenerated silk fibroin nanofiber, which has a great effect on the early stage of cell attachment behavior. Secondly, a novel tissue engineering scaffold material based on electrospun silk fibroin/nano-hydroxyapatite (nHA) biocomposites is prepared by means of an effective calcium and phosphate (Ca–P) alternate soaking method. nHA is successfully produced on regenerated silk fibroin nanofiber within several min without any pre-treatments. The osteoblastic activities of this novel nanofibrous biocomposites are also investigated by employing osteoblastic-like MC3T3-E1 cell line. The cell functionality such as alkaline phosphatase (ALP) activity is ameliorated on mineralized silk nanofibers. All these results indicate that this silk/nHA biocomposite scaffold material may be a promising biomaterial for bone tissue engineering.

Biocomposite scaffolds containing regenerated silk fibroin and nanohydroxyapatite for bone tissue regeneration

Biocomposite scaffolds containing regenerated silk fibroin and nanohydroxyapatite for bone tissue regeneration

Chitosan scaffolds containing silicon dioxide and zirconia nano particles for bone tissue engineering

A scaffold harboring the desired features such as biodegradation, biocompatibility, porous structure could serve as template for bone tissue engineering. In the present study, chitosan (CS), nano-scaled silicon dioxide (Si) and zirconia (Zr) were combined by freeze drying technique to fabricate a bio-composite scaffold. The bio-composite scaffold (CS/Si/Zr) was characterized by SEM, XRD and FT-IR studies. The scaffold possessed a porous nature with pore dimensions suitable for cell infiltration and colonization. The presence of zirconia in the CS/Si/Zr scaffold decreased swelling and increased biodegradation, protein adsorption and bio-mineralization properties. The CS/Si/Zr scaffold was also found to be non-toxic to rat osteoprogenitor cells. Thus, we suggest that CS/Si/Zr bio-composite scaffold is a potential candidate to be used for bone tissue engineering.

Poly(3-hydroxubutyrate-co-4-hydroxubutyrate)/bacterial cellulose composite porous scaffold: Preparation, characterization and biocompatibility evaluation

► Poly(3-hydroxubutyrate-co-4-hydroxubutyrate)/bacterial cellulose composite as biocomposite. ► Three-dimensional network structure composing of multi-distribution of pore size. ► A great increase in hydrophilicity and mechanical properties for composite scaffold. ► Biodegradability and biocompatibility become better than pure poly(3-hydroxubutyrate-co-4-hydroxubutyrate). Bio-composite scaffolds were prepared by freeze-drying using poly(3-hydroxubutyrate-co-4-hydroxubutyrate) (P(3HB-co-4HB)) and bacterial cellulose (BC) as raw materials and trifluoroacetic acid (TFA) as co-solvent. The characteristics of the composite scaffold were investigated by field emission scanning electron microscopy (FESEM), Fourier transform infrared spectra (FT-IR), X-ray diffraction (XRD), water contact angle measurement and tensile testing. Preliminary biodegradation test was performed for P(3HB-co-4HB) and P(3HB-co-4HB)/BC composite scaffold in buffer solution and enzyme solution. The biocompatibility of the composite scaffold was preliminarily evaluated by cell adhesion studies using Chinese Hamster Lung (CHL) fibroblast cells. The cells incubated with composite scaffold for 48 h were capable of forming cell adhesion and proliferation, which showed better biocompatibility than pure P(3HB-co-4HB) scaffold. Thus, the prepared P(3HB-co-4HB)/BC composite scaffold was bioactive and may be suitable for cell adhesion/attachment suggesting that these scaffolds can be used for wound dressing or tissue-engineering scaffolds.

Preparation, characterization and antimicrobial activity of a bio-composite scaffold containing chitosan/nano-hydroxyapatite/nano-silver for bone tissue engineering

In this study, a bio-composite scaffold containing chitosan/nano-hydroxyapatite/nano-silver particles (CS/nHAp/nAg) was developed by freeze drying technique, followed by introduction of silver ions in controlled amount through reduction phenomenon by functional groups of chitosan. The scaffolds were characterized using SEM, FT-IR, XRD, swelling, and biodegradation studies. The testing of the prepared scaffolds with Gram-positive and Gram-negative bacterial strains showed antibacterial activity. The scaffold materials were also found to be non-toxic to rat osteoprogenitor cells and human osteosarcoma cell line. Thus, these results suggested that CS/nHAp/nAg bio-composite scaffolds have the potential in controlling implant associated bacterial infection during reconstructive surgery of bone.

Evaluation of the Biocompatibility of PLACL/Collagen Nanostructured Matrices with Cardiomyocytes as a Model for the Regeneration of Infarcted Myocardium

AbstractPioneering research suggests various modes of cellular therapeutics and biomaterial strategies for myocardial tissue engineering. Despite several advantages, such as safety and improved function, the dynamic myocardial microenvironment prevents peripherally or locally administered therapeutic cells from homing and integrating of biomaterial constructs with the infarcted heart. The myocardial microenvironment is highly sensitive due to the nanoscale cues that it exerts to control bioactivities, such as cell migration, proliferation, differentiation, and angiogenesis. Nanoscale control of cardiac function has not been extensively analyzed in the field of myocardial tissue engineering. Inspired by microscopic analysis of the ventricular organization in native tissue, a scalable in‐vitro model of nanoscale poly(L‐lactic acid)‐co ‐poly(ϵ ‐caprolactone)/collagen biocomposite scaffold is fabricated, with nanofibers in the order of 594 ± 56 nm to mimic the native myocardial environment for freshly isolated cardiomyocytes from rabbit heart, and the specifically underlying extracellular matrix architecture: this is done to address the specificity of the underlying matrix in overcoming challenges faced by cellular therapeutics. Guided by nanoscale mechanical cues provided by the underlying random nanofibrous scaffold, the tissue constructs display anisotropic rearrangement of cells, characteristic of the native cardiac tissue. Surprisingly, cell morphology, growth, and expression of an interactive healthy cardiac cell population are exquisitely sensitive to differences in the composition of nanoscale scaffolds. It is shown that suitable cell–material interactions on the nanoscale can stipulate organization on the tissue level and yield novel insights into cell therapeutic science, while providing materials for tissue regeneration.

Fabrication of nano‐hydroxyapatite on electrospun silk fibroin nanofiber and their effects in osteoblastic behavior

In this study, a novel tissue engineering scaffold material of electrospun silk fibroin/nano-hydroxyapatite (nHA) biocomposite was prepared by means of an effective calcium and phosphate (Ca-P) alternate soaking method. nHA was successfully produced on regenerated silk fibroin nanofiber as a substrate within several minutes without any pretreatments. The morphologies of both nonmineralized and mineralized nanofibers were analyzed using a field-emission scanning electron microscopy (FESEM). The crystallographic phases of the nHA were analyzed using X-ray diffraction (XRD). Fourier transform infrared (FTIR) spectrophotometer and thermogravimetry analyses (TGA) were employed to determine the type of functional groups and the amount of nHA presenting in the silk/nHA biocomposite nanofibers, respectively. The osteoblastic activities of this novel nanofibrous biocomposite scaffold were also investigated by employing osteoblastic-like MC3T3-E1 cell line. The cell functionality such as alkaline phosphatase (ALP) activity was ameliorated on mineralized nanofibers. All these results indicated that this silk/nHA biocomposite scaffold material may be a promising biomaterial for bone tissue engineering.

Inorganic/Organic Biocomposite Cryogels for Regeneration of Bony Tissues

The present work focuses on the physical, mechanical and in vitro properties of porous inorganic/organic biocomposite scaffolds of polyvinyl alcohol–tetraethylorthosilicate–alginate–calcium oxide (PTAC). These scaffolds are prepared by means of cryogelation technology and are intended for bone tissue engineering applications. The biocomposite cryogels have much more favorable physical and biological properties compared to the previous work of our group on the same composition in the form of pellets and foams. The optimized and heat-treated PTAC biocomposite cryogels show homogenous porosity and good mechanical properties and also exhibit the formation of a hydroxyapatite-like layer on their surface on coming in contact with simulated body fluid (SBF). Furthermore, the biocomposite cryogels showed good biocompatibility with L929 fibroblasts. Also, the influence of pre-soaking in SBF to that of non-soaked scaffolds was compared in terms of proliferation of MG-63 osteoblast-like osteosarcoma cells on these scaffolds and it was found that the pre-soaking caused a decrease in cell proliferation. Finally, the response of human osteoblasts on these scaffolds was analyzed by MTT assay, scanning electron microscopy, energy dispersive X-ray spectroscopy and micro X-ray computing tomography. The cells revealed good biocompatibility with the biocomposite cryogels and were mostly present as cell sheets on the surface with thick bundles of collagenous extracellular matrix during initial period of incubation. During later phases, the formation of calcium phosphate-like mineral deposits was observed on the surface of the cryogels suggesting a high potential of the biocomposite cryogels towards bone regeneration. Therefore, the PTAC biocomposite cryogels, due to their favorable properties and high biocompatibility with human osteoblasts can be suggested as potential scaffolds for bone tissue engineering applications.

Cell adhesive and growth behavior on electrospun nanofibrous scaffolds by designed multifunctional composites

Nanostructured biocomposite scaffolds of poly(l-lactide) (PLLA) blended with collagen (coll) or hydroxyapatite (HA), or both for tissue engineering application, were fabricated by electrospinning. The electrospun scaffolds were characterized for the morphology, chemical and tensile properties by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), water contact angle (WCA), Fourier transform infrared (FTIR) measurement, and tensile testing. Electrospun biocomposite scaffolds of PLLA and collagen or (and) HA in the diameter range of 200–700nm mimic the nanoscale structure of the extracellular matrix (ECM) with a well-interconnection pore network structure. The presence of collagen in the scaffolds increased their hydrophility, and enhanced cell attachment and proliferation, while HA improved the tensile properties of the scaffolds. The biocompatibility of the electrospun scaffolds and the viability of contacting cells were evaluated by 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) nuclear staining and by fluorescein diacetate (FDA) and propidium iodide (PI) double staining methods. The results support the conclusion that 293T cells grew well on composite scaffolds. Compared with pure PLLA scaffolds a greater density of viable cells was seen on the composites, especially the PLLA/HA/collagen scaffolds.

Characterization and biocompatibility of nanohybrid scaffold prepared via in situ crystallization of hydroxyapatite in chitosan matrix

Hydroxyapatite (HAP) precursor solution was first mixed with an acetic acid chitosan (CS) solution. The mixture was then lyophilized to form the original scaffold, which stored the HAP precursors. The nano HAP crystallized homogeneously from the CS matrix during the alkaline treatment to form a nanohybrid scaffold. Scanning electron microscopy (SEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR) were used to investigate the fabrication process of the nanohybrid scaffold. XRD results showed that the in situ deposited mineral (HAP) in the scaffold has phase structure similar to natural bone. FTIR and XPS results indicated that CS's hydroxyl group, amino and amide regulated the nano HAP crystallization process, which resulted in the nano homogeneous distribution of nano HAP and provided nano topographical features for the nanohybrid scaffold. MTT testing and SEM images of human bone mesenchymal stem cells (hBMSCs) cultures revealed the attachment and growth of hBMSCs in the biocomposite scaffold. Cell morphology and viability data showed that the nanohybrid composite scaffold is suitable for use in bioapplications.

Electrospinning of PLGA/gelatin randomly-oriented and aligned nanofibers as potential scaffold in tissue engineering

Electrospinning technique can be used to produce the three-dimensional nanofibrous scaffold similar to natural extracellular matrix, which satisfies particular requirements of tissue engineering scaffold. Randomly-oriented and aligned poly(lactic-co-glycolic acid) (PLGA) and PLGA/gelatin biocomposite scaffolds were successfully produced by electrospinning in the present study. The resulting nanofibrous scaffolds exhibited smooth surface and high porous structure. Blending PLGA with gelatin enhanced the hydrophilicity but decreased the average fiber diameter and the mechanical properties of the scaffolds under the same electrospinning condition. The cell culture results showed that the elongation of the osteoblast on the aligned nanofibrous scaffold was parallel to the fiber arrangement and the cell number was similar to that of randomly-oriented scaffold, indicating that the aligned nanofibrous scaffold provide a beneficial approach for the bone regeneration.

Augmentation of bone defect healing using a new biocomposite scaffold: An in vivo study in sheep

Previous studies support resorbable biocomposites made of poly(L-lactic acid) (PLA) and beta-tricalcium phosphate (TCP) produced by supercritical gas foaming as a suitable scaffold for tissue engineering. The present study was undertaken to demonstrate the biocompatibility and osteoconductive properties of such a scaffold in a large animal cancellous bone model. The biocomposite (PLA/TCP) was compared with a currently used beta-TCP bone substitute (ChronOS, Dr. Robert Mathys Foundation), representing a positive control, and empty defects, representing a negative control. Ten defects were created in sheep cancellous bone, three in the distal femur and two in the proximal tibia of each hind limb, with diameters of 5 mm and depths of 15 mm. New bone in-growth (osteoconductivity) and biocompatibility were evaluated using microcomputed tomography and histology at 2, 4 and 12 months after surgery. The in vivo study was validated by the positive control (good bone formation with ChronOS) and the negative control (no healing with the empty defect). A major finding of this study was incorporation of the biocomposite in bone after 12 months. Bone in-growth was observed in the biocomposite scaffold, including its central part. Despite initial fibrous tissue formation observed at 2 and 4 months, but not at 12 months, this initial fibrous tissue does not preclude long-term application of the biocomposite, as demonstrated by its osteointegration after 12 months, as well as the absence of chronic or long-term inflammation at this time point.