Hydrogel/bioactive glass composites for bone regeneration applications: Synthesis and characterisation

Due to the deficiencies of current commercially available biological bone grafts, alternative bone graft substitutes have come to the forefront of tissue engineering in recent times. The main challenge for scientists in manufacturing bone graft substitutes is to obtain a scaffold that has sufficient mechanical strength and bioactive properties to promote formation of new tissue. The ability to synthesise hydrogel based composite scaffolds using photopolymerisation has been demonstrated in this study. The prepared hydrogel based composites were characterised using techniques including Fourier Transform Infrared Spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), Energy-dispersive X-ray spectrometry (EDX), rheological studies and compression testing. In addition, gel fraction, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), porosity and swelling studies of the composites were carried out. It was found that these novel hydrogel bioglass composite formulations did not display the inherent brittleness that is typically associated with bioactive glass based bone graft materials and exhibited enhanced biomechanical properties compared to the polyethylene glycol hydrogel scaffolds along. Together, the combination of enhanced mechanical properties and the deposition of apatite on the surface of these hydrogel based composites make them an ideal candidate as bone graft substitutes in cancellous bone defects or low load bearing applications.     

Development of Porous Hydroxyapatite Scaffolds

Porous objects of hydroxyapatite (HA) were prepared by mixing sodium chloride as channeling agent with ultra high molecular weight polyethylene (UHMWPE) as a binder and hydroxyapatite powder. After hot isostatic pressing (HIP) and dissolving the channeling agent, the bioceramic showed an interconnecting porosity with pore diameters in the range of 160-220 µm. The method can be used for any material that is insoluble in water. Such objects are promising for bone regeneration because the interconnecting porosity in implants provides a good environment for bone attachment and in-growth.     

浸浆法制备生物多孔陶瓷

自然骨的主要无机成分是羟基磷灰石，由于纯羟基磷灰石对于人体组织有良好的生物相容性，被广泛使用为骨植入材料，为了得到孔径和孔隙率合适的羟基磷灰石框架，本文采用多孔聚氨脂泡沫浸浆法制备，实验表明，该方法可以有效的获得需要的羟基磷灰石框架。     

Fabrication and development of artificial osteochondral constructs based on cancellous bone/hydrogel hybrid scaffold

Using tissue engineering techniques, an artificial osteochondral construct was successfully fabricated to treat large osteochondral defects. In this study, porcine cancellous bones and chitosan/gelatin hydrogel scaffolds were used as substitutes to mimic bone and cartilage, respectively. The porosity and distribution of pore size in porcine bone was measured and the degradation ratio and swelling ratio for chitosan/gelatin hydrogel scaffolds was also determined in vitro. Surface morphology was analyzed with the scanning electron microscope (SEM). The physicochemical properties and the composition were tested by using an infrared instrument. A double layer composite scaffold was constructed via seeding adipose-derived stem cells (ADSCs) induced to chondrocytes and osteoblasts, followed by inoculation in cancellous bones and hydrogel scaffolds. Cell proliferation was assessed through Dead/Live staining and cellular activity was analyzed with IpWin5 software. Cell growth, adhesion and formation of extracellular matrix in composite scaffolds blank cancellous bones or hydrogel scaffolds were also analyzed. SEM analysis revealed a super porous internal structure of cancellous bone scaffolds and pore size was measured at an average of 410 ± 59 μm while porosity was recorded at 70.6 ± 1.7 %. In the hydrogel scaffold, the average pore size was measured at 117 ± 21 μm and the porosity and swelling rate were recorded at 83.4 ± 0.8 % and 362.0 ± 2.4 %, respectively. Furthermore, the remaining hydrogel weighed 80.76 ± 1.6 % of the original dry weight after hydration in PBS for 6 weeks. In summary, the cancellous bone and hydrogel composite scaffold is a promising biomaterial which shows an essential physical performance and strength with excellent osteochondral tissue interaction in situ. ADSCs are a suitable cell source for osteochondral composite reconstruction. Moreover, the bi-layered scaffold significantly enhanced cell proliferation compared to the cells seeded on either single scaffold. Therefore, a bi-layered composite scaffold is an appropriate candidate for fabrication of osteochondral tissue.     

Three dimensional printed macroporous polylactic acid/hydroxyapatite composite scaffolds for promoting bone formation in a critical-size rat calvarial defect model

We have explored the applicability of printed scaffold by comparing osteogenic ability and biodegradation property of three resorbable biomaterials. A polylactic acid/hydroxyapatite (PLA/HA) composite with a pore size of 500 μm and 60% porosity was fabricated by three-dimensional printing. Three-dimensional printed PLA/HA, β-tricalcium phosphate (β-TCP) and partially demineralized bone matrix (DBM) seeded with bone marrow stromal cells (BMSCs) were evaluated by cell adhesion, proliferation, alkaline phosphatase activity and osteogenic gene expression of osteopontin (OPN) and collagen type I (COL-1). Moreover, the biocompatibility, bone repairing capacity and degradation in three different bone substitute materials were estimated using a critical-size rat calvarial defect model in vivo. The defects were evaluated by micro-computed tomography and histological analysis at four and eight weeks after surgery, respectively. The results showed that each of the studied scaffolds had its own specific merits and drawbacks. Three-dimensional printed PLA/HA scaffolds possessed good biocompatibility and stimulated BMSC cell proliferation and differentiation to osteogenic cells. The outcomes in vivo revealed that 3D printed PLA/HA scaffolds had good osteogenic capability and biodegradation activity with no difference in inflammation reaction. Therefore, 3D printed PLA/HA scaffolds have potential applications in bone tissue engineering and may be used as graft substitutes in reconstructive surgery.     

纳米羟基磷灰石/聚合物多孔复合支架材料

为提高骨组织工程支架材料的力学性能,改善其生物活性,综合天然与合成高分子的优点,采用溶液共混相分离法制备出聚己内酯(PCL)-壳聚糖(CS)多孔支架材料, 并进一步采用离心注浆法填充具有生物活性的纳米羟基磷灰石(HA)-聚乙烯醇(PVA)复合浆料, 制备了n-HA-PVA/PCL-CS复合多孔支架材料, 改善了PCL-CS支架材料力学性能。采用扫描电子显微镜、红外光谱、元素分析、孔隙率和抗压强度试验对材料进行了表征。结果表明, PCL-CS支架材料的内部具有蜂窝状的相互贯通的孔隙结构,孔隙率可以达到60%～80%。CS含量越大,孔隙率越大,而抗压强度越小。填充后的n-HA-PVA/PCL-CS复合多孔支架材料,孔隙率有所下降,但仍大于60%,而其弹性模量可提高至25.71 MPa。      

Preparation of a novel biodegradable nanocomposite scaffold based on poly (3-hydroxybutyrate)/bioglass nanoparticles for bone tissue engineering

One of the most important challenges in composite scaffolds is pore architecture. In this study, poly (3-hydroxybutyrate) with 10% bioglass nanoparticles was prepared by the salt leaching processing technique, as a nanocomposite scaffold. The scaffolds were characterized by SEM, FTIR and DTA. The SEM images demonstrated uniformed porosities of appropriate sizes (about 250–300 μm) which are interconnected. Furthermore, higher magnification SEM images showed that the scaffold possesses less agglomeration and has rough surfaces that may improve cell attachment. In addition, the FTIR and DTA results showed favorable interaction between polymer and bioglass nanoparticles which improved interfaces in the samples. Moreover, the porosity of the scaffold was assessed, and the results demonstrated that the scaffold has uniform and high porosity in its structure (about 84%). Finally it can be concluded that this scaffold has acceptable porosity and morphologic character paving the way for further studies to be conducted from the perspective of bone tissue engineering.     

In-situ hardening hydroxyapatite-based scaffold for bone repair

Musculoskeletal conditions are becoming a major health concern because of an aging population and sports- and traffic-related injuries. While sintered hydroxyapatite implants require machining, calcium phosphate cement (CPC) bone repair material is moldable, self-hardens in situ, and has excellent osteoconductivity. In the present work, new approaches for developing strong and macroporous scaffolds of CPC were tested. Relationships were determined between scaffold porosity and strength, elastic modulus and fracture toughness. A biocompatible and biodegradable polymer (chitosan) and a water-soluble porogen (mannitol) were incorporated into CPC: Chitosan to make the material stronger, fast-setting and anti-washout; and mannitol to create macropores. Flexural strength, elastic modulus, and fracture toughness were measured as functions of mannitol mass fraction in CPC from 0% to 75%. After mannitol dissolution in a physiological solution, macropores were formed in CPC in the shapes of the original entrapped mannitol crystals, with diameters of 50 μm to 200 μm for cell infiltration and bone ingrowth. The resulting porosity in CPC ranged from 34.4% to 83.3% volume fraction. At 70.2% porosity, the hydroxyapatite scaffold possessed flexural strength (mean ± sd; n = 6) of (2.5 ± 0.2) MPa and elastic modulus of (0.71 ± 0.10) GPa. These values were within the range for sintered porous hydroxyapatite and cancellous bone. Predictive equations were established by regression power-law fitting to the measured data (R² > 0.98) that described the relationships between scaffold porosity and strength, elastic modulus and fracture toughness. In conclusion, a new graft composition was developed that could be delivered during surgery in the form of a paste to harden in situ in the bone site to form macroporous hydroxyapatite. Compared to conventional CPC without macropores, the increased macroporosity of the new apatite scaffold may help facilitate implant fixation and tissue ingrowth.     

高强度力学性能丝素/羟基磷灰石多孔支架材料的初步制备

以丝素和羟基磷灰石为基材,磷酸盐缓冲液为溶剂,戊二醛为交联剂制备丝素/羟基磷灰石支架材料,对丝素生物材料在骨材料中的开发应用有积极意义。研究表明,丝素/羟基磷灰石复合材料具有结晶结构和β构象。当丝素与羟基磷灰石的比例为6/4时,支架材料的弹性模量和压缩强度达到最大,分别为48.087 MPa和1.427 MPa,孔隙率...     

Hybrid composite scaffolds prepared by sol–gel method for bone regeneration

This study reports the synthesis and characterization of hydroxyapatite (HA)/polycaprolactone (PCL) hybrid composite materials synthesized by sol–gel method. The fabrication of scaffolds was performed by salt-leaching technique using NaCl as porogen agent. In the first step, the physico-chemical characterization of composite material was performed to evaluate the composition and the interaction between the organic/inorganic phases. In the second step, optimized scaffolds were bioactivated on the surface. The combined effect of scaffold morphology and surface bioactivity is ideal for bone tissue engineering, supporting bone cells adhesion, proliferation and differentiation. Here, a combined strategy involving biomimetic approach, using a supersaturated Simulated Body Fluid (SBF), and salt-leaching technique has been developed to grow hydroxyapatite in composite scaffolds able to regenerate the natural bone.     

Personalised bone tissue engineering scaffold with controlled architecture using fractal tool paths in layered manufacturing

The scaffolds for bone tissue engineering should consider the functional requirements such as the external shape of the replacement, porosity for vessel and nutrient conduit, and stiffness in order to avoid stress shielding and to stimulate growth of the new tissue. Layered manufacturing (LM) has shown great promise in fabricating such porous bone scaffold. The present work proposes a biomimetic design and LM of patient- and site-specific controlled porosity scaffolds for optimised mechanical properties for repair and regeneration of bone. Correlation models between porosity and modulus for bone, and known biomaterials processable by LM are used to estimate the site-specific porosity requirements in the scaffold model. A novel method for generating a tool path using space-filling fractal curves eliminates representation difficulties associated with LM of porous objects. A representative study of a hydroxyapatite scaffold for a cortical bone defect site in human femur is presented to illustrate the methodology.     

Comparative study of PCL-HAp and PCL-bioglass composite scaffolds for bone tissue engineering

The aim of this work is to compare the effect of hydroxyapatite (HAp) or bioglass (BG) nanoparticles in a polycaprolactone composite scaffold aimed to bone regeneration. To allow a comparison of the influence of both types of fillers, scaffolds made of PCL or composites containing up to 20 % by weight HAp or BG were obtained. Scaffolds showed acceptable mechanical properties for its use and high interconnected porosity apt for cellular colonization. To study the effect of the different materials on pre-osteoblast cells differentiation, samples with 5 % mineral reinforcement, were cultured for up to 28 days in osteogenic medium. Cells proliferated in all scaffolds. Nevertheless, differentiation levels for the selected markers were higher in pure PCL scaffolds than in the composites; inclusion of bioactive particles showed no positive effects on cell differentiation. In osteogenic culture conditions, the presence of bioactive particles is thus not necessary in order to observe good differentiation.     

骨组织工程用纳米羟基磷灰石/ 壳聚糖多孔支架材料的制备及性能表征

以16.7%(质量分数)的柠檬酸水溶液作溶剂,通过粒子沥滤法制备了 n HA/CS多孔材料,并对其进行了IR、XRD、SEM、孔隙率及力学性能测试。结果表明n HA/CS复合材料中羟基磷灰石呈弱结晶状态,复合前后两组分的化学组成未发生显著变化,但两相间发生了相互作用。多孔材料呈高度多孔结构,孔壁上富含微孔,孔间贯通性高;复合材料/致孔剂质量比为1时,多孔材料的孔隙率为 53%,其抗压强度可达17 MPa左右,可以满足组织工程支架材料的要求。     

壳聚糖/羟基磷灰石表面修饰聚己内酯多孔骨支架的制备及性能

采用选择性激光烧结技术构建多孔聚己内酯(PCL)骨支架,用原位合成的方法制得壳聚糖/羟基磷灰石(CS/HA)悬浮液,并采用真空浸泡、低速离心和冷冻凝胶的方法使CS/HA黏附在PCL支架的表面,以改善骨支架的生物相容性和细胞增殖活性。通过X射线衍射(XRD)和扫描电子显微镜(SEM)观测复合支架的物相和形貌,测量支架的压缩强度和杨氏模量,测量支架表面的水接触角,并通过体外细胞实验研究复合支架的生物学性能。实验结果表明,原位合成的方法制得了羟基磷灰石(HA);CS/HA凝胶与PCL骨支架表面黏附良好;CS/HA改善了PCL支架表面的亲水性,提升了骨支架的生物相容性和细胞增殖活性。     

3D-Cultivation of bone marrow stromal cells on hydroxyapatite scaffolds fabricated by dispense-plotting and negative mould technique

The main principle of a bone tissue engineering (BTE) strategy is to cultivate osteogenic cells in an osteoconductive porous scaffold. Ceramic implants for osteogenesis are based mainly on hydroxyapatite (HA), since this is the inorganic component of bone. Rapid Prototyping (RP) is a new technology in research for producing ceramic scaffolds. This technology is particularly suitable for the fabrication of individually and specially tailored single implants. For tissue engineering these scaffolds are seeded with osteoblast or osteoblast precursor cells. To supply the cultured osteoblastic cells efficiently with nutrition in these 3D-geometries a bioreactor system can be used. The aim of this study was to analyse the influence of differently fabricated HA-scaffolds on bone marrow stromal cells. For this, two RP-techniques, dispense-plotting and a negative mould method, were used to produce porous ceramics. The manufactured HA-scaffolds were then cultivated in a dynamic system (bioreactor) with an osteoblastic precursor cell line. In our study, the applied RP-techniques give the opportunity to design and process HA-scaffolds with defined porosity, interconnectivity and 3D pore distribution. A higher differentiation of bone marrow stromal cells could be detected on the negative mould fabricated scaffolds, while cell proliferation was higher on the dispense-plotted scaffolds. Nevertheless, both scaffold types can be used in tissue engineering applications.     

Biodegradable polycaprolactone scaffold with controlled porosity obtained by modified particle-leaching technique

Scaffold with controlled porosity constitute a cornerstone in tissue engineering, as a physical support for cell adhesion and growth. In this work, scaffolds of polycaprolactone were synthesized by a modified particle leaching method in order to control porosity and pore interconnectivity; the aim is to observe their influence on the mechanical properties and, in the future, on cell adhesion and proliferation rates. Low molecular weight PEMA beads with an average size of 200 μm were sintered with various compression rates in order to obtain the templates (negatives of the scaffolds). Then the melt polycaprolactone was injected into the porous template under nitrogen pressure in a custom made device. After cooling and solidifying of the melt polymer, the porogen was removed by selective dissolution in ethanol. The porosity and morphology of the scaffold were studied as well as the mechanical properties. Porosities from 60% to 85% were reached; it was found that pore interconnectivity logically increases with increasing porosity, and that mechanical strength decreases with increasing porosity. Because of their interesting properties and interconnected structure, these scaffolds are expected to find useful applications as a cartilage or bone repair material.     

Preparation of porous scaffold from hydroxyapatite powders

Hydroxyapatite (HA) powder was prepared by wet chemical method. The hydroxyapatite phase was stable up to 1250 °C without decomposition to beta-tricalcium phosphate. Interconnected porous hydroxyapatite scaffold resembling trabecular bone structure was developed from polymeric replica sponge method. The prepared scaffold has 60 vol.% porosity having a major fraction of ~ 50–125 μm pore diameter. The pore content, pore morphology, pore interconnectivity of scaffold and their compressive strength were dependent on the solid loading and binder content. In-vitro bioactivity and bioresorbability confirmed the feasibility of the developed scaffolds.     

Australian Coral as a Biomaterial: Characteristics

In order to produce effective implants, the materials used must be biocompatible. Hydroxyapatite (HAp) is a bioactive material similar to the mineral component of teeth and bone which is often used for orbital implants and bone graft applications. HAp can be manufactured from corals via hydrothermal conversion. Coral is particularly useful as a starting material for hydroxyapatite production because of its porous nature. When a porous structure is used tissue ingrowth can occur readily and hence an excellent mechanical bond can be achieved. A large pore size and a high degree of pore interconnections are desirable implant properties. In the present paper a comparison of the properties of four different species of Australian coral has been made to determine the most favourable species to use as a starting material for hydrothermal conversion.     

Finite element predictions compared to experimental results for the effective modulus of bone tissue engineering scaffolds fabricated by selective laser sintering

A current challenge in bone tissue engineering is to create scaffolds with suitable mechanical properties, high porosity, full interconnectivity and suitable pore size. In this paper, polyamide and polycaprolactone scaffolds were fabricated using a solid free form technique known as selective laser sintering. These scaffolds had fully interconnected pores, minimized strut thickness, and a porosity of approximately 55%. Tensile and compression tests as well as finite element analysis were carried out on these scaffolds. It was found that the values predicted for the effective modulus by the FE model were much higher than the actual values obtained from experimental results. One possible explanation for this discrepancy, viz. the surface roughness of the scaffold and the presence of micropores in the scaffold struts, was investigated with a view to making recommendations on improving FE model configurations for accurate effective property predictions.     

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.