Performance of degradable composite bone repair products made via three-dimensional fabrication techniques

This study analyzed the in vivo performance of composite degradable bone repair products fabricated using the TheriForm process, a solid freeform fabrication (SFF) technique, in a rabbit calvarial defect model at 8 weeks. Scaffolds were composed of polylactic-co-glycolic acid (PLGA) polymer with 20% w/w beta-tricalcium phosphate (beta-TCP) ceramic with engineered macroscopic channels, a controlled porosity gradient, and a controlled pore size for promotion of new bone ingrowth. Scaffolds with engineered macroscopic channels and a porosity gradient had higher percentages of new bone area compared to scaffolds without engineered channels. These scaffolds also had higher percentages of new bone area compared to unfilled control defects, suggesting that scaffold material and design combinations could be tailored to facilitate filling of bony defects. This proof-of-concept study demonstrated that channel size, porosity, and pore size can be controlled and used to influence new bone formation and calvarial defect healing.     

MicroCT analysis of hydroxyapatite bone repair scaffolds created via three-dimensional printing for evaluating the effects of scaffold architecture on bone ingrowth

Recent studies have shown that it is now possible to construct tissue-engineered bone repair scaffolds with tight pore size distributions and controlled geometries using 3-D Printing techniques (3DP). This study evaluated two hydroxyapatite (HA) 8-mm diameter discs with controlled architectures in a rabbit trephine defect at 8 and 16 weeks using a 2 x 2 factorial design. Input parameters were time and scaffold void volume at two levels. Three output variables were extracted from MicroCT data: bone volume ingrowth with respect to total region of interest, bone volume ingrowth with respect to available ingrowth volume, and soft tissue volume. The experiment measured two groups--Group 1: 500-microm x 500-microm channels parallel to the scaffold's long axis and penetrating up 3-mm from the bottom. Group 2: 800-microm x 800-microm struts spaced 500 microm apart set perpendicularly to each other in each printed layer. Rendered 3-dimensional MicroCT scans and undecalcified histological slides of implants revealed good integration with the surrounding tissue, and a sizeable amount of bone ingrowth into the device. Factorial analysis revealed that the effects of time were the greatest determinant of soft tissue ingrowth, while time and its interaction with void volume were the greatest determinants of bone volume ingrowth with respect to both total and available volume.     

In vivo study on hydroxyapatite scaffolds with trabecular architecture for bone repair

The objective of this research was to investigate the bone formation and angio-conductive potential of hydroxyapatite (HA) scaffolds closely matched to trabecular bone in a canine segmental defect after 3 and 12 weeks post implantation. Histomorphometric comparisons were made between naturally forming trabecular bone (control) and defects implanted with scaffolds fabricated with micro-size (M-HA) and nano-size HA (N-HA) ceramic surfaces. Scaffold architecture was similar to trabecular bone formed in control defects at 3 weeks. No significant differences were identified between the two HA scaffolds; however, significant bone in-growth was observed by 12 weeks with 43.9 +/- 4.1% and 50.4 +/- 8.8% of the cross-sectional area filled with mineralized bone in M-HA and N-HA scaffolds, respectively. Partially organized, lamellar collagen fibrils were identified by birefringence under cross-polarized light at both 3 and 12 weeks post implantation. Substantial blood vessel infiltration was identified in the scaffolds and compared with the distribution and diameter of vessels in the surrounding cortical bone. Vessels were less numerous but significantly larger than native cortical Haversian and Volkmann canals reflecting the scaffold architecture where open spaces allowed interconnected channels of bone to form. This study demonstrated the potential of trabecular bone modeled, highly porous and interconnected, HA scaffolds for regenerative orthopedics.     

Solid freeform fabrication of bone tissue engineering scaffolds

INTRODUCTION　　 Tissue engineering is a promising approach to large segmental bone repair fortrauma,replacement surgery,skeletal deficiency or abnormal development. Thefabrication of boneregeneration scaffoldswith appropriate bone conductive property,bone inductive property,biodegradation property and mechanical properties is thecrux of this approach. Traditional scaffold fabrication technologies include phaseseparation technology,salt-leaching technology and gas-induced foaming technolo-…     

Freeze casting of hydroxyapatite scaffolds for bone tissue engineering

Although extensive efforts have been put into the development of porous scaffolds for bone regeneration, with encouraging results, all porous materials have a common limitation: the inherent lack of strength associated with porosity. Hence, the development of porous hydroxyapatite scaffolds has been hindered to non-load bearing applications. We report here how freeze casting can be applied to synthesize porous scaffolds exhibiting unusually high compressive strength, e.g. up to 145 MPa for 47% porosity and 65 MPa for 56% porosity. The materials are characterized by well-defined pore connectivity along with directional and completely open porosity. Various parameters affecting the porosity and compressive strength have been investigated, including initial slurry concentration, freezing rate, and sintering conditions. The implications and potential application as bone substitute are discussed. These results might open the way for hydroxyapatite-based materials designed for load-bearing applications. The biological response of these materials is yet to be tested.     

Synthesis of macroporous hydroxyapatite scaffolds for bone tissue engineering

A novel method of preparing macroporous hydroxyapatite (HA) by dual-phase mixing was developed: HA slurry and Polymethylmethacrylate (PMMA) resin were mixed together at the volume ratio of 1:1. After pyrolytic removal of the PMMA phase, HA with an open porous structure was obtained. In this way, the porosity of the ceramic was limited to 50%. Attempts to increase the porosity by adding more PMMA resin were confronted with the technical hurdle of sample collapse during the pyrolysis process. To increase the porosity and to improve pore interconnection, an extra foaming step was introduced before the polymerization of PMMA resin. Three foaming agent systems were tried, based on the reactions of citric acid and (bi)carbonate salts: sodium bicarbonate, calcium carbonate, and ammonium bicarbonate. Although all the three foaming agents were able to increase the porosity up to 70%, keeping all the pores interconnected throughout, only ammonium bicarbonate system turned out to be applicable to make HA scaffolds or implants, because both NaHCO(3) and CaCO(3) systems caused alkalic residues in the final ceramic. The porous HA samples were fully characterized by FTIR, XRD, ESEM (EDX), and optical microscopy.     

Solid freeform fabrication of three-dimensional scaffolds for engineering replacement tissues and organs

Most tissue engineering (TE) strategies for creating functional replacement tissues or organs rely on the application of temporary three-dimensional scaffolds to guide the proliferation and spread of seeded cells in vitro and in vivo. The characteristics of TE scaffolds are major concerns in the quest to fabricate ideal scaffolds. This paper identifies essential structural characteristics and the pre-requisites for fabrication techniques that can yield scaffolds that are capable of directing healthy and homogeneous tissue development. Emphasis is given to solid freeform (SFF), also known as rapid prototyping, technologies which are fast becoming the techniques of choice for scaffold fabrication with the potential to overcome the limitations of conventional manual-based fabrication techniques. SFF-fabricated scaffolds have been found to be able to address most, if not all the macro- and micro-architectural requirements for TE applications. This paper reviews the application/potential application of state-of-the-art SFF fabrication techniques in creating TE scaffolds. The advantages and limitations of the SFF techniques are compared. Related research carried out worldwide by different institutions, including the authors' research are discussed.     

Silk as a biocohesive sacrificial binder in the fabrication of hydroxyapatite load bearing scaffolds

Limitations of current clinical methods for bone repair continue to fuel the demand for a high strength, bioactive bone replacement material. Recent attempts to produce porous scaffolds for bone regeneration have been limited by the intrinsic weakness associated with high porosity materials. In this study, ceramic scaffold fabrication techniques for potential use in load-bearing bone repairs have been developed using naturally derived silk from Bombyx mori. Silk was first employed for ceramic grain consolidation during green body formation, and later as a sacrificial polymer to impart porosity during sintering. These techniques allowed preparation of hydroxyapatite (HA) scaffolds that exhibited a wide range of mechanical and porosity profiles, with some displaying unusually high compressive strength up to 152.4 ± 9.1 MPa. Results showed that the scaffolds exhibited a wide range of compressive strengths and moduli (8.7 ± 2.7 MPa to 152.4 ± 9.1 MPa and 0.3 ± 0.1 GPa to 8.6 ± 0.3 GPa) with total porosities of up to 62.9 ± 2.7% depending on the parameters used for fabrication. Moreover, HA-silk scaffolds could be molded into large, complex shapes, and further machined post-sinter to generate specific three-dimensional geometries. Scaffolds supported bone marrow-derived mesenchymal stem cell attachment and proliferation, with no signs of cytotoxicity. Therefore, silk-fabricated HA scaffolds show promise for load bearing bone repair and regeneration needs.     

Effect of different hydroxyapatite incorporation methods on the structural and biological properties of porous collagen scaffolds for bone repair

Scaffolds which aim to provide an optimised environment to regenerate bone tissue require a balance between mechanical properties and architecture known to be conducive to enable tissue regeneration, such as a high porosity and a suitable pore size. Using freeze‐dried collagen‐based scaffolds as an analogue of native ECM, we sought to improve the mechanical properties by incorporating hydroxyapatite (HA) in different ways while maintaining a pore architecture sufficient to allow cell infiltration, vascularisation and effective bone regeneration. Specifically we sought to elucidate the effect of different hydroxyapatite incorporation methods on the mechanical, morphological, and cellular response of the resultant collagen‐HA scaffolds. The results demonstrated that incorporating either micron‐sized (CHA scaffolds) or nano‐sized HA particles (CnHA scaffolds) prior to freeze‐drying resulted in moderate increases in stiffness (2.2‐fold and 6.2‐fold, respectively, vs. collagen‐glycosaminoglycan scaffolds, P < 0.05, a scaffold known to support osteogenesis), while enabling good cell attachment, and moderate mesenchymal stem cell (MSC)‐mediated calcium production after 28 days'' culture (2.1‐fold, P < 0.05, and 1.3‐fold, respectively, vs. CG scaffolds). However, coating of collagen scaffolds with a hydroxyapatite precipitate after freeze‐drying (CpHA scaffolds) has been shown to be a highly effective method to increase the compressive modulus (26‐fold vs. CG controls, P < 0.001) of scaffolds while maintaining a high porosity (~ 98%). The coating of the ligand‐dense collagen structure results in a lower cell attachment level (P < 0.05), although it supported greater cell‐mediated calcium production (P < 0.0001) compared with other scaffold variants after 28 days'' culture. The comparatively good mechanical properties of these high porosity scaffolds is obtained partially through highly crosslinking the scaffolds with both a physical (DHT) and chemical (EDAC) crosslinking treatment. Control of scaffold microstructure was examined via alterations in freezing temperature. It was found that the addition of HA prior to freeze‐drying generally reduced the pore size and so the CpHA scaffold fabrication method offered increased control over the resulting scaffolds microstructure. These findings will help guide future design considerations for composite biomaterials and demonstrate that the method of HA incorporation can have profound effects on the resulting scaffold structural and biological response.     

Evaluation of three-dimensional scaffolds made of blends of hydroxyapatite and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) for bone reconstruction

Hydroxyapatite (HAP) was blended into poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) to make films and scaffolds. After HAP blending, mechanical properties of PHB including compressive elastic modulus and maximum stress showed improvement and osteoblast responses including cell growth and alkaline phosphatase activity were also strengthened. On the other hand, scaffolds made of PHBHHx blended with HAP had an adverse effect. No remarkable change on degradation of PHB or PHBHHx blended with HAP, respectively, was observed in simulated body fluid. Scanning electron microscopy examination revealed that osteoblast responses to HAP incorporation may be related to surface morphology and to the exposed HAP particles on polymer surface. All these results indicated that the blending of HAP particles into PHBHHx scaffolds fabricated by salt leaching was unable to either strengthen its mechanical properties or enhance osteoblast responses. Although HAP is bioactive and osteoconductive, its blending with PHBHHx did not generate a better performance on bone reconstruction.     

Fabrication of three-dimensional polycaprolactone/hydroxyapatite tissue scaffolds and osteoblast-scaffold interactions in vitro

Computer-aided tissue-engineering approach was used to develop a novel precision extrusion deposition (PED) process to directly fabricate Polycaprolactone (PCL) and composite PCL/hydroxyapatite (PCL-HA) tissue scaffolds. The process optimization was carried out to fabricate both PCL and PCL-HA (25% concentration by weight of HA) with a controlled pore size and internal pore structure of the 0 degrees /90 degrees pattern. Two groups of scaffolds having 60% and 70% porosity and with pore sizes of 450 and 750 microm, respectively, were evaluated for their morphology and compressive properties using scanning electron microscopy (SEM) and mechanical testing. Our results suggested that inclusion of HA significantly increased the compressive modulus from 59 to 84 MPa for 60% porous scaffolds and from 30 to 76 MPa for 70% porous scaffolds. In vitro cell-scaffolds interaction study was carried out using primary fetal bovine osteoblasts to assess the feasibility of scaffolds for bone tissue-engineering application. The cell proliferation and differentiation were calculated by Alamar Blue assay and by determining alkaline phosphatase activity. The osteoblasts were able to migrate and proliferate over the cultured time for both PCL as well as PCL-HA scaffolds. Our study demonstrated the viability of the PED process to the fabricate PCL and PCL-HA composite scaffolds having necessary mechanical property, structural integrity, controlled pore size and pore interconnectivity desired for bone tissue engineering.     

Preparation and histological evaluation of biomimetic three-dimensional hydroxyapatite/chitosan-gelatin network composite scaffolds

A novel biodegradable hydroxyapatite/chitosan-gelatin network (HA/CS-Gel) composite of similar composition to that of normal human bone was prepared as a three-dimensional biomimetic scaffold by phase separation method for bone tissue engineering. Changing the solid content and the compositional variables of the original mixtures allowed control of the porosities and densities of the scaffolds. The HA granules were dispersed uniformly in the organic network with intimate interface contact via pulverizing and ultrasonically treating commercial available HA particles. Scaffolds of 90.6% porosity were used to examine the proliferation and functions of the cells in this three-dimensional microenvironment by culturing neonatal rat caldaria osteoblasts. Histological and immunohistochemical staining and scanning electron microscopy observation indicated that the osteoblasts attached to and proliferated on the scaffolds. Extracellular matrices including collagen I and proteoglycan-like substrate were synthesized, while osteoid and bone-like tissue formed during the culture period. Furthermore, the cell/scaffold constructs had good biomineralization effect after 3 weeks in culture.     

Poly(lactide-co-glycolide)/hydroxyapatite composite scaffolds for bone tissue engineering

Biodegradable polymer/bioceramic composite scaffolds can overcome the limitations of conventional ceramic bone substitutes such as brittleness and difficulty in shaping. However, conventional methods for fabricating polymer/bioceramic composite scaffolds often use organic solvents (e.g., the solvent casting and particulate leaching (SC/PL) method), which might be harmful to cells or tissues. Furthermore, the polymer solutions may coat the ceramics and hinder their exposure to the scaffold surface, which may decrease the likelihood that the seeded osteogenic cells will make contact with the bioactive ceramics. In this study, a novel method for fabricating a polymer/nano-bioceramic composite scaffold with high exposure of the bioceramics to the scaffold surface was developed for efficient bone tissue engineering. Poly(D,L-lactic-co-glycolic acid)/nano-hydroxyapatite (PLGA/HA) composite scaffolds were fabricated by the gas forming and particulate leaching (GF/PL) method without the use of organic solvents. The GF/PL method exposed HA nanoparticles at the scaffold surface significantly more than the conventional SC/PL method does. The GF/PL scaffolds showed interconnected porous structures without a skin layer and exhibited superior enhanced mechanical properties to those of scaffolds fabricated by the SC/PL method. Both types of scaffolds were seeded with rat calvarial osteoblasts and cultured in vitro or were subcutaneously implanted into athymic mice for eight weeks. The GF/PL scaffolds exhibited significantly higher cell growth, alkaline phosphatase activity, and mineralization compared to the SC/PL scaffolds in vitro. Histological analyses and calcium content quantification of the regenerated tissues five and eight weeks after implantation showed that bone formation was more extensive on the GF/PL scaffolds than on the SC/PL scaffolds. Compared to the SC/PL scaffolds, the enhanced bone formation on the GF/PL scaffolds may have resulted from the higher exposure of HA nanoparticles at the scaffold surface, which allowed for direct contact with the transplanted cells and stimulated the cell proliferation and osteogenic differentiation. These results show that the biodegradable polymer/bioceramic composite scaffolds fabricated by the novel GF/PL method enhance bone regeneration compared with those fabricated by the conventional SC/PL method.     

Human bone marrow stem cell-encapsulating calcium phosphate scaffolds for bone repair

Due to its injectability and excellent osteoconductivity, calcium phosphate cement (CPC) is highly promising for orthopedic applications. However, a literature search revealed no report on human bone marrow mesenchymal stem cell (hBMSC) encapsulation in CPC for bone tissue engineering. The aim of this study was to encapsulate hBMSCs in alginate hydrogel beads and then incorporate them into CPC, CPC–chitosan and CPC–chitosan–fiber scaffolds. Chitosan and degradable fibers were used to mechanically reinforce the scaffolds. After 21 days, that the percentage of live cells and the cell density of hBMSCs inside CPC-based constructs matched those in alginate without CPC, indicating that the CPC setting reaction did not harm the hBMSCs. Alkaline phosphate activity increased by 8-fold after 14 days. Mineral staining, scanning electron microscopy and X-ray diffraction confirmed that apatitic mineral was deposited by the cells. The amount of hBMSC-synthesized mineral in CPC–chitosan–fiber matched that in CPC without chitosan and fibers. Hence, adding chitosan and fibers, which reinforced the CPC, did not compromise hBMSC osteodifferentiation and mineral synthesis. In conclusion, hBMSCs were encapsulated in CPC and CPC–chitosan–fiber scaffolds for the first time. The encapsulated cells remained viable, osteodifferentiated and synthesized bone minerals. These self-setting, hBMSC-encapsulating CPC-based constructs may be promising for bone tissue engineering applications.     

3D打印钛合金孔隙支架骨长入影响因素的分析

BACKGROUND: With the development of three-dimensional (3D) printing technology, 3D printed porous titanium scaffolds as bone substitutes have become a research hotspot. OBJECTIVE: To introduce and discuss the effects of each parameter of 3D printed porous titanium scaffolds on bone ingrowth, and to sum out the optimal parameters for bone ingrowth. METHODS: The first author retrieved PubMed, Springerlink and Medline databases with “three-dimensional (3D) printing, scaffold, titanium, bone ingrowth” as keywords for relevant articles published from 2006 to 2016. 125 articles were retrieved initially, and finally 42 eligible articles were included for analysis. RESULTS AND CONCLUSION: Pore size, porosity, pore structures and surface modifications of 3D printed porous titanium scaffolds all make effects on bone ingrowth or osteoblasts in scaffolds. Scaffolds with appropriate pore size and porosity can promote the vascularization and provide adequate nutrition and oxygen supplement, to ensure high cell viability. Regulations of cell performances, such as cell attachment, proliferation and differentiation, are also affected by pore structures and nano-scale surface modification. Herein, a detailed combination of the parameters, as mentioned above, can create a better porous scaffold for better bone ingrowth. Hence, the high-stability interface between bone and scaffolds may be obtained through the parameter adjustment.     

The mechanical properties and osteoconductivity of hydroxyapatite bone scaffolds with multi-scale porosity

The relative osteoconductivity and the change in the mechanical properties of hydroxyapatite (HA) scaffolds with multi-scale porosity were compared to scaffolds with a single pore size. Non-microporous (NMP) scaffolds contained only macroporosity (250-350 microm) and microporous (MP) scaffolds contained both macroporosity and microporosity (2-8 microm). Recombinant human bone morphogenetic protein-2 (rhBMP-2) was incorporated into all scaffolds via gelatin microspheres prior to implantation into the latissimus dorsi muscle of Yorkshire pigs. After 8 weeks, only the MP scaffolds contained bone. The result demonstrates the efficacy of the MP scaffolds as drug carriers. Implanted and as-fabricated scaffolds were compared using histology, microcomputed tomography, scanning electron microscopy, and compression testing. Implanted scaffolds exhibited a stress-strain response similar to that of cancellous bone with strengths between those of cancellous and cortical bone. The strength and stiffness of implanted NMP scaffolds decreased by 15% and 46%, respectively. Implanted MP scaffolds lost 30% of their strength and 31% of their stiffness. Bone arrested crack propagation effectively in MP scaffolds. The change in mechanical behavior is discussed and the study demonstrates the importance of scaffold microporosity on bone ingrowth and on the mechanical behavior of HA implant materials.     

Multiscale three-dimensional scaffolds for soft tissue engineering via multimodal electrospinning

A novel (scalable) electrospinning process was developed to fabricate bio-inspired multiscale three-dimensional scaffolds endowed with a controlled multimodal distribution of fiber diameters and geared towards soft tissue engineering. The resulting materials finely mingle nano- and microscale fibers together, rather than simply juxtaposing them, as is commonly found in the literature. A detailed proof of concept study was conducted on a simpler bimodal poly(ε-caprolactone) (PCL) scaffold with modes of fiber distribution at 600 nm and 3.3 μm. Three conventional unimodal scaffolds with mean diameters of 300 nm and 2.6 and 5.2 μm, respectively, were used as controls to evaluate the new materials. Characterization of the microstructure (i.e. porosity, fiber distribution and pore structure) and mechanical properties (i.e. stiffness, strength and failure mode) indicated that the multimodal scaffold had superior mechanical properties (Young’s modulus ～40 MPa and strength ～1 MPa) in comparison with the controls, despite the large porosity (～90% on average). A biological assessment was conducted with bone marrow stromal cell type (mesenchymal stem cells, mTERT-MSCs). While the new material compared favorably with the controls with respect to cell viability (on the outer surface), it outperformed them in terms of cell colonization within the scaffold. The latter result, which could neither be practically achieved in the controls nor expected based on current models of pore size distribution, demonstrated the greater openness of the pore structure of the bimodal material, which remarkably did not come at the expense of its mechanical properties. Furthermore, nanofibers were seen to form a nanoweb bridging across neighboring microfibers, which boosted cell motility and survival. Lastly, standard adipogenic and osteogenic differentiation tests served to demonstrate that the new scaffold did not hinder the multilineage potential of stem cells.     

壳聚糖-脱细胞真皮三维材料作为骨组织工程支架材料的研究

目的观察MC3T3-El成骨前体细胞在壳聚糖-脱细胞真皮三维支架材料上的黏附情况,并评价其细胞相容性。方法通过冷冻干燥制备壳聚糖-脱细胞真皮三维支架材料,并测试其孔隙率、密度和吸水率,通过扫描电镜分析支架的微观形貌。采用体外培养细胞的方法,将MC3T3-E1细胞直接接种到壳聚糖-脱细胞真皮三维支架材料上,培养2,3,4,5h,各时间点各取3个样品,测定细胞在支架上的黏附率,确定最佳的细胞贴壁时间。将细胞接种到支架上,共培养1,3,5,7,9,11,13d,采用MTS方法绘制细胞增殖曲线,组织化学染色观察细胞形态,并利用材料试验机测试不同时间材料细胞复合物的压缩弹性模量。结果壳聚糖-脱细胞真皮材料具有连通的多孔结构,孔隙率为92.8%,密度为97.96g/L,吸水率为(2169±100)%。细胞相容性实验显示,成骨细胞易于在支架材料上黏附、增殖。结论壳聚糖-脱细胞真皮材料具有连通的孔隙,孔径较均匀,MC3T3-El成骨前体细胞易在壳聚糖-脱细胞真皮三维支架材料上黏附、增殖,表明该支架材料具有良好的细胞相容性。     

3D scaffolds for bone marrow stem cell support in bone repair

Bone tissue repair is one of the major concerns of regenerative medicine. The current need for tissue replacements has necessitated the development of a new science termed 'bone tissue engineering'. The basic organization of bone tissue requires the design and fabrication of a porous 3D structure or 'scaffold' to contain the bone-forming cells. This scaffold should be formulated from biocompatible, osteoconductive materials that are not immunoreactive. 3D scaffolds provide the necessary support for cells to proliferate and maintain their capacity to differentiate and scaffolds containing bone marrow-derived osteoprogenitors can be employed within implants to enhance bone repair. The complex construct is intended to mimic the native in vivo microenvironment and this demands construction of bioactive scaffolds that are also capable of supporting vascularization as well as cell proliferation and osteogenic differentiation. 3D bioactive scaffolds containing committed osteoprogenitors can provide a promising surgical tool for bone tissue engineering directed at orthopedic and cranio-maxillofacial clinical applications.