Fabrication and in vitro evaluations with osteoblast-like MG-63 cells of porous hyaluronic acid-gelatin blend scaffold for bone tissue engineering applications

In this study, hyaluronic acid–gelatin (HyA–Gel) scaffolds were prepared with HyA:Gel ratios of 15:85, 50:50, and 85:15 with the goal of obtaining a porous biocompatible scaffold for bone tissue engineering applications. Scanning electron microscopy and Fourier-transform infrared spectroscopy were done to characterize the morphological orientations of the scaffolds. The biocomposite structure was highly porous and the pores in the scaffolds were interconnected. The compressive strength of the scaffold was 7.39 ± 0.2 MPa for the HyA–Gel when fabricated at a ratio of 15:85. To assess the biocompatibility and cell behavior on the HyA–Gel biocomposite, the proliferation of MG-63 osteoblast cell on the scaffolds was examined using the MTT assay, optical microscopy, and confocal microscopy. Collagen type I and osteopontin expression of cells cultured on the scaffolds were examined using immunoblotting. The scaffolds fabricated with a 15:85—HyA:Gel ratio showed excellent biocompatibility, good mechanical properties, and high porosity, which suggest that the highly porous scaffold holds great promise for use in bone tissue engineering applications.     

Biomimetic mineralization of electrospun poly(lactic-co-glycolic acid)/multi-walled carbon nanotubes composite scaffolds in vitro

Biomimetic mineralization is an effective method to improve the biocompatibility and bone inductivity of certain materials. In this study, composite scaffolds composed of poly(lactic-co-glycolic acid) (PLGA) and multi-walled carbon nanotubes (MWNTs) were prepared by electrospinning. Subsequently, the scaffolds were immersed in a simulated body fluid (1.5 × SBF) at 37 °C for 7, 14 and 21 days for biomimetic mineralization. Scanning electron microscopy, Raman spectroscopy, and X-ray diffraction were used for characterization. It was found that the electrospun scaffolds had extremely resemblant structural morphology to the natural extracellular matrix. After mineralization, apatite crystals were deposited on the PLGA/MWNTs composite scaffolds. The mineralized PLGA/MWNTs composites may be potentially useful in tissue engineering applications, particularly as scaffolds for bone tissue regeneration.     

Development of nano-sized hydroxyapatite reinforced composites for tissue engineering scaffolds

Nano-sized hydroxyapatite (nanoHA) reinforced composites, mimicking natural bone, were produced. Examination by transmission electron microscopy revealed that the nanoHA particles had a rod-like morphology, 20–30 nm in width and 50–80 nm in length. The phase composition of hydroxyapatite was confirmed by X-ray diffraction. The nanoHA particles were incorporated into poly-2-hydroxyethylmethacrylate (PHEMA)/polycaprolactone (PCL) matrix to make new nanocomposites: nanoHA-PHEMA/PCL. Porous nanocomposite scaffolds were then produced using a porogen leaching method. The interconnectivity of the porous structure of the scaffolds was revealed by non-destructive X-ray microtomography. Porosity of 84% was achieved and pore sizes were approximately around 300–400 μm. An in vitro study found that the nanocomposites were bioactive as indicated by the formation of a bone-like apatite layer after immersion in simulated body fluid. Furthermore, the nanocomposites were able to support the growth and proliferation of primary human osteoblast (HOB) cells. HOB cells developed a well organized actin cytoskeletal protein on the nanocomposite surface. The results demonstrate the potential of the nanocomposite scaffolds for tissue engineering applications for bone repair.     

Injectable and Crosslinkable PLGA‐Based Microribbons as 3D Macroporous Stem Cell Niche

Poly(lactide‐co‐glycolide) (PLGA) has been widely used as a tissue engineering scaffold. However, conventional PLGA scaffolds are not injectable, and do not support direct cell encapsulation, leading to poor cell distribution in 3D. Here, a method for fabricating injectable and intercrosslinkable PLGA microribbon‐based macroporous scaffolds as 3D stem cell niche is reported. PLGA is first fabricated into microribbon‐shape building blocks with tunable width using microcontact printing, then coated with fibrinogen to enhance solubility and injectability using aqueous solution. Upon mixing with thrombin, firbornogen‐coated PLGA microribbons can intercrosslink into 3D scaffolds. When subject to cyclic compression, PLGA microribbon scaffolds exhibit great shock‐absorbing capacity and return to their original shape, while conventional PLGA scaffolds exhibit permanent deformation after one cycle. Using human mesenchymal stem cells (hMSCs) as a model cell type, it is demonstrated that PLGA μRB scaffolds support homogeneous cell encapsulation, and robust cell spreading and proliferation in 3D. After 28 days of culture in osteogenic medium, hMSC‐seeded PLGA μRB scaffolds exhibit an increase in compressive modulus and robust bone formation as shown by staining of alkaline phosphatase, mineralization, and collagen. Together, the results validate PLGA μRBs as a promising injectable, macroporous, non‐hydrogel‐based scaffold for cell delivery and tissue regeneration applications.     

β-Tricalcium-phosphate stimulates the differentiation of dental follicle cells

The use of dental progenitor cells is a straightforward strategy for regenerative dentistry. For example a cell based therapy with dental follicle cells (DFCs) could be a novel therapeutic strategy for the regeneration of oral tissues in the future. For the regeneration of large bone defects for example dental progenitor cells have to be combined with bone substitutes as scaffolds. This study therefore investigated cell attachment (scanning electron microscopy), cell vitality/proliferation (WST-1 assay) and cell differentiation (under in vitro conditions) of human DFCs on synthetic β-tricalcium phosphate (TCP). DFCs showed considerable cell attachment and proliferation on TCP. Moreover, TCP stimulates osteogenic differentiation in comparison to DFCs with a standard protocol. Here, for example, the osteoblast marker bone sialoprotein (BSP) was highly expressed on TCP, but almost absent in differentiated DFCs without TCP. In conclusion, our study shows that TCP is an excellent scaffold for DFCs for oral tissue regeneration.     

Ca-deficient hydroxyapatite/polylactide nanocomposites with chemically modified interfaces by high pressure consolidation at room temperature

Hydroxyapatite (HAP) is a close synthetic analog of the bone mineral and is often considered as a material for bone graft substitutes and tissue engineering scaffolds. Despite its attractive bioactive properties low-fracture toughness limits the use of HAP ceramics to a number of non-load-bearing applications. To obtain a more adequate mechanical behavior, HAP is often combined with polymers based on lactic and glycolic acids or polycaprolactone using hot pressing. In such composite materials, the compatibility and bonding strength of HAP–polymer interfaces are critical parameters that must be controlled and improved. This may be achieved, for example, by covalent immobilization of organic moieties on the ceramic particles surface. In this work, the surface of calcium-deficient hydroxyapatite (CDHAP) was modified by reaction with hexamethylene diisocyanate (HDI) in a non-aqueous suspension. Composites of CDHAP–HDI with polylactide (PLA) were high pressure consolidated at room temperature at 2.5 GPa yielding up to 90% theoretical density. The effects of total organic fraction and modification extent on compression strength were studied. Materials with high extent of modification and high organic content exhibited compressive strength of ~295 MPa, much higher than reported in other studies. These materials are suitable candidates for load bearing orthopedic applications.     

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.     

Synthesis and characterization of cholesterol-poly(ethylene glycol)-poly(D,L-lactic acid) copolymers for promoting osteoblast attachment and proliferation

A novel cholesterol-poly(ethylene glycol)-poly(D,L-lactic acid) copolymer (CPEG-PLA) has been synthesized as a potential surface additive for promoting osteoblast attachment and proliferation. The gel permeation chromatography (GPC) and nuclear magnetic resonance spectroscopy (NMR) results indicated the product had expected structure with low polydispersities in the range of 1.1–1.5. By blending the poly(D,L-lactic acid) (PLA) with CPEG-PLA, the surface of modified PLA membrane was investigated by atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and contact angle. The results revealed the enrichment of PEG chain on the surface. Osteoblast cell line (MC3T3) was chosen to test the cell behavior on modified PLA membranes. The osteoblast test about cell attachment, proliferation, cell viability and cell morphology investigation on CPEG-PLA modified PLA substrates showed the CPEG-PLA with 15 and 5 ethylene glycol units promoted osteoblast attachment and growth, while the CPEG-PLA with 30 ethylene glycol units prevent osteoblast adhesion and proliferation. This simple surface treatment method may have potentials for tissue engineering and other biomedical applications.     

Application of K/Sr co-doped calcium polyphosphate bioceramic as scaffolds for bone substitutes

Ion doping is one of the most important methods to modify the properties of bioceramics for better biodegrade abilities, biomechanical properties, and biocompatibilities. This paper presents a novel ion doping method applied in calcium polyphosphate (CPP)-based bioceramic scaffolds substituted by potassium and strontium ions (K/Sr) to form (K/Sr–CPP) scaffolds for bone tissue regeneration. The microstructure and crystallization of the scaffolds were detected by scanning electron microscopy and X-ray diffraction. Compressive strength and degradation tests were assessed to evaluate the mechanical and chemical stabilities of K/Sr–CPP in vitro. The cell biocompatibility was measured with respect to the cytotoxicity of the extractions of scaffolds. Muscle pouches and bone implantation were performed to evaluate the biodegradability and osteoconductivity of the scaffolds. The results indicated that the obtained K/Sr–CPP scaffolds had a single beta-CPP phase. The unit cell volume and average grain size increased but the crystallization decreased after the ions were doped into the CPP structure. The K/Sr–CPP scaffolds yielded a higher compressive strength and a better degradation property than the pure CPP scaffold. The MTT assay and in vivo results reveal that the K/Sr–CPP scaffolds exhibited a better cell biocompatibility and a tissue biocompatibility than CPP and hydroxyapatite scaffolds. This study proves the potential applications of K/Sr–CPP scaffolds in bone repair.     

Porous poly (lactic-co-glycolide) microsphere sintered scaffolds for tissue repair applications

In this paper, a new route to preparing porous poly (lactic-co-glycolide) (PLGA) scaffolds for bone tissue repair applications was developed. Novel porous PLGA scaffolds were fabricated via microsphere sintered technique and gas forming technique. Ammonium bicarbonate was used to regulate porosity of these porous scaffolds. Porosity of the scaffolds, and cell attachment, viability and proliferation on the scaffolds were evaluated. The results indicated that PLGA porous scaffolds were with the porosity from around 30% to 95% by regulating ammonium bicarbonate content from 0 to 10%. We also found that PLGA porous microsphere scaffolds benefited cell attachment and viability. Taken together, the achieved porous scaffolds have controlled porosity and also support mesenchymal stem cell proliferation, which could serve as potential scaffolds for bone repair applications.     

Degradation behavior of hydrophilized PLGA scaffolds prepared by melt-molding particulate-leaching method: Comparison with control hydrophobic one

Porous PLGA/PVA scaffolds as hydrophilized PLGA scaffolds for tissue engineering applications were fabricated by a novel melt-molding particulate leaching method (non-solvent method). The prepared scaffolds exhibited highly porous and open-cellular pore structures with almost same surface and interior porosities (pore size, 200–300 μ m; porosity, about 90%). The in vitro degradation behavior of the PLGA and PLGA/PVA scaffolds was compared at 37^∘C in PBS (pH 7.4) with and without the solution change everyday to see the effect of solution pH as well as scaffold hydrophilicity on the degradation behavior. The changes in dimension, molecular weight, mechanical properties (maximum load and modulus), and morphology of the scaffolds were examined with degradation time. The degradation behavior of the PLGA and PLGA/PVA scaffolds was further investigated in vivousing a rat model (subcutaneously implantation). It was observed that both PLGA and PLGA/PVA scaffolds in decreasing pH condition (PBS no change) showed faster degradation than those in constant pH condition (PBS change everyday), owing to the enhanced intramolecular depolymerization by the increment of chain hydrophilicity caused by carboxylate groups as well as the autocatalysis of carboxylic acids accumulated in the solution by the cleavage of PLGA backbone ester bonds. The scaffolds in vivo condition also showed faster degradation than those in vitro, probably due to the aid of foreign body giant cells or enzymes. The PLGA/PVA scaffold showed slightly faster degradation than the PLGA scaffold for both in vitro and in vivo conditions. Author to whom all correspondence should be addressed.     

Basic research on aw-AC/PLGA composite scaffolds for bone tissue engineering

Recently, it has become important to develop effective material to be used as scaffolds for bone tissue engineering. Therefore, we fabricated new three-dimensional (3D) scaffolds consisting of biodegradable poly(d,l-lactide-co-glycolic acid)(PLGA)(75/25) with anti-washout type AC (aw-AC) particles. The aim of this study was to evaluate this new scaffold concerning its basic properties and biocompatibility. The obtained scaffolds were observed with scanning electron microscopy (SEM), and measured for porosity, shrinkage and biaxial compressive strengths. It was shown that PLGA with aw-AC composite scaffolds (aw-AC/PL) showed a greater strength and stability than PLGA scaffolds (PL). Also, the mass reduction of aw-AC/PL during incubation decreased compared to that of PL. The number of MC3T3-E1 cell in PL and aw-AC/PL was counted at 5 h, 1 week, and 2 weeks after cell seeding. As a result, aw-AC/PL exhibited a superior performance in terms of attachment and proliferation compared to PL. Histologically, aw-AC/PL showed an excellent response toward soft tissues. Therefore, it was shown that aw-AC/PL was more biocompatible than PL. In conclusion, it was strongly suggested that aw-AC/PL was more useful for cell transplantation than PL in bone tissue engineering.     

Robotic dispensing of composite scaffolds and in vitro responses of bone marrow stromal cells

The development of bioactive scaffolds with a designed pore configuration is of particular importance in bone tissue engineering. In this study, bone scaffolds with a controlled pore structure and a bioactive composition were produced using a robotic dispensing technique. A poly(ε-caprolactone) (PCL) and hydroxyapatite (HA) composite solution (PCL/HA = 1) was constructed into a 3-dimensional (3D) porous scaffold by fiber deposition and layer-by-layer assembly using a computer-aided robocasting machine. The in vitro tissue cell compatibility was examined using rat bone marrow stromal cells (rBMSCs). The adhesion and growth of cells onto the robotic dispensed scaffolds were observed to be limited by applying the conventional cell seeding technique. However, the initially adhered cells were viable on the scaffold surface. The alkaline phosphatase activity of the cells was significantly higher on the HA–PCL than on the PCL and control culture dish, suggesting that the robotic dispensed HA–PCL scaffold should stimulate the osteogenic differentiation of rBMSCs. Moreover, the expression of a series of bone-associated genes, including alkaline phosphatase and collagen type I, was highly up-regulated on the HA–PCL scaffold as compared to that on the pure PCL scaffold. Overall, the robotic dispensed HA–PCL is considered to find potential use as a bioactive 3D scaffold for bone tissue engineering. Seok-Jung Hong and Ishik Jeong contributed equally.     

Indirect rapid prototyping of biphasic calcium phosphate scaffolds as bone substitutes: influence of phase composition,macroporosity and pore geometry on mechanical properties

While various materials have been developed for bone substitute and bone tissue engineering applications over the last decades, processing techniques meeting the high demands of scaffold shaping are still under development. Individually adapted and mechanically optimised scaffolds can be derived from calcium phosphate (CaP-) ceramics via rapid prototyping (RP). In this study, porous ceramic scaffolds with a periodic pattern of interconnecting pores were prepared from hydroxyapatite, β-tricalcium phosphate and biphasic calcium phosphates using a negative-mould RP technique. Moulds predetermining various pore patterns (round and square cross section, perpendicular and 60° inclined orientation) were manufactured via a wax printer and subsequently impregnated with CaP-ceramic slurries. Different pore patterns resulted in macroporosity values ranging from about 26.0–71.9 vol% with pore diameters of approximately 340 μm. Compressive strength of the specimens (1.3–27.6 MPa) was found to be mainly influenced by the phase composition as well as the macroporosity, both exceeding the influence of the pore geometry. A maximum was found for scaffolds with 60 wt% hydroxyapatite and 26.0 vol% open porosity. It has been shown that wax ink-jet printing allows to process CaP-ceramic into scaffolds with highly defined geometry, exhibiting strength values that can be adjusted by phase composition and pore geometry. This strength level is within and above the range of human cancellous bone. Therefore, this technique is well suited to manufacture scaffolds for bone tissue engineering.     

Increased osteoblast functions on nanophase titania dispersed in poly-lactic-co-glycolic acid composites

The design of nanophase titania/poly-lactic-co-glycolic acid (PLGA) composites offers an exciting approach to combine the advantages of a degradable polymer with nano-size ceramic grains to optimize physical and biological properties for bone regeneration. Importantly, nanophase titania mimics the size scale of constituent components of bone since it is a nanostructured composite composed of nanometre dimensioned hydroxyapatite well dispersed in a mostly collagen matrix. For these reasons, the objective of the present in vitro study was to investigate osteoblast (bone-forming cell) adhesion and long-term functions on nanophase titania/PLGA composites. Since nanophase titania tended to significantly agglomerate when added to polymers, different sonication output powers were applied in this study to improve titania dispersion. Results demonstrated that the dispersion of titania in PLGA was enhanced by increasing the intensity of sonication and that greater osteoblast adhesion correlated with improved nanophase titania dispersion in PLGA. Moreover, results correlated better osteoblast long-term functions, such as alkaline phosphatase activity and calcium-containing mineral deposition, on nanophase titania/PLGA composites compared to plain PLGA. In fact, the greatest collagen production by osteoblasts occurred when cultured on nanophase titania sonicated in PLGA at the highest powers. In this manner, the present study demonstrates that PLGA composites with well dispersed nanophase titania can enhance osteoblast functions necessary for improved bone tissue engineering applications.     

Fabrication of mineralized electrospun PLGA and PLGA/gelatin nanofibers and their potential in bone tissue engineering

Surface mineralization is an effective method to produce calcium phosphate apatite coating on the surface of bone tissue scaffold which could create an osteophilic environment similar to the natural extracellular matrix for bone cells. In this study, we prepared mineralized poly(d,l-lactide-co-glycolide) (PLGA) and PLGA/gelatin electrospun nanofibers via depositing calcium phosphate apatite coating on the surface of these nanofibers to fabricate bone tissue engineering scaffolds by concentrated simulated body fluid method, supersaturated calcification solution method and alternate soaking method. The apatite products were characterized by the scanning electron microscopy (SEM), Fourier transform-infrared spectroscopy (FT-IR), and X-ray diffractometry (XRD) methods. A large amount of calcium phosphate apatite composed of dicalcium phosphate dihydrate (DCPD), hydroxyapatite (HA) and octacalcium phosphate (OCP) was deposited on the surface of resulting nanofibers in short times via three mineralizing methods. A larger amount of calcium phosphate was deposited on the surface of PLGA/gelatin nanofibers rather than PLGA nanofibers because gelatin acted as nucleation center for the formation of calcium phosphate. The cell culture experiments revealed that the difference of morphology and components of calcium phosphate apatite did not show much influence on the cell adhesion, proliferation and activity.     

Development of poly (lactic-co-glycolic acid)-collagen scaffolds for tissue engineering

Collagen as an important extra-cellular matrix (ECM) in many tissues is weakly antigenic and the structure of collagen sponges is highly porous with interconnected pores effective for cell infiltration and mass transfer of oxygen and nutrients. Its application as a scaffold is limited by poor mechanical strength and rapid biodegradation. In this paper, we attempt to graft hydrolyzed PLGA fiber surfaces with collagen by N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) in combination with N-hydroxysuccinimide (NHS), and then embed these collagen-grafted PLGA fibers in collagen sponge to form a hybrid PLGA-collagen scaffold. For further stability, we cross-linked the collagen in the scaffold and used it in rat liver cell cultivation. The scaffold was characterized by mechanical micro-tester, scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Results showed that (1) the scaffolds exhibited isotropic and interconnected porous structure; (2) the compression modulus of this scaffold was enhanced 50 fold compared to the collagen scaffolds. The cell attachment and cytotoxicity of this scaffold were studied. Cell attachment was improved remarkably and the cytotoxicity of the hybrid PLGA-collagen scaffold was lower than that of the un-grafted PLGA-collagen scaffolds using alamarBlue™ assay normalized to the DNA content in each scaffold. This new hybrid scaffold has potential applications for tissue engineering.     

The fundamental parameters of chitosan in polymer scaffolds affecting osteoblasts (MC3T3-E1)

The aim of this study was to investigate the degree of deacetylation (DD) and molecular weight (MW) of chitosan within chitosan–collagen scaffolds on mouse osteoblasts (MC3T3-E1). The chitosan–collagen scaffolds were fabricated by freeze-drying technique. The studies on cell attachment and proliferation, alkaline phosphatase (ALP) activity, cell morphology, and mineralized nodule formation by osteoblasts on scaffolds were investigated. No statistically significant difference was found on cell attachment, but the chitosan–collagen scaffolds with low-DD chitosan had a statistically significantly (P < 0.05) higher proliferative effect and ALP activity than those scaffolds with high-DD chitosan, regardless of molecular weight. Scanning electron images demonstrated that MC3T3-E1 cells grew well on all test scaffolds; on the contrary, mineralized nodule formation was not found. In conclusion, the DD of chitosan is a crucial factor for MC3T3-E1 cells and it should be considered in further applications for bone tissue engineering.     

Needle-like nano hydroxyapatite/poly(l-lactide acid) composite scaffold for bone tissue engineering application

In this paper, a new nano-hydroxyapatite / poly (l-lactide acid) (nHAP/PLLA) composite scaffold comprising needle-like nHAP particles was prepared. In the first step, the identification and morphology of chemically synthesized HAP particles were determined by XRD, EDX, FTIR and SEM analyses. The needle-like nHAP particles with an average size of approximately 30–60 nm in width and 100–400 nm in length were found similar to needle-like bone nano apatites in terms of chemical composition and morphology. In the second step, nHAP and micro-sized HAP (mHAP) particles were used to fabricate HAP filled PLLA (HAP/PLLA) composites scaffolds using solid–liquid phase separation method. The porosity of scaffolds was up to 85%, and their average macropore diameter was in the range of 64–175 µm. FTIR and XRD analyses showed the presence of molecular interactions and chemical linkages between HAP particles and PLLA matrix. The compressive strength of nanocomposite scaffolds could high up to 8.46 MPa while those of pure PLLA and microcomposite scaffolds were 1.79 and 4.61 MPa, respectively. The cell affinity and cytocompatibility of the nanocomposite scaffold were found to be higher than those of pure PLLA and microcomposite scaffolds. Based on the results, the newly developed nHAP/PLLA composite scaffold is comparable with cancellous bone in terms of microstructure and mechanical strength, so it may be a suitable alternative for bone tissue engineering applications.     

Feasibility, tailoring and properties of polyurethane/bioactive glass composite scaffolds for tissue engineering

This research work aims to propose highly porous polymer/bioactive glass composites as potential scaffolds for hard-tissue and soft-tissue engineering. The scaffolds were prepared by impregnating an open-cells polyurethane sponge with melt-derived particles of a bioactive glass belonging to the SiO₂–P₂O₅–CaO–MgO–Na₂O–K₂O system (CEL2). Both the starting materials and the composite scaffolds were investigated from a morphological and structural viewpoint by X-ray diffraction analysis and scanning electron microscopy. Tensile mechanical tests, carried out according to international ISO and ASTM standards, were performed by using properly tailored specimens. In vitro tests by soaking the scaffolds in simulated body fluid (SBF) were also carried out to assess the bioactivity of the porous composites. It was found that the composite scaffolds were highly bioactive as after 7 days of soaking in SBF a HA layer grew on their surface. The obtained polyurethane/CEL2 composite scaffolds are promising candidates for tissue engineering applications.