Novel hydroxyapatite/chitosan bilayered scaffold for osteochondral tissue-engineering applications: Scaffold design and its performance when seeded with goat bone marrow stromal cells

Recent studies suggest that bone marrow stromal cells are a potential source of osteoblasts and chondrocytes and can be used to regenerate damaged tissues using a tissue-engineering (TE) approach. However, these strategies require the use of an appropriate scaffold architecture that can support the formation de novo of either bone and cartilage tissue, or both, as in the case of osteochondral defects. The later has been attracting a great deal of attention since it is considered a difficult goal to achieve. This work consisted on developing novel hydroxyapatite/chitosan (HA/CS) bilayered scaffold by combining a sintering and a freeze-drying technique, and aims to show the potential of such type of scaffolds for being used in TE of osteochondral defects. The developed HA/CS bilayered scaffolds were characterized by Fourier transform infra-red spectroscopy, X-ray diffraction analysis, micro-computed tomography, and scanning electron microscopy (SEM). Additionally, the mechanical properties of HA/CS bilayered scaffolds were assessed under compression. In vitro tests were also carried out, in order to study the water-uptake and weight loss profile of the HA/CS bilayered scaffolds. This was done by means of soaking the scaffolds into a phosphate buffered saline for 1 up to 30 days. The intrinsic cytotoxicity of the HA scaffolds and HA/CS bilayered scaffolds extract fluids was investigated by carrying out a cellular viability assay (MTS test) using Mouse fibroblastic-like cells. Results have shown that materials do not exert any cytotoxic effect. Complementarily, in vitro (phase I) cell culture studies were carried out to evaluate the capacity of HA and CS layers to separately, support the growth and differentiation of goat marrow stromal cells (GBMCs) into osteoblasts and chondrocytes, respectively. Cell adhesion and morphology were analysed by SEM while the cell viability and proliferation were assessed by MTS test and DNA quantification. The chondrogenic differentiation of GBMCs was evaluated measuring the glucosaminoglycans synthesis. Data showed that GBMCs were able to adhere, proliferate and osteogenic differentiation was evaluated by alkaline phosphatase activity and immunocytochemistry assays after 14 days in osteogenic medium and into chondrocytes after 21 days in culture with chondrogenic medium. The obtained results concerning the physicochemical and biological properties of the developed HA/CS bilayered scaffolds, show that these constructs exhibit great potential for their use in TE strategies leading to the formation of adequate tissue substitutes for the regeneration of osteochondral defects.     

Self-joining of zirconia/hydroxyapatite composites using plastic deformation process

A plastic deformation process was demonstrated to self-join 3 mol.% yttria partially stabilized zirconia (3Y-TZP)/hydroxyapatite (HA) composites. The 3Y-TZP/40 vol.% HA composites were fabricated by conventional ceramic processing by cold pressing premixed 3Y-TZP and HA powders into pellets. Densification (≈90%) of composites was achieved by sintering composite powder compacts at 1450 °C for 5 h. Optimum self-joining of 3Y-TZP/40 vol.% HA composites was obtained at 1300 °C for a strain rate of 5 × 10⁻⁵/s. The flow stress during joining was 40 MPa. Microstructural and mechanical characterizations of the joint interface demonstrated that there were no discernible differences between the joint and the composite material away from the interface.     

复合纳米人工骨的注射、凝固及其机械性能研究

目的 构建半水硫酸钙和纳米羟基磷灰石为主的复合人工骨材料并对其注射性能、凝固性能和机械强度的影响因素进行考察.方法 测试不同液固比条件下复合材料的注射特性,25℃和37℃时分别测试不同液固比、不同二水硫酸钙促凝剂条件下的材料初、终凝时间和压缩强度,均与纯硫酸钙作对比.结果 液固比0.50以上时注射性能满意.无论何种液/固比,复合材料的凝固时间均较硫酸钙延长,37℃下的凝固时间较25℃下延长.一定范围内促凝剂用量过大或过小均使凝固时间延长.液固比越大或促凝剂用量越高,材料压缩强度越低.纳米磷灰石含量增大则材料强度降低.结论 合理掌握纳米磷灰石的比例,液固比和促凝剂的用量,是开发可注射纳米人工骨的关键.     

骨髓间充质干细胞复合羟基磷灰石/磷酸三钙植骨材料的体外研究

目的研究兔骨髓间充质干细胞(BMSCs)在羟基磷灰石/磷酸三钙(HA/TCP)植骨材料上的黏附增殖情况。方法抽取兔股骨骨髓,进行贴壁培养BMSCs。在成骨诱导液中诱导BMSCs,于7d用钙钴法检测碱性磷酸酶活性,10d进行茜素红矿化结节染色;在成脂诱导液中诱导BMSCs,于21d进行油红O染色。将BMSCs接种到HA/TCP植骨材料上,加入成骨诱导液,采用倒置显微镜、荧光显微镜及扫描电镜检测,并采用四氮唑蓝(MTT)比色法测定HA/TCP植骨材料上BMSCs的增殖情况。结果BMSCs在成骨诱导液中7d碱性磷酸酶呈强阳性,10d矿化结节染色呈橘红色;在成脂诱导液中,21d油红O染色呈阳性。BMSCs在HA/TCP植骨材料上孔隙周围及孔隙内生长良好并大量增殖。MTT分析结果显示,HA/TCP对BMSCs的体外增殖无抑制作用。结论BMSCs与HA/TCP植骨材料有良好的生物相容性。     

Substrate geometry directs the in vitro mineralization of calcium phosphate ceramics

Repetitive concavities on the surface of bone implants have recently been demonstrated to foster bone formation when implanted at ectopic locations in vivo. The current study aimed to evaluate the effect of surface concavities on the surface mineralization of hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) ceramics in vitro. Hemispherical concavities with different diameters were prepared at the surface of HA and β-TCP sintered disks: 1.8 mm (large concavity), 0.8 mm (medium concavity) and 0.4 mm (small concavity). HA and β-TCP disks were sintered at 1100 or 1200 °C and soaked in simulated body fluid for 28 days at 37 °C; the mineralization process was followed by scanning electron microscopy, energy-dispersive spectroscopy, X-ray diffraction and calcium quantification analyses. The results showed that massive mineralization occurred exclusively at the surface of HA disks treated at 1200 °C and that nucleation of large aggregates of calcium phosphate started specifically inside small concavities instead of on the planar surface of the disks. Regarding the effect of concavity diameter size on surface mineralization, it was observed that small concavities induce 124- and 10-fold increased mineralization compared to concavities of large or medium size, respectively. The results of this study demonstrated that (i) in vitro surface mineralization of calcium phosphate ceramics with surface concavities starts preferentially within the concavities and not on the planar surface, and (ii) concavity size is an effective parameter to control the spatial position and extent of mineralization in vitro.     

A novel injectable,cohesive and toughened Si-HPMC (silanized-hydroxypropyl methylcellulose) composite calcium phosphate cement for bone substitution

This study reports on the incorporation of the self-setting polysaccharide derivative hydrogel (silanized-hydroxypropyl methylcellulose, Si-HPMC) into the formulation of calcium phosphate cements (CPCs) to develop a novel injectable material for bone substitution. The effects of Si-HPMC on the handling properties (injectability, cohesion and setting time) and mechanical properties (Young’s modulus, fracture toughness, flexural and compressive strength) of CPCs were systematically studied. It was found that Si-HPMC could endow composite CPC pastes with an appealing rheological behavior at the early stage of setting, promoting its application in open bone cavities. Moreover, Si-HPMC gave the composite CPC good injectability and cohesion, and reduced the setting time. Si-HPMC increased the porosity of CPCs after hardening, especially the macroporosity as a result of entrapped air bubbles; however, it improved, rather than compromised, the mechanical properties of composite CPCs, which demonstrates a strong toughening and strengthening effect. In view of the above, the Si-HPMC composite CPC may be particularly promising as bone substitute material for clinic application.     

Preparation and characterization of homogeneous chitosan–polylactic acid/hydroxyapatite nanocomposite for bone tissue engineering and evaluation of its mechanical properties

Homogeneous nanocomposites composed of hydroxyapatite and chitosan in the presence of polylactic acid were synthesized by a novel in situ precipitation method. The morphological and compositional properties of composites were investigated. Hydroxyapatite nanoparticles in a special rod-like shape with a diameter of about 50 nm and a length of about 300 nm were distributed homogeneously within the chitosan–polylactic acid matrix. The interaction between the organic matrix and the inorganic crystallite and the formation mechanism of the rod-like nanoparticles were also studied. The results suggested that the formation of the special rod-like nanoparticles could be controlled by a multiple-order template effect. High-resolution images showed that the rod-like inorganic particles were composed of randomly orientated subparticles about 10 nm in diameter. The mechanical properties of the composites were evaluated by measuring their compressive strength and elastic modulus. The data indicated that the addition of polylactic acid can make homogeneous composites scaffold resist significantly higher stress. The elastic modulus of the composites was also improved by the addition of polylactic acid, which can make them more beneficial for surgical applications.     

Tibial bone tunnel widening is reduced by polylactate/hydroxyapatite interference screws compared to metal screws after ACL reconstruction with hamstring grafts

Composite interference screws containing calcium phosphate for anterior cruciate ligament graft fixation could improve implant/bone integration and thereby reduced tunnelwidening and graft slippage. The present study investigated the effect of polylactate/hydroxyapatite interference screw (HA/PLLA) screw used for tibial graft fixation on tunnel widening and clinical outcomes compared with a metal interference screw. We hypothesized less tibial tunnelwidening with HA/PLLA screws compared to metal screws. Hundred patients with HA/PLLA screw tibial fixation was compared with 100 patients with metal screw tibial fixation. Tibial tunnel widening was measured on AP and lateral radiographs taken at 12 months follow-up. Clinical outcome was assessed by objective and subjective international Knee Documentation Committee (IKDC) scores, Noyes Sports Activity and Occupational Rating scores and KT-1000 knee laxity measurements. Tibial tunnel widening at the level of the metal screw group was 36% and 38% on AP and lateral radiographs respectively. Tunnel widening was lower in the HA/PLLA group with mean tunnel widening of 30% and 32% (p = 0.012 and 0.018) on AP and lateral radiographs respectively. No differences were found for any of the clinical scores or for anterior knee laxity. The use of a polylactate/hydroxyapatite interference screw resulted in less tibial tunnel widening than a metal screw around the screw but did not affect clinical outcome or objective knee laxity.     

A physical approach to modify the hydraulic reactivity of α-tricalcium phosphate powder

A microsized α-tricalcium phosphate (α-TCP) powder was calcined at various temperatures (350 °C < T < 800 °C) for various durations (1–24 h) and the resulting physico-chemical and reactivity changes were measured. Without calcination, the α-TCP powder started reacting within minutes after contacting a 0.2 M Na₂HPO₄ solution as measured by isothermal calorimetry. The overall reaction was finished within a few days. After calcination at 350 °C  T  550 °C for 24 h, no significant changes in the crystalline composition, crystallite size, particle size or specific surface area were noticed. However, the powder reactivity was progressively changed. More specifically, the hydraulic reaction of the powders calcined at 500 and 550 °C only started after 2–3 h whereas the overall hydraulic reaction was only slightly postponed, suggesting that physical or chemical changes had occurred at the particle surface. As mainly physical changes were detected at the particle surface during calcination at 500 °C, it was speculated that the appearance of this reaction delay (= induction time) was due to the disappearance of surface defects during the calcination step, i.e. to the need to create surface defects to induce dissolution and hence reaction.     

The promotion of osteointegration under diabetic conditions using chitosan/hydroxyapatite composite coating on porous titanium surfaces

Composited Chitosan/Hydroxyapatite (CS/HA) material coated on titanium surface (cTi) is a promising approach to produce biomaterials with better osseointegration capacity, but its bio-performance under diabetic conditions and the mechanisms involved remain elusive. We propose that the alterations in the Wnt/β-catenin pathway may play a role in mediating the improvement effect of cTi on diabetes-induced impaired implant osteointegration. To confirm the hypothesis, primary rat osteoblasts incubated on Ti and cTi were subjected to normal serum (NS), diabetic serum (DS), DS + Wnt3a (a specific Wnt agonist) and DS + Dkk1 (a specific Wnt antagonist) treatment. In vivo study was performed on diabetic sheep implanted with Ti or cTi into the bone defect on crista iliaca. Results showed that diabetes depressed osteoblast function evidenced by impaired cell adhesion and morphology, decreased cell proliferation and ALP activity, and higher apoptotic rate on Ti. Importantly, both cTi and Wnt3a treatment ameliorated osteoblastic dysfunction and apoptosis under diabetic condition. Implantation with cTi significantly improved osteointegration evidenced by Micro-CT and histological examinations compared with Ti. Moreover, the aforementioned promotive effects afforded by cTi were abolished by blocking Wnt pathway with Dkk1. Our study explicitly demonstrates that CS/HA composite material improves diabetes-induced impaired osteointegration of Ti via the reactivation of Wnt/β-catenin pathway and provides a target point for biomaterial modification to attain better clinical performance in diabetic patients.