Mineralised collagen—an artificial,extracellular bone matrix—improves osteogenic differentiation of bone marrow stromal cells

In the field of bone tissue engineering there is a high demand on bone graft materials which promote bone formation. By combination of collagen type I with nanocrystalline hydroxyapatite (HA) we generated a resorbable material which structure and composition is close to those of the extracellular bone matrix. This nanocomposit material was produced in a biomimetic process in which collagen fibril assembly and mineralisation with hydroxyapatite occur simultaneously. In this study the proliferation and osteogenic differentiation of human bone marrow-derived stromal cells (hBMSC) on membranes of biomimetically mineralised collagen type I was investigated. To this end, we optimised biochemical assays for determination of cell number and alkaline phosphatase activity corresponding to the special properties of this biomaterial. For cell experiments hBMSC were seeded on the mineralised collagen membranes and cultivated over 35 days, both in static and perfusion culture, in the presence and absence of dexamethasone, β-glycerophosphate and ascorbate. Compared to cells grown on tissue culture polystyrene we found attenuated proliferation rates, but markedly increased activity of alkaline phosphatase on the mineralised collagen indicating its promoting effect on the osteogenic differentiation of hBMSC. Therefore this bone-like material may act as a suitable artificial extracellular matrix for bone tissue engineering. Perfusion of the 2D cell matrix constructs with cell culture medium did not improve proliferation and osteogenic differentiation of the hBMSC. Anne Bernhardt and Anja Lode contributed equally to this paper     

Mineralised tissues as nanomaterials: analysis by atomic force microscopy

Mineralised tissues, such as bone, consist of two material phases: collagen protein fibrils that form the structural models upon which the mineral, calcium hydroxyapatite, is subsequently deposited. Collagen and mineral are removed in a three-dimensional manner by osteoclasts during bone turnover in skeletal growth or repair, and matrix proteins are replaced by the synthetic activity of osteoblasts and then calcify. The resolution of atomic force microscopy and use of unmodified, fully calcified samples has enabled the imaging of the overall bone and dentine structure, including collagen and mineral phases. Mineral crystals, in the diameter size range of 225 nm up to 1.4 microm, were found in unmodified bone and dentine respectively. D-banded collagen is observed in dentine after acid treatment and in bone after osteoclast-mediated matrix resorption; axial periodicity values of approximately 67 and 69 nm are observed, respectively. These experimental approaches have enabled the structure of mineralised tissues to be examined in native samples and will facilitate the study of bone structure in important clinical disorders of the skeleton, such as osteoporosis.     

Preparation and characterization of bioactive collagen/wollastonite composite scaffolds

A novel biodegradable collagen/wollastonite composite was prepared as three-dimensional scaffolds by freeze-drying method. Scanning electron microscope (SEM) micrographs of scaffolds showed a continuous structure of interconnected pores, and pore size was about 100 m. The tensile strength of the scaffolds was improved by incorporation of wollastonite and the in vitro bioactivity of the scaffolds was evaluated by examining the hydroxyapatite (HA) deposition on their surface in simulated body fluid (SBF). After soaking in SBF for 7 days, collagen reconstituted to fibers and HA nodules formed on collagen fibers. The result suggests that the incorporation of wollastonite could improve the mechanical strength and the in vitro bioactivity of the composite. The scaffolds could be a potential biomaterial for bone tissue engineering.     

Controlling ion release from bioactive glass foam scaffolds with antibacterial properties

Bioactive glass scaffolds have been produced, which meet many of the criteria for an ideal scaffold for bone tissue engineering applications, by foaming sol-gel derived bioactive glasses. The scaffolds have a hierarchical pore structure that is very similar to that of cancellous bone. The degradation products of bioactive glasses have been found to stimulate the genes in osteoblasts. This effect has been found to be dose dependent. The addition of silver ions to bioactive glasses has also been investigated to produce glasses with bactericidal properties. This paper discusses how changes in the hierarchical pore structure affect the dissolution of the glass and therefore its bioactivity and rate of ion delivery and demonstrates that silver containing bioactive glass foam scaffolds can be synthesised. It was found that the rate of release of Si and Ca ions was more rapid for pore structures with a larger modal pore diameter, although the effect of tailoring the textural porosity on the rate of ion release was more pronounced. Bioactive glass scaffolds, containing 2 mol% silver, released silver ions at a rate that was similar to that which has previously been found to be bactericidal but not high enough to be cytotoxic to bone cells.     

Biomimetic Nanosilica–Collagen Scaffolds for In Situ Bone Regeneration: Toward a Cell‐Free,One‐Step Surgery

Current approaches to fabrication of nSC composites for bone tissue engineering (BTE) have limited capacity to achieve uniform surface functionalization while replicating the complex architecture and bioactivity of native bone, compromising application of these nanocomposites for in situ bone regeneration. A robust biosilicification strategy is reported to impart a uniform and stable osteoinductive surface to porous collagen scaffolds. The resultant nSC composites possess a native‐bone‐like porous structure and a nanosilica coating. The osteoinductivity of the nSC scaffolds is strongly dependent on the surface roughness and silicon content in the silica coating. Notably, without the use of exogenous cells and growth factors (GFs), the nSC scaffolds induce successful repair of a critical‐sized calvarium defect in a rabbit model. It is revealed that topographic and chemical cues presented by nSC scaffolds could synergistically activate multiple signaling pathways related to mesenchymal stem cell recruitment and bone regeneration. Thus, this facile surface biosilicification approach could be valuable by enabling production of BTE scaffolds with large sizes, complex porous structures, and varied osteoinductivity. The nanosilica‐functionalized scaffolds can be implanted via a cell/GF‐free, one‐step surgery for in situ bone regeneration, thus demonstrating high potential for clinical translation in treatment of massive bone defects.     

Novel 3D scaffold with enhanced physical and cell response properties for bone tissue regeneration,fabricated by patterned electrospinning/electrospraying

Developing three dimensional scaffolds mimicking the nanoscale structure of native extracellular matrix is a key parameter in tissue regeneration. In this study, we aimed to introduce a novel 3D structures composed of nanofibers (NF) and micro particles (MP) and compare their efficiency with 2D nanofibrous scaffold. The conventional nanofibrous PCL scaffolds are 2D mats fabricated by the electrospinning technique, whereas the NF/MP and patterned NF/MP PCL scaffolds are three dimensional structures fabricated by a modified electrospinning/electrospraying technique. The mentioned method was carried out by varying the electrospinning solution parameters and use of a metal mesh as the collector. Detailed fabrication process and morphological properties of the fabricated structures is discussed and porosity, pore size and PBS solution absorption value of the prepared structures are reported. Compared with the 2D structure, 3D scaffolds possessed enhanced porosity and pore size which led to the significant increase in their water uptake capacity. In vitro cell experiments were carried out on the prepared structures by the use of MG-63 osteosarcoma cell line. The fabricated 3D structures offered significantly increased cell attachment, spread and diffusion which were confirmed by SEM analysis. In vitro cytocompatibility assessed by MTT colorimetric assay indicated a continuous cell proliferation over 21 days on the innovative 3D structure, while on 2D mat cell proliferation stopped at early time points. Enhanced osteogenic differentiation of the seeded MG-63 cells on 3D scaffold was confirmed by the remarkable ALP activity together with increased and accelerated calcium deposition on this structure compared to 2D mat. Massive and well distributed bone minerals formed on patterned 3D structure were shown by EDX analysis. In comparison between NF/MP quasi-3D and Patterned NF/MP 3D scaffolds, patterned structures proceeded in all of the above properties. As such, the innovative Patterned NF/MP 3D scaffold could be considered as a proper bone graft substitute for bone tissue regeneration.     

An <Emphasis Type="Italic">in vivo</Emphasis> study of a bone grafting material consisting of hydroxyapatite and reconstituted collagen

This study aims to evaluate the performance of our recently developed microspheres of hydroxyapatite/reconstituted collagen as a bone grafting material. The microspheres were fabricated into a circular disc and implanted in a pre-drilled hole in a rats calvaria. The bone tissue had regenerated and grown into the disc bone graft 4 weeks following implantation. After 16 weeks of implantation, the regenerated bone had integrated with the remaining material and made close contact with it. The disc had been completely absorbed with almost no visible bone graft left after 24 weeks of implantation. In contrast, a hydroxyapatite disc still remained intact on the 24th week after implantation. These results suggested that the hydroxyapatite/reconstituted collagen microsphere can be used as an excellent bone grafting material.     

骨组织工程用硅胶/掺锶β-磷酸三钙/硫酸钙复合多孔支架的制备与性能研究

以掺锶β-磷酸三钙/硫酸钙为原料,利用搅拌喷雾干燥法制备出掺锶β-磷酸三钙/硫酸钙复合小球,再将硅胶与制备的复合小球复合,通过在模具中堆垛聚集的方法,制备出硅胶/掺锶β-磷酸三钙/硫酸钙复合生物支架。采用XRD,SEM,FT-IR等方法分析制得复合多孔支架的成分、形貌以及结构特征,并研究复合生物支架的降解性、孔隙率、力学性能和细胞毒性等。结果表明:该复合多孔生物支架具有一定的不规则孔洞结构,小球与小球之间的孔隙约为0.2~1mm,而每个小球上也有大量的微孔,孔径在50~200μm之间,且平均孔隙率达到62%,基本能满足骨组织工程支架对孔隙率的要求;该复合多孔支架无细胞毒性,其降解周期约为80天,抗压强度约为0.1MPa,因此该支架在非承重骨组织修复方面具有良好的应用前景。     

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.     

Glass,Ceramic, Polymeric,and Composite Scaffolds with Multiscale Porosity for Bone Tissue Engineering

Porosity affects performance of scaffolds for bone tissue engineering both in vitro and in vivo. Macropores (i.e., pores with a diameter >100 μm) are essential for cellular infiltration; micropores (i.e., pores with a diameter of 1–10 μm) promote cell adhesion and facilitate nutrient absorption. Scaffolds containing both macropores and micropores exploit the advantages of both pore sizes and have excellent osteogenic properties. Nanopores (i.e., pores with a diameter of 1–50 nm) can be included as well, to improve cell–material interactions by further enhancing the surface area of the scaffold. This article reviews fabrication techniques and properties of scaffolds with multiscale porosity, focusing on glass, ceramic, polymeric, and composite scaffolds. After discussing the structure of bone and how it inspired scaffolds for bone tissue engineering, pore nomenclature is introduced. Then, the techniques used to induce multiscale porosity, the nature of the pores created, and the effects of scaffold porosity on mechanical properties and biological activity of the scaffolds are discussed. The review concludes by providing an outlook for this field, including advancements that are made possible by computational modeling and artificial intelligence.     

Providing osteogenesis conditions to mesenchymal stem cells using bioactive nanocomposite bone scaffolds

Development of bone scaffolds with excellent osteogenic potential is highly important for stem cell-based bone engineering. Here we developed novel scaffolds made of poly(lactic acid) (PLA) biopolymer with bioactive glass nanocomponent. In vitro bone bioactivity and osteogenic potential of the nanocomposite scaffolds were determined using bone marrow mesenchymal stem cells. Glass nanocomponent was evenly embedded within the PLA matrix while preserving the scaffold pore structure. Simulated body fluid (SBF) test revealed rapid induction of bone mineral-like apatite over the surface of the nanocomposite scaffold, which was not readily observed in the PLA. Cells adhered well onto the nanocomposite scaffold and multiplied during culture period. Nanocomposite scaffold significantly stimulated alkaline phosphatase (ALP) activity and the expression of bone-associated genes (collagen I, ALP, osteopontin and osteocalcin) with respect to PLA. Western blot analysis confirmed the osteogenic protein level was also higher on the nanocomposite scaffold. Results suggest that the nanocomposite scaffolds provide favorable conditions for osteogenesis of MSCs and thus find potential uses in bone tissue engineering.     

分级多孔壳聚糖/聚乳酸复合支架的制备及骨修复性能

为了仿生莲藕内部的贯穿大孔结构,以生物相容性好的壳聚糖(CS)作为基质材料,利用冰粒致孔、石蜡模具和冰模具成型3种成型方法制备了分级多孔CS支架材料,然后与力学强度较高的聚乳酸(PLLA)复合,制备网络互穿CS/PLLA复合支架。通过SEM、压缩强度测试和兔股骨髁骨缺损模型对CS/PLLA复合材料的形貌、力学强度和骨修复性能进行了表征。结果表明:利用冰模具制备的CS/PLLA复合支架能可控、批量制备,具有微米-毫米分级多孔结构,大孔孔径约为2mm,内部均匀分布着孔径约为60μm的贯穿微孔,并在微孔内形成密集的PLLA絮状网络结构。干态复合材料的压缩强度和模量分别比纯CS支架的提高了6倍和15倍。体内植入实验结果表明,CS/PLLA复合材料能够促进骨缺损的愈合,并随着新骨的形成,复合材料逐渐被降解吸收。     

Scaffolds with a standardized macro-architecture fabricated from several calcium phosphate ceramics using an indirect rapid prototyping technique

Calcium phosphate ceramics, commonly applied as bone graft substitutes, are a natural choice of scaffolding material for bone tissue engineering. Evidence shows that the chemical composition, macroporosity and microporosity of these ceramics influences their behavior as bone graft substitutes and bone tissue engineering scaffolds but little has been done to optimize these parameters. One method of optimization is to place focus on a particular parameter by normalizing the influence, as much as possible, of confounding parameters. This is difficult to accomplish with traditional fabrication techniques. In this study we describe a design based rapid prototyping method of manufacturing scaffolds with virtually identical macroporous architectures from different calcium phosphate ceramic compositions. Beta-tricalcium phosphate, hydroxyapatite (at two sintering temperatures) and biphasic calcium phosphate scaffolds were manufactured. The macro- and micro-architectures of the scaffolds were characterized as well as the influence of the manufacturing method on the chemistries of the calcium phosphate compositions. The structural characteristics of the resulting scaffolds were remarkably similar. The manufacturing process had little influence on the composition of the materials except for the consistent but small addition of, or increase in, a beta-tricalcium phosphate phase. Among other applications, scaffolds produced by the method described provide a means of examining the influence of different calcium phosphate compositions while confidently excluding the influence of the macroporous structure of the scaffolds.     

Porous ceramic bone scaffolds for vascularized bone tissue regeneration

Hydroxyapatite scaffolds with a multi modal porosity designed for use in tissue engineering of vascularized bone graft substitutes were prepared by three dimensional printing. Depending on the ratio of coarse (mean particle size 50 microm) to fine powder (mean particle size 4 microm) in the powder granulate and the sintering temperature total porosity was varied from 30% to 64%. While macroscopic pore channels with a diameter of 1 mm were created by CAD design, porosity structure in the sintered solid phase was governed by the granulate structure of the printing powder. Scaffolds sintered at 1,250 degrees C were characterized by a bimodal pore structure with intragranular pores of 0.3-0.4 microm and intergranular pores of 20 microm whereas scaffolds sintered at 1,400 degrees C exhibit a monomodal porosity with a maximum of pore size distribution at 10-20 microm. For in-vivo testing, matrices were implanted subcutaneously in four male Lewis rats. Scaffolds with 50% porosity and an average pore size of approximately 18 microm were successfully transferred to rats and vascularized within 4 weeks.     

Controlling the processing of collagen-hydroxyapatite scaffolds for bone tissue engineering

Scaffolds are an important aspect of the tissue engineering approach to tissue regeneration. This study shows that it is possible to manufacture scaffolds from type I collagen with or without hydroxyapatite (HA) by critical point drying. The mean pore sizes of the scaffolds can be altered from 44 to 135 μm depending on the precise processing conditions. Such pore sizes span the range that is likely to be required for specific cells. The mechanical properties of the scaffolds have been measured and behave as expected of foam structures. The degradation rate of the scaffolds by collagenase is independent of pore size. Dehydrothermal treatment (DHT), a common method of physically crosslinking collagen, was found to denature the collagen at a temperature of 120^∘C resulting in a decrease in the scaffold’s resistance to collagenase. Hybrid scaffold structures have also been manufactured, which have the potential to be used in the generation of multi-tissue interfaces. Microchannels are neatly incorporated via an indirect solid freeform fabrication (SFF) process, which could aid in reducing the different constraints commonly observed with other scaffolds.     

Comparative evaluation of a biomimic collagen/hydroxyapatite/β-tricaleium phosphate scaffold in alveolar ridge preservation with Bio-Oss Collagen

Bone scaffolds are critical in current implant and periodontal regeneration approaches. In this study, we prepared a novel composite type-I collagen and hydroxyapatite (HA)/β-tricaleium phosphate (TCP) scaffold (CHTS) by incorporating type-I collagen and bovine calcined bone granules, prepared as a mixture of 50% HA and 50% TCP, by freeze drying. We then characterized the CHTS and determined its cytotoxic effects. Additionally, ridge preservation experiments were carried out to evaluate the clinical effects of the CHTS. The results demonstrated that the composite scaffolds had good surface morphology and no cytotoxicity. Additionally, an in vivo experiment in an animal model showed that the CHTS performed equally as well as Bio-Oss Collagen, a widely used bone graft in ridge preservation. These findings revealed that the CHTS, which contained natural constituents of bone, could be used as a scaffold for bone regeneration and clinical use.     

Ectopic bone formation in cell-seeded poly(ethylene oxide)/poly(butylene terephthalate) copolymer scaffolds of varying porosity

Scaffolds from poly(ethylene oxide) and poly(butylene terephthalate), PEOT/PBT, with a PEO molecular weight of 1,000 and a PEOT content of 70 weight% (1000PEOT70PBT30) were prepared by leaching salt particles (425–500 μm). Scaffolds of 73.5, 80.6 and 85.0% porosity were treated with a CO₂ gas plasma and seeded with rat bone marrow stromal cells (BMSCs). After in vitro culture for 7 days (d) in an osteogenic medium the scaffolds were subcutaneously implanted for 4 weeks in nude mice. Poly(d, l-lactide) (PDLLA) and biphasic calcium phosphate (BCP) scaffolds were included as references. After 4 weeks (wks) all scaffolds showed ectopic formation of bone and bone marrow. For the scaffolds of different porosities, no significant differences were observed in the relative amounts of bone (7–9%) and bone marrow (6–11%) formed, even though micro computed tomography (μ-CT) data showed considerable differences in accessible pore volume and surface area. 1000PEOT70PBT30 scaffolds with a porosity of 85% could not maintain their original shape in vivo. Surprisingly, 1000PEOT70PBT30 scaffolds with a porosity of 73.5% showed cartilage formation. This cartilage formation is most likely due to poorly accessible pores in the scaffolds, as was observed in histological sections. μ-CT data showed a considerably smaller accessible pore volume (as a fraction of the total volume) than in 1000PEOT70PBT30 scaffolds of 80.6 and 85.0% porosity. BMSC seeded PDLLA (83.5% porosity) and BCP scaffolds (29% porosity) always showed considerably more bone and bone marrow formation (bone marrow formation is approximately 40%) and less fibrous tissue ingrowth than the 1000PEOT70PBT30 scaffolds. The scaffold material itself can be of great influence. In more hydrophobic and rigid scaffolds like the PDLLA or BCP scaffolds, the accessibility of the pore structure is more likely to be preserved under the prevailing physiological conditions than in the case of hydrophilic 1000PEOT70PBT30 scaffolds. Scaffolds prepared from other PEOT/PBT polymer compositions, might prove to be more suited.     

Robotic dispensing of composite scaffolds and in vitro responses of bone marrow stromal cells

The development of bioactive scaffolds with a designed pore configuration is of particular importance in bone tissue engineering. In this study, bone scaffolds with a controlled pore structure and a bioactive composition were produced using a robotic dispensing technique. A poly(ε-caprolactone) (PCL) and hydroxyapatite (HA) composite solution (PCL/HA = 1) was constructed into a 3-dimensional (3D) porous scaffold by fiber deposition and layer-by-layer assembly using a computer-aided robocasting machine. The in vitro tissue cell compatibility was examined using rat bone marrow stromal cells (rBMSCs). The adhesion and growth of cells onto the robotic dispensed scaffolds were observed to be limited by applying the conventional cell seeding technique. However, the initially adhered cells were viable on the scaffold surface. The alkaline phosphatase activity of the cells was significantly higher on the HA–PCL than on the PCL and control culture dish, suggesting that the robotic dispensed HA–PCL scaffold should stimulate the osteogenic differentiation of rBMSCs. Moreover, the expression of a series of bone-associated genes, including alkaline phosphatase and collagen type I, was highly up-regulated on the HA–PCL scaffold as compared to that on the pure PCL scaffold. Overall, the robotic dispensed HA–PCL is considered to find potential use as a bioactive 3D scaffold for bone tissue engineering. Seok-Jung Hong and Ishik Jeong contributed equally.     

Stiffness Improvement of 45S5 Bioglass®‐Based Scaffolds Through Natural and Synthetic Biopolymer Coatings: An Ultrasonic Study

Due to its excellent bioactivity, 45S5 Bioglass^® is being highly considered in tissue engineering scaffold development. In order to enhance vascularization promoting tissue growth, these scaffolds typically have a highly interconnected porous structure with a porosity between 80 and >90%. Often, Bioglass^®‐based scaffolds of such a high porosity have insufficient stiffness. In order to increase the stiffness of Bioglass^®‐based scaffolds fabricated by the foam replica method, the herein investigated scaffolds were coated with a number of different biopolymers, including: collagen, gelatin, polycaprolactone (PCL), alginate and poly(l ‐lactic acid). The resulting stiffness gain was quantified by means of ultrasonic measurements. Accordingly, PCL and collagen coatings increased the scaffold stiffness, as compared to uncoated scaffolds, by 58 and 38%, respectively; while no remarkable stiffness increase was recorded for the other coatings. Additionally, scanning electron microscopy images of polymer coated scaffolds revealed that PCL coatings had not clogged the scaffold's micropores, which is deemed essential for cell seeding and to enable in‐growth of bone tissue. Thus, the application of PCL coatings represents a promising strategy for mechanical competence enhancement of Bioglass^®‐based scaffolds for bone tissue engineering.     

基于Micro-CT不同多孔结构陶瓷支架的建模及其贯通性与液流分布分析

贯通性是骨组织工程支架的重要参数, 决定包含蛋白和细胞的体液渗入和组织生长。本研究采用Micro-CT技术对三种不同工艺(球粒堆积、蜡球造孔、纤维堆积)构建的羟基磷灰石多孔支架进行断层扫描, 并从三方面研究支架的贯通性: (1)通过影像重建定量分析支架孔隙的三维贯通结构; (2)统计分析比较三种支架在贯流方向上的孔隙率变化; (3)有限元模拟支架的内部液流分布情况。结果表明, 球粒堆积支架与蜡球造孔支架孔隙率分布较均匀, 而纤维堆积支架孔隙分布较杂乱。液流模拟(流速分布)发现, 球粒堆积支架与蜡球造孔支架中液体流动均匀, 但是蜡球造孔支架孔壁近表面区域存在大量“漩涡流”, 不利于支架内细胞与液流之间的物质交换, 该结果有可能解释球粒堆积支架体内成骨性优于蜡球造孔支架的动物体内实验结果。