3D printing of polycaprolactone/bioactive glass composite scaffolds for in situ bone repair

3D printing technology can fabricate customized scaffolds based on patient-derived medical images, so it has attracted much attention in the field of developing bone repair scaffolds. Polycaprolactone (PCL) is a suitable polymer for preparing bone repair scaffolds because of its good biocompatibility, thermal stability, excellent mechanical properties and degradable properties. However, PCL is a bioinert material and cannot induce new bone formation at the defect site. In this study, the bioactive material 58s bioactive glass was mixed into PCL to form PCL/bioactive glass composite material. The results of contact angle showed that the hydrophilicity of the scaffold was significantly enhanced with the increase of bioactive glass content. In vitro experiment results showed that, with the increase of bioactive glass content, cell adhesion and proliferation were enhanced, the expression levels of Runx2 and Collagen I(COL-I) were upregulated. The experimental results of in vivo radial defect repair in rats also showed that the effect of bone repair was improved with the increase of bioactive glass content. In conclusion, PCL customized bone repair scaffold containing 20% bioactive glass has widely potential used in the field of clinical bone repair.     

A modified pultrusion process for preparing composites reinforced with continuous fibers and aligned hydroxyapatite nano needles

Of importance to orthopedic procedures are repair materials that can carry significant loads without excessive deformation. In addition, these materials need to be biocompatible, bioabsorbable, and conducive to the replacement of the repair material with native bone. To meet these requirements, we have designed composites using two biocompatible/bioabsorbable polymers, poly(l ‐lactic acid) (PLLA) in fiber form and polycaprolactone (PCL) filled in a carefully designed fashion with nano needles of hydroxyapatite (HA). Discussed in this article is a modified pultrusion process that proved useful for producing suitable composites. In this process, the feed to the pultrusion die is a stabilized suspension of the HA needles in a PCL/MEK solution, whereas the fiber “bundle” is a single yarn containing 360 PLLA fibers. The small scale is needed to maximize the content of HA in the final composite and assure uniformity. Process variables studied include the ratio of HA to PCL in the suspension, the solids content of the suspension and various geometrical variables in the die design that influence the shear‐rate profile along the fiber path. The goal of the pultrusion step is to fill all interstices between the fibers with a PCL matrix that is reinforced with highly aligned HA needles. POLYM. COMPOS., 36:931–938, 2015. © 2014 Society of Plastics Engineers     

Enhancing Osteoconduction of PLLA-Based Nanocomposite Scaffolds for Bone Regeneration Using Different Biomimetic Signals to MSCs

In bone engineering, the adhesion, proliferation and differentiation of mesenchymal stromal cells rely on signaling from chemico-physical structure of the substrate, therefore prompting the design of mimetic "extracellular matrix"-like scaffolds. In this study, three-dimensional porous poly-L-lactic acid (PLLA)-based scaffolds have been mixed with different components, including single walled carbon nanotubes (CNT), micro-hydroxyapatite particles (HA), and BMP2, and treated with plasma (PT), to obtain four different nanocomposites: PLLA + CNT, PLLA + CNTHA, PLLA + CNT + HA + BMP2 and PLLA + CNT + HA + PT. Adult bone marrow mesenchymal stromal cells (MSCs) were derived from the femur of orthopaedic patients, seeded on the scaffolds and cultured under osteogenic induction up to differentiation and mineralization. The release of specific metabolites and temporal gene expression profiles of marrow-derived osteoprogenitors were analyzed at definite time points, relevant to in vitro culture as well as in vivo differentiation. As a result, the role of the different biomimetic components added to the PLLA matrix was deciphered, with BMP2-added scaffolds showing the highest biomimetic activity on cells differentiating to mature osteoblasts. The modification of a polymeric scaffold with reinforcing components which also work as biomimetic cues for cells can effectively direct osteoprogenitor cells differentiation, so as to shorten the time required for mineralization.     

Morphology,wettability, and mechanical properties of polycaprolactone/hydroxyapatite composite scaffolds with interconnected pore structures fabricated by a mini‐deposition system

A new mini‐deposition system (MDS) was developed to fabricate scaffolds with interconnected pore structures and anatomical geometry for bone tissue engineering. Polycaprolactone/hydroxyapatite (PCL/HA) composites with varying hydroxyapatite (HA) content were adopted to manufacture scaffolds by using MDS with a porosity of 54.6%, a pore size of 716 μm in the x‐y plane, and 116 μm in the z direction. The water uptake ratio and compressive modulus of PCL/HA composite scaffold increase from 8 to 39% and from 26.5 to 49.8 MPa, respectively, as the HA content increases from 0 to 40%. PCL/HA composite scaffolds have better wettability and mechanical properties than pure PCL scaffold. A PCL/HA composite scaffold for mandible bone repair was successfully fabricated with both interconnected pore structures and anatomical shape to demonstrate the versatility of MDS. POLYM. ENG. SCI., 2012. © 2012 Society of Plastics Engineers     

Biocompatibility and strengthening of porous hydroxyapatite scaffolds using poly(l-lactic acid) coating

Hydroxyapatite (HA) is a well-known biocompatible bone substitute. Porous HA is more resorbable and osteoconductive compared with non-porous HA, and has been studied both experimentally and clinically. However, the mechanical strength of porous HA scaffolds is known to be weak. In this study, we developed a porous HA scaffold coated with a synthetic biodegradable polymer, poly(l-lactic acid) (PLLA), to strengthen the scaffold. PLLA-coated HA pellets were used to investigate the in vitro proliferation and alkaline phosphatase (ALP) activity of osteoblasts. PLLA-coated porous HA scaffolds were observed using scanning electron microscopy to investigate surface characteristics, porosity, and mechanical strength. PLLA coating concentration varied from 2 to 10 wt%. Osteoblast proliferation was higher in HA samples coated with PLLA compared with non-coated. ALP activity was highest on 8 wt% PLLA-coating after 3 days and on 4 wt% and 6 wt% PLLA after 9 and 12 days. Porous HA scaffolds with higher concentrations of PLLA were found to have a smoother, flatter surface. This enhanced proliferation and attachment of osteoblasts onto the porous HA scaffold. PLLA solution at a concentration of 10 wt% decreased scaffold porosity to half that of HA scaffolds with no PLLA coating. Scaffold mechanical strength was increased two-fold with a PLLA concentration of 2 wt%. Based on in vitro experimentation, it can be concluded that PLLA-coating on porous HA scaffolds enhances both the biocompatibility and the mechanical strength.     

生物质基含硅骨修复复合支架材料的制备、特性及评价

模仿天然骨的精密结构制备有机-无机复合骨修复支架材料已成为骨组织工程发展的重要方向。生物质材料如胶原、明胶、壳聚糖、丝素蛋白等由于具有优良的生物学性能而得到广泛关注。含硅生物活性材料由于具有良好的骨传导性和骨诱导性,成为骨修复支架材料中重要的无机组分。本文主要介绍了粉体复合和原位复合两种骨支架材料组分的复合技术,阐述了冷冻干燥、静电纺丝、仿生矿化以及3D打印等骨支架材料结构的构建策略,着重总结了生物质基含硅骨修复支架材料研究进展,阐明当前骨支架材料制备的难点在于支架材料的力学性能和多孔性结构以及生物降解性能与新骨生成速率之间的匹配性问题,并对骨支架材料的发展进行了展望。     

The effect of BMP‐2 and VEGF loading of gelatin‐pectin‐BCP scaffolds to enhance osteoblast proliferation

A composite scaffold of gelatine (Gel)‐pectin (Pec)‐biphasic calcium phosphate (BCP) was successfully fabricated. Growth factors such as bone morphogenetic protein‐2 (BMP‐2) and vascular endothelial growth factor (VEGF) were loaded into the Gel‐Pec‐BCP hydrogel scaffolds by freeze‐drying. The surface morphology was investigated by scanning electron microscopy, and BCP dispersion in the hydrogel scaffolds was measured by energy dispersive and X‐ray diffraction spectroscopy. The results obtained from Fourier transform infrared spectroscopy and quantitative measurements showed successfully loading of BMP‐2 and VEGF into the Gel‐Pec‐BCP hydrogel scaffolds. In addition MC3T3‐E1 preosteoblasts were cultivated on the three types of scaffolds to investigate the effects of BMP‐2 and VEGF on cell viability and proliferation. The Gel‐Pec‐BCP scaffolds loaded with VEGF and BMP‐2 demonstrated more cell spreading and proliferation compared to those of the Gel‐Pec‐BCP scaffolds. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41241.     

Surface‐engineered hydroxyapatite nanocrystal/ poly(ε‐caprolactone) hybrid scaffolds for bone tissue engineering

To achieve novel polymer/bioceramic composite scaffolds for use in materials for bone tissue engineering, we prepared organic/inorganic hybrid scaffolds composed of biodegradable poly(ε‐caprolactone) (PCL) and hydroxyapatite (HA), which has excellent biocompatibility with hard tissues and high osteoconductivity and bioactivity. To improve the interactions between the scaffolds and osteoblasts, we focused on surface‐engineered, porous HA/PCL scaffolds that had HA molecules on their surfaces and within them because of the biochemical affinity between the biotin and avidin molecules. The surface modification of HA nanocrystals was performed with two different methods. Using Fourier transform infrared, X‐ray diffraction, and thermogravimetric analysis measurements, we found that surface‐modified HA nanocrystals prepared with an ethylene glycol mediated coupling method showed a higher degree of coupling (%) than those prepared via a direct coupling method. HA/PCL hybrid scaffolds with a well‐controlled porous architecture were fabricated with a gas‐blowing/particle‐leaching process. All HA/PCL scaffold samples exhibited approximately 80–85% porosity. As the HA concentration within the HA/PCL scaffolds increased, the porosity of the HA/PCL scaffolds gradually decreased. The homogeneous immobilization of biotin‐conjugated HA nanocrystals on a three‐dimensional, porous scaffold was observed with confocal microscopy. According to an in vitro cytotoxicity study, all scaffold samples exhibited greater than 80% cell viability, regardless of the HA/PCL composition or preparation method. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Bone morphogenetic protein‐2 immobilization on porous PCL‐BCP‐Col composite scaffolds for bone tissue engineering

The objective of this study was to develop novel porous composite scaffolds for bone tissue engineering through surface modification of polycaprolactone–biphasic calcium phosphate‐based composites (PCL–BCP). PCL–BCP composites were first fabricated with salt‐leaching method followed by aminolysis. Layer by layer (LBL) technique was then used to immobilize collagen (Col) and bone morphogenetic protein (BMP‐2) on PCL–BCP scaffolds to develop PCL–BCP–Col–BMP‐2 composite scaffold. The morphology of the composite was examined by scanning electron microscopy (SEM). The efficiency of grafting of Col and BMP‐2 on composite scaffold was measured by X‐ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR). Both XPS and FTIR confirmed that Col and BMP‐2 were successfully immobilized into PCL–BCP composites. MC3TC3‐E1 preosteoblasts cells were cultivated on composites to determine the effect of Col and BMP‐2 immobilization on cell viability and proliferation. PCL–BCP–Col–BMP‐2 showed more cell attachment, cell viability, and proliferation bone factors compared to PCL–BCP‐Col composites. In addition, in vivo bone formation study using rat models showed that PCL–BCP–Col–BMP‐2 composites had better bone formation than PCL–BCP‐Col scaffold in critical size defect with 4 weeks of duration. These results suggest that PCL–BCP–Col–BMP‐2 composites can enhance bone regeneration in critical size defect in a rat model with 4 weeks of duration. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134, 45186.     

Three-dimensional printing of a β-tricalcium phosphate scaffold with dual bioactivities for bone repair

β-Tricalcium phosphate (β-TCP) had been widely used in the field of bone defect repair because of its osteoconduction and osteoinduction properties. However, for critical-sized bone defects, β-TCP scaffolds need to be functionalised to enhance osteoinduction and antibacterial activity. In this study, we proposed a protocol for mimicking a mussel adhesion mechanism to immobilise bone morphoprotein 2 mimetic peptide (BMP2-MP) and Ornithodoros savignyi (OS) on a three-dimensionally printed β-TCP scaffold. BMP2-MP and the OS polypeptides containing the YKYKY tail were converted into 3,4‐dihydroxyphenylalanine (DOPA) molecules via hydroxylase. The surface morphology and phase composition of the different scaffolds were analysed via scanning electron microscopy and X-ray diffraction. In addition, the binding activity of BMP2-MP and OS containing the DOPA tail to the scaffold were evaluated. The antibacterial activity of the different scaffolds was studied in vitro by performing bacteriostatic experiments against Escherichia coli and Staphylococcus aureus. The osteoinduction capability of the different scaffolds was evaluated by detecting osteogenesis-associated genes via quantitative polymerase chain reaction and by determining alkaline phosphatase expression levels. Our results demonstrated that introduction of the DOPA tail enhanced the binding capability of BMP2-MP and OS with the β-TCP scaffold, thereby enhancing the antibacterial and osteoinduction capabilities of the scaffold. A scaffold with strong antibacterial and osteoinduction capability will have good application prospects in the field of critical-sized bone defect repair.     

Fabrication and properties of 3D printed zirconia scaffold coated with calcium silicate/hydroxyapatite

The scaffold of bone repair needs a variety of material combinations to meet its intended performance; a typical single material such as zirconia has excellent mechanical properties, while hydroxyapatite and calcium silicate are bioactive materials with different degradation rates. In this paper, porous zirconia scaffolds were fabricated using 3D printing technology. The surface of the scaffold was coated by dipping with different contents of calcium silicate and hydroxyapatite to improve the biological activity and mechanical properties. Mechanical tests show that the coating material can effectively fill the pores of the porous scaffold, increasing its compressive strength by an average of 55%. The simulated body fluid (SBF) test showed that the higher calcium silicate in the coating increased the degradation rate. Cell experiments showed that the coated scaffolds exhibited good cytocompatibility and were beneficial to the proliferation and differentiation of cells. In conclusion, coated scaffolds have potential applications in the field of bone repair.     

Site-Directed Immobilization of an Engineered Bone Morphogenetic Protein 2 (BMP2) Variant to Collagen-Based Microspheres Induces Bone Formation In Vivo

For the treatment of large bone defects, the commonly used technique of autologous bone grafting presents several drawbacks and limitations. With the discovery of the bone-inducing capabilities of bone morphogenetic protein 2 (BMP2), several delivery techniques were developed and translated to clinical applications. Implantation of scaffolds containing adsorbed BMP2 showed promising results. However, off-label use of this protein-scaffold combination caused severe complications due to an uncontrolled release of the growth factor, which has to be applied in supraphysiological doses in order to induce bone formation. Here, we propose an alternative strategy that focuses on the covalent immobilization of an engineered BMP2 variant to biocompatible scaffolds. The new BMP2 variant harbors an artificial amino acid with a specific functional group, allowing a site-directed covalent scaffold functionalization. The introduced artificial amino acid does not alter BMP2′s bioactivity in vitro. When applied in vivo, the covalently coupled BMP2 variant induces the formation of bone tissue characterized by a structurally different morphology compared to that induced by the same scaffold containing ab-/adsorbed wild-type BMP2. Our results clearly show that this innovative technique comprises translational potential for the development of novel osteoinductive materials, improving safety for patients and reducing costs.     

Preparation of chitosan-sodium alginate/bioactive glass composite cartilage scaffolds with high cell activity and bioactivity

Chitosan-sodium alginate/bioactive glass (CSB) composite cartilage scaffold with outstanding in vitro mineralization property and cytocompatibility is synthesized by freeze drying method. The effect of bioactive glass (BG) addition on the microstructure, porosity, swelling/degradation ratio, in vitro mineralization property and cytocompatibility of CSB scaffold is investigated by the characterization techniques of SEM, XRD, FTIR and BET. Results showed that CSB composite cartilage scaffold had a three-dimensional (3D) porous structure, and both porosity and average pore size met the requirements of cartilage tissue repair. Among, the typical CSB-1.0 had the largest overall pore size and lowest compressive modulus (1.083 ± 0.002 MPa). As the amount of BG increased, pore volume and porosity of CSB scaffolds gradually decreased, and the swelling and degradation ratios gradually reduced. After immersing in SBF for 3 d, cauliflower like hydroxyapatite (HA) was formed on CSB surface, indicating that the scaffold had good in vitro mineralization property. Moreover, the introduction of BG into the composite scaffold can improve the relative cell viability of MC3T3-E1 cells, and CSB-1.0 has the strongest ability to promote the proliferation of cells. Therefore, the as-obtained CSB scaffold can be used as a strong candidate for cartilage tissue engineering scaffold to meet clinical needs.     

Novel Layered Hydroxyapatite/Tri‐Calcium Phosphate–Zirconia Scaffold Composite with High Bending Strength for Load‐Bearing Bone Implant Application

The integration of biological and mechanical requirements remains a challenge in developing porous hydroxyapatite (HA) and tri‐calcium phosphate (TCP) scaffolds for load‐bearing bone implant application. With the newly developed slip‐deposition and coating‐substrate co‐sintering technique, a strong layered HA/TCP‐zirconia scaffold composite structure was successfully fabricated. The bending strength (321 MPa) of this composite can match upper strength limit of the natural compact bone. The HA‐based scaffold coating has multiple scale porous structures with pore size ranging 1–10 and 20–50 μm. The zirconia‐based substrate is also porous with submicropores. Focus ion beam micrographs show most of the micropores in the coating are interconnected. Microindentation and primarily adhesive strength tests demonstrate that the scaffold coating strongly bonds with the zirconia based substrate. In vitro cell culture study indicates that the coatings have no cytotoxicity. It is evident that the strong layered HA–zirconia scaffold composite offers new implant options for bone repairs requiring immediate load bearing capacity.     

Electroactive Hydroxyapatite/Carbon Nanofiber Scaffolds for Osteogenic Differentiation of Human Adipose-Derived Stem Cells

Traditional bone defect treatments are limited by an insufficient supply of autologous bone, the immune rejection of allogeneic bone grafts, and high medical costs. To address this medical need, bone tissue engineering has emerged as a promising option. Among the existing tissue engineering materials, the use of electroactive scaffolds has become a common strategy in bone repair. However, single-function electroactive scaffolds are not sufficient for scientific research or clinical application. On the other hand, multifunctional electroactive scaffolds are often complicated and expensive to prepare. Therefore, we propose a new tissue engineering strategy that optimizes the electrical properties and biocompatibility of carbon-based materials. Here, a hydroxyapatite/carbon nanofiber (HAp/CNF) scaffold with optimal electrical activity was prepared by electrospinning HAp nanoparticle-incorporated polyvinylidene fluoride (PVDF) and then carbonizing the fibers. Biochemical assessments of the markers of osteogenesis in human adipose-derived stem cells (h-ADSCs) cultured on HAp/CNF scaffolds demonstrate that the material promoted the osteogenic differentiation of h-ADSCs in the absence of an osteogenic factor. The results of this study show that electroactive carbon materials with a fibrous structure can promote the osteogenic differentiation of h-ADSCs, providing a new strategy for the preparation and application of carbon-based materials in bone tissue engineering.     

Comparison of osteogenesis in poly(L-lactic acid)-coated and non-coated porous hydroxyapatite scaffolds

To improve the biocompatibility and mechanical strength of porous hydroxyapatite (HA) scaffolds that have high osteoconduction, we coated them with 1, 5, or 10 wt% poly(L-lactic acid) (PLLA). In the 5 and 10 wt% PLLA-coated groups, osteoblast proliferation rates were higher than those in non-coated and 1 wt% PLLA-coated groups. The alkaline phosphatase (ALP) activity was highest in the 5 wt% PLLA-coated group. In addition, the porosity was highest in the non-coated HA group (82 %), whereas it was 72, and 41 % in the 5, and 10 wt% PLLA-coated groups, respectively. Scaffold strength increased in proportion to the concentration of PLLA coating. Overall, the 5 wt% PLLA scaffold had best characteristics when considering osteoblast proliferation, ALP activity, porosity, and compressive strength. We next tested this scaffold in an in vivo alveolar defect rabbit model. Multi-detector row computed tomography showed that non-coated and 5 wt% PLLA-coated HA groups had a similar high density. Adequate osteogenesis was observed by histological analysis in the non-coated HA and 5 wt% PLLA-coated HA groups. In addition, sufficient mineralization was observed in the coated scaffolds by X-ray fluorescence spectroscopy without significant adverse effects. Therefore, based on our findings, a PLLA-coated porous HA scaffold may be a suitable bone substitute for the correction of bone defects.     

3D mesoporous bioactive glass/silk/chitosan scaffolds and their compatibility with human adipose-derived stromal cells

Human adipose-derived stem/stromal cells (hASCs) have been popularly studied as cell-based therapy in the field of regenerative medicine due to their ability to differentiate into several cell types. In this study, in order to improve the mechanical strength and bioactivity of scaffolds for bone tissue engineering, three types of mesoporous bioactive glasses with different shapes and compositions were dispersed in the silk fibroin/chitosan (SF/CS)-based scaffolds, which were fabricated with a combination of freezing and lyophilization. The characteristic and physical properties of these composite scaffolds were evaluated. The biocompatibility was also assessed through hASCs in vitro tests. Both Alamar Blue® and Live/Dead assay® revealed that the spherical mesoporous bioactive glass doped scaffolds enhanced cell viability and proliferation. Furthermore, the addition of spherical mesoporous bioactive glass into SF/CS scaffolds encouraged hASC osteogenic differentiation as well. These results suggested that this composite scaffold can be applicable material for bone regeneration.     

In vitro evaluation of electrospun gelatin‐bioactive glass hybrid scaffolds for bone regeneration

Organic–inorganic hybrid materials, composed of phases that interact on a nanoscale and a microstructure that mimics the extracellular matrix, can potentially provide attractive scaffolds for bone regeneration. In the present study, hybrid scaffolds of gelatin and bioactive glass (BG) with a fibrous microstructure were prepared by a combined sol–gel and electrospinning technique and evaluated in vitro. Structural and chemical analyses showed that the fibers consisted of gelatin and BG that were covalently linked by 3‐glycidoxypropyltrimethoxysilane to form a homogeneous phase. Immersion of the gelatin–BG hybrid scaffolds in a simulated body fluid (SBF) at 37°C resulted in the formation of a hydroxyapatite (HA)‐like material on the surface of the fibers within 12 h, showing the bioactivity of the scaffolds. After 5 days in SBF, the surface of the hybrid scaffolds was completely covered with an HA‐like layer. The gelatin–BG hybrid scaffolds had a tensile strength of 4.3 ± 1.2 MPa and an elongation to failure of 168 ± 14%, compared to values of 0.5 ± 0.2 MPa and 63 ± 2% for gelatin scaffolds with a similar microstructure. The hybrid scaffolds supported the proliferation of osteoblastic MC3T3‐E1 cells, alkaline phosphatase activity, and mineralization during in vitro culture, showing their biocompatibility. The results indicate that these gelatin–BG hybrid scaffolds prepared by a combination of sol–gel processing and electrospinning have potential for application in bone regeneration. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

Preparation and characterization of plla composite scaffolds by ScCO2‐induced phase separation

Composite Scaffolds have received much attention in the tissue engineering, and how to choose the materials has become the research focus in this field. Supercritical CO₂ (ScCO₂)‐induced phase separation process was employed to prepare porous poly‐L ‐lactide (PLLA) composite scaffolds. An experiment system was set up for the purpose of investigating the effects of such parameters as the mass ratios of PLLA to polyethylene glycol (PEG) and PLLA to β‐TCP on porosity and compressive strength of composite scaffolds. The obtained composite scaffolds were characterized in many ways. Scanning electron microscopy was used to examine the morphology and pore size; porosity was analyzed by pycnometer; and the compressive strength was recorded by texture analyzer. The results indicated that the porosity was increased with the addition of PEG, and the highest porosity of PLLA/PEG composite scaffolds was 92% with the mass ratio of PLLA to PEG of 1:0.05; the compressive strength was increased with the addition of β‐TCP, and the highest compressive strength of PLLA/β‐TCP composite scaffolds was 1.76 MPa with the mass ratio of PLLA to β‐TCP of 1:0.1. POLYM. COMPOS., 2012. © 2012 Society of Plastics Engineers