Multilevel surface engineering of nanostructured TiO2 on carbon-fiber-reinforced polyetheretherketone

As an implantable material, carbon-fiber-reinforced polyetheretherketone (CFRPEEK) possesses an adjustable elastic modulus similar to that of cortical bone and is a prime candidate to replace metallic surgical implants. However, the bioinertness and poor osteogenic properties of CFRPEEK limit its clinical application as orthopedic implants. In this work, titanium ions are introduced energetically into CFRPEEK by plasma immersion ion implantation (PIII). Scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS) reveal the formation of nanopores with the side wall and bottom embedded with ∼20 nm TiO₂ nanoparticles on the CFRPEEK surface. Nanoindentation measurements confirm the stability and improved elastic resistance of the structured surfaces. In vitro cell adhesion, viability assay, and real-time PCR analyses disclose enhanced adhesion, proliferation, and osteo-differentiation of rat bone mesenchymal stem cells (bMSCs). The multilevel structures on CFRPEEK also exhibit partial antibacterial activity to Staphylococcus aureus and Escherichia coli. Our results indicate that a surface with multifunctional biological properties can be produced by multilevel surface engineering and application of CFRPEEK to orthopedic and dental implants can be broadened and expedited based on this scheme.     

Synergistic effects of dual Zn/Ag ion implantation in osteogenic activity and antibacterial ability of titanium

Zinc (Zn) and silver (Ag) are co-implanted into titanium by plasma immersion ion implantation. A Zn containing film with Ag nanoparticles (Ag NPs) possessing a wide size distribution is formed on the surface and the corrosion resistance is improved due to the micro-galvanic couples formed by the implanted Zn and Ag. Not only are the initial adhesion, spreading, proliferation and osteogenic differentiation of rBMSCs observed from the Zn/Ag implanted Ti in vitro, but also bacteria killing is achieved both in vitro and in vivo. Electrochemical polarization and ion release measurements suggest that the excellent osteogenic activity and antibacterial ability of the Zn/Ag co-implanted titanium are related to the synergistic effect resulting from the long-range interactions of the released Zn ions and short-range interactions of the embedded Ag NPs. The Zn/Ag co-implanted titanium offers both excellent osteogenic activity and antibacterial ability and has large potential in orthopedic and dental implants.     

Cytocompatibility,osseointegration, and bioactivity of three-dimensional porous and nanostructured network on polyetheretherketone

Porous biomaterials with the proper three-dimensional (3D) surface network can enhance biological functionalities especially in tissue engineering, but it has been difficult to accomplish this on an important biopolymer, polyetheretherketone (PEEK), due to its inherent chemical inertness. In this study, a 3D porous and nanostructured network with bio-functional groups is produced on PEEK by sulfonation and subsequent water immersion. Two kinds of sulfonation-treated PEEK (SPEEK) samples, SPEEK-W (water immersion and rinsing after sulfonation) and SPEEK-WA (SPEEK-W with further acetone rinsing) are prepared. The surface characteristics, in vitro cellular behavior, in vivo osseointegration, and apatite-forming ability are systematically investigated by X-ray photoelectron spectroscopy, Fourier-transform infrared spectroscopy, scanning electron microscopy, cell adhesion and cell proliferation assay, real-time RT-PCR analysis, micro-CT evaluation, push-out tests, and immersion tests. SPEEK-WA induces pre-osteoblast functions including initial cell adhesion, proliferation, and osteogenic differentiation in vitro as well as substantially enhanced osseointegration and bone-implant bonding strength in vivo and apatite-forming ability. Although SPEEK-W has a similar surface morphology and chemical composition as SPEEK-WA, its cytocompatibility is inferior due to residual sulfuric acid. Our results reveal that the pre-osteoblast functions, bone growth, and apatite formation on the SPEEK surfaces are affected by many factors, including positive effects introduced by the 3D porous structure and SO₃H groups as well as negative ones due to the low pH environment. Surface functionalization broadens the use of PEEK in orthopedic implants.     

光固定明胶修饰的聚醚醚酮(PEEK)表面促进细胞增殖及成骨性研究

目的聚醚醚酮(PEEK)具有与骨骼相似的弹性模量,但PEEK作为骨修复材料的应用受到其表面生物惰性及缺乏促成骨性的限制。为了解决这个问题,我们通过表面修饰光固定明胶增强PEEK生物活性。方法我科研团队合成的光固定明胶可通过紫外照射粘附于多种材料表面,并可增强材料的生物相容性。进行SEM、静态水接触角、细胞增殖、细胞形态、碱性磷酸酶分化等表征学及细胞学系统研究。结果研究表明光固定明胶可固定于PEEK表面,改变了PEEK等高分子材料的表面性质。在细胞学研究中,光固定明胶改性后相对于普通PEEK,成骨细胞增殖、伸展、基质分泌及分化能力明显增强。结论通过光固定明胶对PEEK表面进行改性,可明显增强其生物活性,是一种有潜力的骨科内植入物及医疗器械材料。     

Immunomodulation by mesenchymal stem cells combats the foreign body response to cell-laden synthetic hydrogels

The implantation of non-biological materials, including scaffolds for tissue engineering, ubiquitously leads to a foreign body response (FBR). We recently reported that this response negatively impacts fibroblasts encapsulated within a synthetic hydrogel and in turn leads to a more severe FBR, suggesting a cross-talk between encapsulated cells and inflammatory cells. Given the promise of mesenchymal stem cells (MSCs) in tissue engineering and recent evidence of their immunomodulatory properties, we hypothesized that MSCs encapsulated within poly(ethylene glycol) (PEG) hydrogels will attenuate the FBR. In vitro, murine MSCs encapsulated within PEG hydrogels attenuated classically activated primary murine macrophages by reducing gene expression and protein secretion of pro-inflammatory cytokines, most notably tumor necrosis factor-α. Using a COX2 inhibitor, prostaglandin E2 (PGE2) was identified as a mediator of MSC immunomodulation of macrophages. In vivo, hydrogels laden with MSCs, osteogenically differentiating MSCs, or no cells were implanted subcutaneously into C57BL/6 mice for 28 days to assess the impact of MSCs on the fibrotic response of the FBR. The presence of encapsulated MSCs reduced fibrous capsule thickness compared to acellular hydrogels, but this effect diminished with osteogenic differentiation. The use of MSCs prior to differentiation in tissue engineering may therefore serve as a dynamic approach, through continuous cross-talk between MSCs and the inflammatory cells, to modulate macrophage activation and attenuate the FBR to implanted synthetic scaffolds thus improving the long-term tissue engineering outcome.     

A theranostic agent to enhance osteogenic and magnetic resonance imaging properties of calcium phosphate cements

With biomimetic biomaterials, like calcium phosphate cements (CPCs), non-invasive assessment of tissue regeneration is challenging. This study describes a theranostic agent (TA) to simultaneously enhance both imaging and osteogenic properties of such a bone substitute material. For this purpose, mesoporous silica beads were produced containing an iron oxide core to enhance bone magnetic resonance (MR) contrast. The same beads were functionalized with silane linkers to immobilize the osteoinductive protein BMP-2, and finally received a calcium phosphate coating, before being embedded in the CPC. Both in vitro and in vivo tests were performed. In vitro testing showed that the TA beads did not interfere with essential material properties like cement setting. Furthermore, bioactive BMP-2 could be efficiently released from the carrier-beads. In vivo testing in a femoral condyle defect rat model showed long-term MR contrast enhancement, as well as improved osteogenic capacity. Moreover, the TA was released during CPC degradation and was not incorporated into the newly formed bone. In conclusion, the described TA was shown to be suitable for longitudinal material degradation and bone healing studies.     

Stimulation of bone growth following zinc incorporation into biomaterials

Rapid development of zinc biology has broadened the applications of Zn-incorporated biomaterials to tissue engineering but also raised concerns about the long-term safety of released Zn²⁺ ions. Clinical success hinges on the amount of incorporated zinc and subsequent optimized release sufficient to stimulate osseointegration. In this study, zinc is incorporated into the sub-surface of TiO₂ coatings by plasma immersion ion implantation and deposition (PIII&D). The Zn-implanted coatings show significant improvement compared to the “bulk-doped” coatings prepared by plasma electrolyte oxidation in terms of osteogenesis in vitro and in vivo. Molecular and cellular osteogenic activities demonstrate that rBMSCs cultured on the Zn-implanted coatings have higher ALP activity and up-regulated osteogenic-related genes (OCN, Col-I, ALP, Runx2) compared to the bulk-doped Zn coatings and controls. In vivo osseointegration studies conducted for 12 weeks on the rat model show early-stage new bone formation and the bone contact ratio (12 week) on the Zn-implanted coating is larger. The ZnT1 and ZIP1 gene expression studies demonstrate that the Zn-implanted coatings can better stimulate bone growth with reduced Zn release than those doped with zinc throughout the coatings.     

Anti-infective and osteointegration properties of silicon nitride,poly(ether ether ketone), and titanium implants

Silicon nitride (Si₃N₄) is an industrial ceramic used in spinal fusion and maxillofacial reconstruction. Maximizing bone formation and minimizing bacterial infection are desirable attributes in orthopedic implants designed to adhere to living bone. This study has compared these attributes of Si₃N₄ implants with implants made from two other orthopedic biomaterials, i.e. poly(ether ether ketone) (PEEK) and titanium (Ti). Dense implants made of Si₃N₄, PEEK, or Ti were surgically implanted into matching rat calvarial defects. Bacterial infection was induced with an injection of 1 × 10⁴ Staphylococcus epidermidis. Control animals received saline only. On 3, 7, and 14 days, and 3 months post-surgery four rats per time period and material were killed, and calvariae were examined to quantify new bone formation and the presence or absence of bacteria. Quantitative evaluation of osteointegration to adjacent bone was done by measuring the resistance to implant push-out (n = 8 rats each for Ti and PEEK, and n = 16 rats for Si₃N₄). Three months after surgery in the absence of bacterial injection new bone formation around Si₃N₄ was ～69%, compared with 24% and 36% for PEEK and Ti, respectively. In the presence of bacteria new bone formation for Si₃N₄, Ti, and PEEK was 41%, 26%, and 21%, respectively. Live bacteria were identified around PEEK (88%) and Ti (21%) implants, whereas none were present adjacent to Si₃N₄. Push-out strength testing demonstrated statistically superior bone growth onto Si₃N₄ compared with Ti and PEEK. Si₃N₄ bioceramic implants demonstrated superior new bone formation and resistance to bacterial infection compared with Ti and PEEK.     

The effect of substrate surface nanotopography on the behavior of multipotnent mesenchymal stromal cells and osteoblasts

Hexagonally arranged Gold nanoparticles with controllable diameters and inter-particle distances were deposited on thick SiO₂ layers on top of Si wafers and used as masks during subsequent reactive ion etching. In this way, arrays of nanopillars are obtained with well-defined diameters (10/30 nm), inter-pillar distances (50–120 nm) and heights (20–35 nm), all on the nanoscale. Such nanotopographies served as substrate for multipotent mesenchymal stromal cells (MSC) and human osteoblasts (OB) allowing to study cellular responses to purely topographically patterned interfaces. Focus was put on adhesion, proliferation and differentiation of the cells. It turned out experimentally that adhesion is comparable for both cell types practically independent of topographical details at the substrate surface. Topography induced proliferation enhancement, however, is again independent of geometrical details in case of MSC, but significantly sensitive to pillar height in case of OB with a clear preference towards short nanopillars (20 nm). A high sensitivity to topographic details is also observed for osteogenic differentiation of MSC, in that case with a preference towards higher nanopillars (50 nm). The present experimental data also allow the important conclusion that cell proliferation and differentiation can be optimized simultaneously by fine-tuning nanoscaled topographical parameters.     

液晶/聚氨酯复合基底影响rBMSCs成骨分化的研究

目的:模拟体内组织弹性微环境,构建液晶(OPC/PU)复合基底,探究复合基底弹性模量及液晶相区尺寸对大鼠骨髓间充值干细胞(rBMSCs)成骨分化的影响。方法:通过调节复合膜中液晶含量,制备不同弹性模量的液晶复合基底。采用偏光显微镜观察复合基底表面液晶相区结构;万能测试仪测量复合基底弹性模量;激光共聚焦显微镜观察rBMSCs的铺展、极化和骨架排列;CCK-8法检测rBMSCs的增殖速率;real-time PCR检测复合膜上的成骨分化标记物Ⅰ型胶原和骨桥蛋白的mRNA表达。结果:(1)复合基底中液晶含量增加,液晶相区数量及尺寸增加,复合基底的弹性模量降低,但仍保持在MPa数量级。(2)rBMSCs在液晶含量较低的OPC10-PU和OPC30-PU表面呈现较好的初始黏附、铺展和增殖。(3)成骨诱导初期及中期,rBMSCs在OPC10-PU上展示较高的Ⅰ型胶原和骨桥蛋白基因表达;诱导培养后期,rBMSCs在OPC30-PU和OPC50-PU上呈现出Ⅰ型胶原和骨桥蛋白基因的高表达,成骨分化的基因表达重点也从早中期的Ⅰ型胶原主要表达转变为后期的骨桥蛋白主要表达。结论:复合基底中液晶含量较低时,rBMSCs主要响应于基底弹性诱发的力学刺激产生细胞行为的变化;基底中液晶含量增加,rBMSCs能够感知到液晶的黏弹特性并与其发生强烈的相互作用,此时基底的弹性和液晶相区的黏弹特性可能均对rBMSCs的成骨分化产生重要影响。     

Osseointegration of machined,injection moulded and oxygen plasma modified PEEK implants in a sheep model

Machined and injection moulded polyetheretherketone (PEEK) implants with and without an oxygen plasma modification were prepared and implanted in sheep cancellous and cortical bone. After 4, 12 and 26 weeks, osseointegration was evaluated through mechanical push-out tests and histomorphometry. In the cancellous bone, push-out force increased with time, a trend toward higher force was observed for machined compared to moulded, and oxygen plasma modified compared to unmodified. On-going remodelling of the bone was detected in the periphery of the implants at 4 weeks. Minimal or no inflammation was observed with all the implants at all locations and time-points. Bone-implant contact (BIC) was quantified at all-time points and locations for all the four PEEK implant surfaces. The BIC values ranged from 15 to 75% with an average of 29 ± 13% in the cancellous bone and 25–65% with an average of 50 ± 12% in the cortical bone. In the cortical bone the BIC increased significantly from 4 to 26 weeks. This in vivo study has identified that surface topography of PEEK implants influences osseointegration. In addition, oxygen plasma has the potential to increase bone-implant interface stability. This study provides a unique reference for further modifications and in vivo assessment of PEEK implants.     

RhBMP-2-loaded calcium silicate/calcium phosphate cement scaffold with hierarchically porous structure for enhanced bone tissue regeneration

Calcium phosphate cement scaffold (CPC) has been widely used as bone graft substitutes, but undesirable osteoinductivity and slow degradability greatly hamper their clinic application. To address these problems, a recombinant human bone morphogenetic protein-2 (rhBMP-2)-loaded calcium silicate/calcium phosphate cement scaffold (CSPC) with hierarchical pores was developed in this study. The CSPC scaffold with both interconnected macropores on the order of 200–500 μm and micropores of 2–5 μm was synthesized from CPC and calcium silicate (CS) by a NaCl particulate-leaching method. In vitro cell culture with C2C12 model cells, in vivo ectopic bone formation and rabbit femur cavity defect repair were performed to evaluate the osteogeneic capacity of the CSPC/rhBMP-2 scaffold. CPC, CSPC and CPC/rhBMP-2 scaffolds were parallelly investigated for comparison. The results demonstrated that the hierarchical macro/microporous structure, whether in presence of CS or rhBMP-2, highly favored the adhesion of C2C12 cells and bone in-growth into the CPC-based scaffolds. But, in comparison to the CPC-based scaffolds with CS or rhBMP-2 alone, the CSPC/rhBMP-2 scaffold strongly promoted osteogenic differentiation in vitro and osteogenetic efficacy in vivo. Further studies demonstrated that Si ions derived from CSPC contributed mainly to maintain the conformation of rhBMP-2 and thus stimulate the synergistic action of CS and rhBMP-2 in osteogenic differentiation and osteoinductivity. Additionally, the incorporation of CS was also beneficial for the dissolution of the scaffold. Those results suggest that the CSPC has superior properties for incorporation of rhBMP-2 and our developed CSPC/rhBMP-2 scaffold have great potential for future use in bone tissue regeneration.     

The nanoscale geometry of TiO2 nanotubes influences the osteogenic differentiation of human adipose-derived stem cells by modulating H3K4 trimethylation

Nanostructured materials can direct stem cell lineage commitment solely by their various, but controllable, geometric cues, which would be very important for their future application in bone tissue engineering and bone regeneration. However, the mechanisms by which nano-geometric cues dictate the osteogenic differentiation of stem cells remain unclear. Epigenetics is central to cellular differentiation, a process that regulates heritable and long-lasting alterations in gene expression without changing the DNA sequence. Here, we explored the varied osteogenic behaviors of human adipose-derived stem cells (hASCs) on titanium dioxide (TiO₂) nanotube arrays of different diameters. Both in vitro and in vivo studies demonstrated that the nanoscale geometry influenced cellular differentiation and TiO₂ nanotubes with a diameter of 70 nm was the optimal dimension for the osteogenic differentiation of hASCs. Moreover, we observed that TiO₂ nanotubes promoted the osteogenic differentiation of hASCs by upregulating methylation level of histone H3 at lysine 4 (H3K4) in the promoter regions of osteogenic genes Runx2 and osteocalcin, by inhibiting demethylase retinoblastoma binding protein 2 (RBP2). These results revealed, for the first time, the epigenetic mechanism by which nanotopography directs stem cell fate.     

An annulus fibrosus closure device based on a biodegradable shape-memory polymer network

Injuries to the intervertebral disc caused by degeneration or trauma often lead to tearing of the annulus fibrosus (AF) and extrusion of the nucleus pulposus (NP). This can compress nerves and cause lower back pain. In this study, the characteristics of poly(d,l-lactide-co-trimethylene carbonate) networks with shape-memory properties have been evaluated in order to prepare biodegradable AF closure devices that can be implanted minimally invasively. Four different macromers with (d,l-lactide) to trimethylene carbonate (DLLA:TMC) molar ratios of 80:20, 70:30, 60:40 and 40:60 with terminal methacrylate groups and molecular weights of approximately 30 kg mol⁻¹ were used to prepare the networks by photo-crosslinking. The mechanical properties of the samples and their shape-memory properties were determined at temperatures of 0 °C and 40 °C by tensile tests- and cyclic, thermo-mechanical measurements. At 40 °C all networks showed rubber-like behavior and were flexible with elastic modulus values of 1.7–2.5 MPa, which is in the range of the modulus values of human annulus fibrosus tissue. The shape-memory characteristics of the networks were excellent with values of the shape-fixity and the shape-recovery ratio higher than 98 and 95%, respectively. The switching temperatures were between 10 and 39 °C. In vitro culture and qualitative immunocytochemistry of human annulus fibrosus cells on shape-memory films with DLLA:TMC molar ratios of 60:40 showed very good ability of the networks to support the adhesion and growth of human AF cells. When the polymer network films were coated by adsorption of fibronectin, cell attachment, cell spreading, and extracellular matrix production was further improved. Annulus fibrosus closure devices were prepared from these AF cell-compatible materials by photo-polymerizing the reactive precursors in a mold. Insertion of the multifunctional implant in the disc of a cadaveric canine spine showed that these shape-memory devices could be implanted through a small slit and to some extent deploy self-sufficiently within the disc cavity.     

Surface topography enhances differentiation of mesenchymal stem cells towards osteogenic and adipogenic lineages

Surface topography impacts on cell growth and differentiation, but it is not trivial to generate defined surface structures and to assess the relevance of specific topographic parameters. In this study, we have systematically compared in vitro differentiation of mesenchymal stem cells (MSCs) on a variety of groove/ridge structures. Micro- and nano-patterns were generated in polyimide using reactive ion etching or multi beam laser interference, respectively. These structures affected cell spreading and orientation of human MSCs, which was also reflected in focal adhesions morphology and size. Time-lapse demonstrated directed migration parallel to the nano-patterns. Overall, surface patterns clearly enhanced differentiation of MSCs towards specific lineages: 15 μm ridges increased adipogenic differentiation whereas 2 μm ridges enhanced osteogenic differentiation. Notably, nano-patterns with a periodicity of 650 nm increased differentiation towards both osteogenic and adipogenic lineages. However, in absence of differentiation media surface structures did neither induce differentiation, nor lineage-specific gene expression changes. Furthermore, nanostructures did not affect the YAP/TAZ complex, which is activated by substrate stiffness. Our results provide further insight into how structuring of tailored biomaterials and implant interfaces – e.g. by multi beam laser interference in sub-micrometer scale – do not induce differentiation of MSCs per se, but support their directed differentiation.     

Differential regulation of osteogenic differentiation of stem cells on surface roughness gradients

Tissue engineering using scaffold-cell constructs holds the potential to develop functional strategies to regenerate bone. The interface of orthopedic implants with the host tissues is of great importance for its later performance. Thus, the optimization of the implant surface in a way that could stimulate osteogenic differentiation of mesenchymal stem cells (MSCs) is of significant therapeutic interest. The effect of surface roughness of polycaprolactone (PCL) on the osteogenic differentiation of human bone-marrow MSCs was investigated. We prepared surface roughness gradients of average roughness (Ra) varying from the sub-micron to the micrometer range (∼0.5–4.7 μm), and mean distance between peaks (RS_m) gradually varying from ∼214 μm to 33 μm. We analyzed the degree of cytoskeleton spreading, expression of alkaline phosphatase, collagen type 1 and mineralization. The response of cells to roughness divided the gradient into three groups of elicited stem cell behavior: 1) faster osteogenic commitment and strongest osteogenic expression; 2) slower osteogenic commitment but strong osteogenic expression, and 3) similar or inferior osteogenic potential in comparison to the control material. The stem-cell modulation by specific PCL roughness surfaces highlights the potential for creating effective solutions for orthopedic applications featuring a clinically relevant biodegradable material.     

Mechanical properties and in vitro response of strontium-containing hydroxyapatite/polyetheretherketone composites

Strontium-containing hydroxyapatite/polyetheretherketone (Sr-HA/PEEK) composites were developed as alternative materials for load-bearing orthopaedic applications. The amount of strontium-containing hydroxyapatite (Sr-HA) incorporated into polyetheretherketone (PEEK) polymer matrix ranged from 15 to 30 vol% and the composites were successfully fabricated by compression molding technique. This study presents the mechanical properties and in vitro human osteoblast-like cell (MG-63) response of the composite material developed. The bending modulus and strength of Sr-HA/PEEK composites were tailored to mimic human cortical bone. PEEK reinforced with 25 and 30 vol% Sr-HA exhibited bending modulus of 9.6 and 10.6 GPa, respectively; alternatively, the bending strengths of the composites were 93.8 and 89.1 MPa, respectively. Based on the qualitative comparison of apatite formation in SBF and quantitative measurement of MG-63-mediated mineralization in vitro, the Sr-HA/PEEK composite was proven to outperform HA/PEEK in providing bioactivity. However, no difference was found in the trend of cell proliferation and alkaline phosphatase activity between different composites. Strontium, in the form of strontium-containing hydroxyapatite (Sr-HA), was confirmed to enhance bioactivity in the PEEK composites.     

A biodegradable microvessel scaffold as a framework to enable vascular support of engineered tissues

A biodegradable microvessel scaffold comprised of distinct parenchymal and vascular compartments separated by a permeable membrane interface was conceptualized, fabricated, cellularized, and implanted. The device was designed with perfusable microfluidic channels on the order of 100 μm to mimic small blood vessels, and high interfacial area to an adjacent parenchymal space to enable transport between the compartments. Poly(glycerol sebacate) (PGS) elastomer was used to construct the microvessel framework, and various assembly methods were evaluated to ensure robust mechanical integrity. In vitro studies demonstrated the differentiation of human skeletal muscle cells cultured in the parenchymal space, a 90% reduction in muscle cell viability due to trans-membrane transport of a myotoxic drug from the perfusate, and microvessel seeding with human endothelial cells. In vivo studies of scaffolds implanted subcutaneously and intraperitoneally, without or with exogenous cells, into nude rats demonstrated biodegradation of the membrane interface and host blood cell infiltration of the microvessels. This modular, implantable scaffold could serve as a basis for building tissue constructs of increasing scale and clinical relevance.