Preparation of porous hydroxyapatite scaffolds

In this work, the sintering and grain growth of hydroxyapatite green bodies are analyzed in order to identify the optimum heat treatments for the preparation of porous hydroxyapatite scaffolds. Sintering in air at temperatures ranging between 1100 and 1200 °C yields dense materials with narrow grain-size distributions. The scaffolds are formed by the infiltration of polymer foams with hydroxyapatite slurries or by robocasting, a novel rapid-prototyping technique. Examples of the microstructures achieved with each approach are presented. It is observed that both techniques can be used to fabricate scaffolds with adequate pore size to promote bone ingrowth.     

Fabrication and characterization of novel hydroxyapatite/porous carbon composite scaffolds

Novel hydroxyapatite (HA)/porous carbon composite scaffolds were prepared by applying sonoelectrodeposition and a subsequent hydrothermal treatment to previous carbonized phenolic resin coated polyurethane sponges. The interconnected pore network and morphology of HA/porous carbon composite scaffolds were determined by scanning electron microscope (SEM), and the whole surface of porous carbons were evenly coated with the deposited HA layer which was confirmed by EDS and XRD. The porosity (83.5 ± 0.3%) and the bulk density (0.297 ± 0.009 g·cm⁻³) of HA/porous carbon scaffolds were detected by the Archimedes method. The compressive and flexural strength of the scaffolds is 1.187 ± 0.064 MPa and 0.607 ± 0.268 MPa, respectively. Compared with the polymeric surface of 24-well cell culture plates, these novel scaffolds significantly promote the proliferation of human osteoblast-like MG-63 cells, indicating that this novel HA/porous carbon composite scaffold could be used for in vitro 3D culture of osteoblasts.     

Effects of osteoblast-like cell seeding on the mechanical properties of porous composite scaffolds

In tissue engineering technology, polymer–ceramics or polymer–polymer composites have been considered as advanced scaffolds having mechanical stability, biocompatibility, cell proliferation, and easy processability. However, the relationship between the mechanical properties and the cell proliferation behavior of such composite scaffolds has not been clarified yet. In this study, two types of composite scaffolds, poly(ethylene terephthalate) (PET) fiber/collagen and β-tricalcium phosphate (β-TCP)/gelatin scaffolds, were investigated. MC3T3-E1 cells were cultured in these scaffolds under appropriate conditions. Compression tests were then periodically conducted to evaluate the compressive elastic modulus. It was found that the modulus of the scaffolds containing cells increased with the cell culture period. It is noted that the modulus of the β-TCP/gelatin with cells was approximately seven times larger than that of the PET fiber/collagen with cells.     

Characterization of porous hydroxyapatite

Hydroxyapatite has been considered for use in the repair of osseous defects for the last 20 years. Recent developments have led to interest in the potential of porous hydroxyapatite as a synthetic bone graft. However, despite considerable activity in this field, regarding assessment of the biological response to such materials, the basic materials characterization is often inadequate. This paper documents the characterization of the chemical composition, mechanical integrity, macro- and microstructure of a porous hydroxyapatite, Endobon ® (E. Merck GmbH), intended for the bone-graft market. Specimens possesed a range of apparent densities from 0.35 to 1.44 g cm^-3. Chemical analysis demonstrated that the natural apatite precursor of Endobon® was not converted to pure hydroxyapatite, but retained many of the ionic substituents found in bone mineral, notably carbonate, sodium and magnesium ions. Investigation of the microstructure illustrated that the struts of the material were not fully dense, but had retained some traces of the network of osteocyte lacunae. Macrostructural analysis demonstrated the complex inter-relationship between the structural features of an open pore structure. Both pore size and connectivity were found to be inversely dependent on apparent density. Furthermore, measurement of pore aspect ratio and orientation demonstrated a relationship between apparent density and the degree of macrostructural anisotropy within the specimens, while, it was also noted that pore connectivity was sensitive to anisotropy. Compression testing demonstrated the effect of apparent density and macrostructural anisotropy on the mechanical properties. An increase in apparent density from 0.38 to 1.25 g cm^-3 resulted in increases in ultimate compressive stress and compressive modulus of 1 to 11 MPa and 0.2 to 3.1 GPa, respectively. Furthermore, anisotropic high density (> 0.9 g cm^-3) specimens were found to possess lower compressive moduli than isotropic specimens with equivalent apparent densities. These results underline the importance of full structural and mechanical characterization of porous ceramic implant materials. ©1999 Kluwer Academic Publishers     

Influence of preparation method on hydroxyapatite porous scaffolds

Hydroxyapatite (HA) is extensively used in medical applications as an artificial bone because of its similarity to the natural components of human bones and for its excellent biocompatibility. The porous structure of HA ceramics is more generally used as a scaffold. Many techniques, which are performed under fluid system, have been applied to fabricate HA porous scaffolds. In this work, polymeric sponge technique was employed in the preparation of HA slurry appropriated for porous ceramic fabrication. Effort for strength improvement was made on porous HA ceramic in several aspects. The effect of HA/water, binder/plasticizer ratios and dispersant content on the rheological properties of HA suspension in combination with the addition of SiC and SiO₂ on the compressive strength of porous bodies were investigated and discussed.     

Highly porous hydroxyapatite scaffolds with elongated pores using stretched polymeric sponges as novel template

This study reports a simple way of improving the compressive strength of highly porous hydroxyapatite (HA) scaffolds by adopting elongated polymeric sponges as a novel template. In this method, as-received polymeric sponges with isotropic pores were stretched uniaxially to 50% elongation at 200 °C for 2 h, and then coated with a HA slurry. The HA-coated sponges were heat-treated at 800 °C for 3 h to remove the polymeric sponges and at 1250 °C for 3 h to sinter the HA walls. The fabricated samples showed a highly anisotropic pore structure with elongated pores parallel to the direction of the elongation of the polymeric sponge. This simple method allowed a highly porous scaffold to have a high compressive strength of 3.8 ± 0.1 MPa at a porosity of 76% when tested parallel to the direction of pore elongation.     

羟基磷灰石基多孔复合支架的制备及其表征

研究利用造孔剂法制备高度贯通的多孔羟基磷灰石(HA)支架,孔隙率约为78%,并利用聚己内酯(PCL)分别复合纳米HA(nHA)或微纳米生物玻璃(nBG)粉末对其进行涂覆改性,粉末的添加量均为10%~40%(质量分数)。4种类型支架分别记为HA、PCL/HA、nHA-PCL/HA和nBG-PCL/HA。实验结果发现,nHA-PCL/HA和nBG-PCL/HA复合支架最大抗压强度分别为1.41~1.98 MPa和1.35~1.78MPa。4类支架矿化实验显示,浸泡21d后nBG-PCL/HA表面促进生成较多的磷灰石矿化物;细胞实验结果显示细胞在4类支架上均生长良好,说明支架具有良好的生物相容性。支架在实验犬背部肌肉组织内植入2个月的组织学检测显示,4种支架内均有新骨形成,尤其是nHA-PCL/HA和nBG-PCL/HA孔内有更多的新生骨组织,说明这两种支架表面复合涂层中的生物活性纳米颗粒对诱导新骨生成具有积极的促进作用。     

Preparation of highly interconnected porous hydroxyapatite scaffolds by chitin gel-casting

In this study, a simple and effective method for producing highly interconnected porous hydroxyapatite (HA) scaffolds was developed by combining gel-casting, particle-leaching and extrusion techniques. Chitin (CT) sol was used to disperse HA particles and wax spheres were introduced as porogens for their excellent deformability. In extrusion process, the accumulated wax spheres in point-to-point contact can transform into surface-to-surface contact by means of the extrusion pressure. Thus, the obtained porous HA scaffolds exhibited an interconnected channel network after leaching out of the porogens. The results showed that the scaffolds prepared by different size of wax spheres exhibited nearly the same volumetric porosity of about 86%, while the compressive strengths decreased as the pore size increased. Therefore, the method developed can be used to effectively tailor the pore size of HA scaffolds while maintaining a high porosity. The highly porous HA scaffolds with excellent interconnectivity are expected to be a promising bone substitute in clinical practice.     

Electrochemistry assisted reacting deposition of hydroxyapatite in porous chitosan scaffolds

Electrochemistry assisted reacting deposition method was employed to prepare porous chitosan/hydroxyapatite (CS/HA) composite scaffold with a new design device using ion exchange membranes to separate calcium salt and phosphate solutions. The results determined from XRD and SEM indicates hydroxyapatite can be electrochemically deposited in the chitosan scaffold using the device. After electrochemistry assisted reacting deposition, the surface of the chitosan scaffold was coated with low crystalline HA, particularly at the frame edge of the scaffold. The pores in the scaffold still kept interconnected well and the deposited hydroxyapatite has a cluster microsphere shape whose size is about 3-5 μm.     

聚乳酸/聚乙二醇/羟基磷灰石多孔骨支架的3D打印制备及其生物相容性

聚乳酸(PLA)是一种应用广泛的生物高分子材料,但在应用过程中存在韧性、亲水性、生物活性差等缺点。用聚乙二醇(PEG)和羟基磷灰石(HA)对PLA进行改性。通过熔融共混制备不同质量比的PLA/PEG/HA复合3D打印线材,并通过分析PLA/PEG/HA线材的力学性能、结晶性能、热性能、流变性能等,筛选更适合熔融沉积成型(FDM)的3D打印成型线材,进而利用3D打印制备精度高的力学性能试样及生物相容性好、细胞可增殖和分化的生物多孔支架。结果表明:PEG的添加提高了PLA的韧性,降低了PLA的熔点。HA的添加则提高PLA/PEG/HA复合材料的弹性模量和冷结晶温度,同时HA也可以改善复合材料的加工性能。SEM与荧光标记结果表明多孔支架与细胞具有良好的生物相容性。生物支架对体外细胞的成功培养,为进一步发掘生物多孔支架在动物体内、生物医学及定制化应用方面提供了潜在可能。     

Microsphere-based scaffolds encapsulating tricalcium phosphate and hydroxyapatite for bone regeneration

Bioceramic mixtures of tricalcium phosphate (TCP) and hydroxyapatite (HAp) are widely used for bone regeneration because of their excellent cytocompatibility, osteoconduction, and osteoinduction. Therefore, we hypothesized that incorporation of a mixture of TCP and HAp in microsphere-based scaffolds would enhance osteogenesis of rat bone marrow stromal cells (rBMSCs) compared to a positive control of scaffolds with encapsulated bone-morphogenic protein-2 (BMP-2). Poly(d,l-lactic-co-glycolic acid) (PLGA) microsphere-based scaffolds encapsulating TCP and HAp mixtures in two different ratios (7:3 and 1:1) were fabricated with the same net ceramic content (30 wt%) to evaluate how incorporation of these ceramic mixtures would affect the osteogenesis in rBMSCs. Encapsulation of TCP/HAp mixtures impacted microsphere morphologies and the compressive moduli of the scaffolds. Additionally, TCP/HAp mixtures enhanced the end-point secretion of extracellular matrix components relevant to bone tissue compared to the “blank” (PLGA-only) microsphere-based scaffolds as evidenced by the biochemical, gene expression, histology, and immunohistochemical characterization. Moreover, the TCP/HAp mixture groups even surpassed the BMP-2 positive control group in some instances in terms of matrix synthesis and gene expression. Lastly, gene expression data suggested that the rBMSCs responded differently to different TCP/HAp ratios presented to them. Altogether, it can be concluded that TCP/HAp mixtures stimulated the differentiation of rBMSCs toward an osteoblastic phenotype, and therefore may be beneficial in gradient microsphere-based scaffolds for osteochondral regeneration.     

Biomechanical assessment of bone ingrowth in porous hydroxyapatite

Porous hydroxyapatite (Endobon®) specimens were implanted into the femoral condyle of New Zealand White rabbits for up to 6 months. After sacrifice, specimens were sectioned for histology and mechanical testing, where the extent of reinforcement by bony ingrowth was assessed by compression testing and fixation was assessed by push-out testing. From histological observations, it was established that the majority of bone ingrowth occurred between 10 day and 5 weeks after implantation and proceeded predominantly from the deep end of the trephined defect, with some integration from the circumferential sides. At 3 months, the implants were fully integrated, exhibiting bony ingrowth, vascularization and bone marrow stroma within the internal macropores. After 5 weeks, the mean ultimate compressive strength of retrieved implants (6.9 MPa) was found to be greater than that of the original implant (2.2 MPa), and by 3 months the fully integrated implants attained a compressive strength of approximately 20 MPa. Push-out testing demonstrated that after 5 weeks in vivo, the interfacial shear strength reached 3.2 MPa, increasing to 7.3 MPa at 3 and 6 months.     

Preparation of highly porous hydroxyapatite from cuttlefish bone

Hydroxyapatite structures for tissue engineering applications have been produced by hydrothermal (HT) treatment of aragonite in the form of cuttlefish bone at 200°C. Aragonite (CaCO₃) monoliths were completely transformed into hydroxyapatite after 48 h of HT treatment. The substitution of CO₃ ²⁻ groups predominantly into the PO₄ ³⁻ sites of the Ca₁₀(PO₄)₆(OH)₂ structure was suggested by FT-IR spectroscopy and Rietveld structure refinement. The intensity of the ν₃PO₄ ³⁻ bands increase, while the intensity of the ν₂CO₃ ²⁻ bands decrease with the duration of HT treatment resulting in the formation of carbonate incorporating hydroxyapatite. The SEM micrographs have shown that the interconnected hollow structure with pillars connecting parallel lamellae in cuttlefish bone is maintained after conversion. Specific surface area (S _BET) and total pore volume increased and mean pore size decreased by HT treatment.     

注射成型羟基磷灰石/聚酰胺6多孔支架的微观结构和力学性能

采用注射成型法制备了羟基磷灰石/聚酰胺6(HA/PA6)多孔支架,用SEM、XRD、DSC等手段表征了支架的组成结构和形貌,检测了其力学性能.研究表明,调节羟基磷灰石(HA)含量、偶氮二甲酰胺(AC)用量和预塑量能有效控制支架的力学性能、孔径大小和孔隙率.HA/PA6支架的孔径在100～500μm左右,孔隙率达到甚至超...     

Bone morphogenic protein-2 (BMP-2) loaded hybrid coating on porous hydroxyapatite scaffolds for bone tissue engineering

In this study, a silica xerogel-chitosan hybrid is utilized as a coating material to incorporate bone morphogenic protein-2 (BMP-2) on a porous hydroxyapatite (HA) scaffold for bone tissue engineering. BMP-2 is known as a therapeutic agent for improving bone regeneration and repair. Silica xerogel-chitosan hybrids have been used for the delivery of a growth factor as well as osteoconductive coatings. The biological properties of the hybrid coating incorporated with BMP-2 were evaluated in terms of the BMP-2 release behavior, osteoblastic cellular responses and in vivo performance. BMP-2 was continuously released from the hybrid coating layer on the porous HA scaffold for up to 6 weeks. The hybrid coating containing BMP-2 showed significantly enhanced osteoblastic cell responses in comparison with the hybrid coating and HA substrate. Consequently, new bone formation was significantly increased within the hybrid coating containing BMP-2. These results reveal that the hybrid coating containing BMP-2 has the potential to be used as a bone implant, whose osteogenic properties are promoted by the release of BMP-2 in a controlled manner for a prolonged period of time.     

Nanocrystalline spherical hydroxyapatite granules for bone repair: in vitro evaluation with osteoblast-like cells and osteoclasts

Conventionally sintered hydroxyapatite-based materials for bone repair show poor resorbability due to the loss of nanocrystallinity. The present study describes a method to establish nanocrystalline hydroxyapatite granules. The material was prepared by ionotropic gelation of an alginate sol containing hydroxyapatite (HA) powder. Subsequent thermal elimination of alginate at 650 °C yielded non-sintered, but unexpectedly stable hydroxyapatite granules. By adding stearic acid as an organic filler to the alginate/HA suspension, the granules exhibited macropores after thermal treatment. A third type of material was achieved by additional coating of the granules with silica particles. Microstructure and specific surface area of the different materials were characterized in comparison to the already established granular calcium phosphate material Cerasorb M^®. Cytocompatibility and potential for bone regeneration of the materials was evaluated by in vitro examinations with osteosarcoma cells and osteoclasts. Osteoblast-like SaOS-2 cells proliferated on all examined materials and showed the typical increase of alkaline phosphatase (ALP) activity during cultivation. Expression of bone-related genes coding for ALP, osteonectin, osteopontin, osteocalcin and bone sialoprotein II on the materials was proven by RT-PCR. Human monocytes were seeded onto the different granules and osteoclastogenesis was examined by activity measurement of tartrate-specific acid phosphatase (TRAP). Gene expression analysis after 23 days of cultivation revealed an increased expression of osteoclast-related genes TRAP, vitronectin receptor and cathepsin K, which was on the same level for all examined materials. These results indicate, that the nanocrystalline granular materials are of clinical interest, especially for bone regeneration.     

骨组织工程多孔支架材料性质及制备技术

多孔性生物可降解支架的选择和制备是组织工程技术成功运用的关键。从骨架的材料要求、常用的骨架材料、骨架的制备技术等几个方面对组织工程和生物降解支架的研究进行了综述 ,并对该研究的前景进行了展望     

Improvement of compressive strength of porous hydroxyapatite scaffolds by adding polystyrene to camphene-based slurries

The compressive strength of porous hydroxyapatite (HA) scaffolds was enhanced by adding polystyrene (PS) polymer as a binder to hydroxyapatite (HA)/camphene slurries. As the PS content was increased from 0 to 20 vol.% in relation to the HA content, the compressive strength was significantly increased from 1.1 ± 0.2 to 2.3 ± 0.5 MPa, while the pore size was decreased from 277 ± 47 to 170 ± 29 µm. The improvement in the compressive strength was mainly attributed to both the suppression of the cracking of the green sample during freeze drying and the mitigation of the formation of micro-pores in the HA walls.     

骨组织工程用多孔HA生物材料的制备

用PVB、NH4HCO3和(NH4)2CO3粒子作造孔剂,制备了骨组织工程用多孔HA生物材料.讨论了烧结工艺和造孔剂含量等对材料结构的影响.研究表明,较佳的烧结工艺为1200℃烧结4h,烧结后样品主要是HA相.造孔剂PVB、(NH4)2CO3、NH4HCO3含量分别为10vol%、15vol%和20vol%时,多孔HA陶瓷拥有大于100μm和5～50 μm的贯通孔,具有较好的孔连通性与孔结构,有利于细胞和组织的生长以及营养输送;其最大孔隙率为50.3%,抗压强度为6.33MPa.     

Histological aspects in bone regeneration of an association with porous hydroxyapatite and bone marrow cells

The osteogenic potential of an association of two kinds of hydroxyapatite (HA) porous ceramics with autologous bone marrow cells cultured with or without dexamethasone (10^-8M) addition in the culture medium and non-cultured rabbit marrow stromal stem cells (MSCs) was tested after 4 weeks of implantation in the dorsal muscles of spine in rabbit. A significantly higher number of rabbits with implants containing bone tissue inside pores were obtained with 10⁷ cells ml^-1 cultured treated with Dex. In the HA porous ceramic using naphtalen as porogen agent, the bone recolonization remains only at the periphery of implants and in the second row of pores, while in the HA porous ceramic using polymethacrylate (PMMA) microbeads as porogen agent, the bone recolonization is observed in the depth of implants. In the PMMA HA group, the Kru¨skal–Wallis variance analysis between the rabbits is significantly different with the percentage of number of occupied pores and occupied pores with bone tissue is different (p < 0.05).