Polysaccharides as scaffolds for bone regeneration

This review is concerned with the use of polysaccharides for bone regeneration. Cellulose, alginate and chitosan have been chosen as examples of the formidable potential of this class of materials. Many others could be cited but our experience and research interests dictated the choice. As in many areas of research we know how they started but we cannot see how and if they will end. Modification of cellulose was our entrance to the world of polysaccharides and a lot of what we know we owe to Charles Baquey, to whom we would like to express our immense gratitude. This chapter is dedicated to him, for his cooperation, unlimited enthusiasm and friendship.

Résumé

Cette revue s'inscrit dans l'utilisation de polysaccharides pour la régénération osseuse. La cellulose, l'alginate et le chitosan ont été choisis comme exemple compte tenu du formidable potentiel de cette classe de matériaux. Beaucoup d'autres auraient pu être cités mais notre expérience et nos intérêts en recherche ont guidé ce choix. La modification de cellulose a été notre introduction dans le monde des « polysaccharides » et la plupart de nos connaissances sont dues à Charles Baquey à qui nous voulons ici exprimer notre immense gratitude. Ce chapitre lui est dédié pour son implication, son enthousiasme illimité et son amitié.     

Novel membrane for guided bone regeneration

Membranes have been clinically used for guided tissue and bone regeneration for decades, but their use in every day clinical practice is rather limited. We developed a biodegradable membrane (InionGTR) composed of polylactide, polyglycolide and trimethylene carbonate aiming to improve the properties of membrane. Before application the membrane is treated with N-methyl-pyrrolidone (NMP) to achieve a rubber like consistency, to allow easy handling and manageability in the clinical setting. After placing the membrane NMP diffuses out from the polymer phase into the water phase. The loss of NMP in the polymer stiffens the membrane up and allows space maintenance in the defect area. In addition the influx and efflux of NMP creates a porous surface on the membrane leading to an improved integration of tissues into the porous surface layers of the InionGTR membrane. Therefore, the use of NMP improves the handling in the clinical setting, and allows tissue integration and space maintenance, both important for the outcome of the treatment.     

Collagen-apatite nanocomposite membranes for guided bone regeneration

Collagen-apatite nanocomposite is regarded as a potential biomaterial because of its composition and structure, which are similar to those of human hard tissues. However, there have been few investigations of its mechanical and biological benefits in direct comparison with a collagen equivalent. Herein, we successfully produced a biomedical membrane made of a nanocomposite, and systemically evaluated the mechanical, chemical, and biological properties of the nanocomposite in comparison with those of pure collagen. The results showed that significant improvements were achieved by the nanocomposite approach, particularly in terms of the mechanical strength and chemical stability. The present findings point to the potential usefulness of the collagen-apatite nanocomposite membrane in the field of guided bone regeneration (GBR).     

Characterization of mineralized collagen-glycosaminoglycan scaffolds for bone regeneration

Mineralized collagen–glycosaminoglycan scaffolds designed for bone regeneration have been synthesized via triple co-precipitation in the absence of a titrant phase. Here, we characterize the microstructural and mechanical properties of these newly developed scaffolds with 50 and 75 wt.% mineral content. The 50 wt.% scaffold had an equiaxed pore structure with isotropic mechanical properties and a Ca–P-rich mineral phase comprised of brushite; the 75 wt.% scaffold had a bilayer structure with a pore size varying in the through-thickness direction and a mineral phase comprised of 67% brushite and 33 wt.% monetite. The compressive stress–strain response of the scaffolds was characteristic of low-density open-cell foams with distinct linear elastic, collapse plateau and densification regimes. The elastic modulus and strength of individual struts within the scaffolds were measured using an atomic force microscopy cantilevered beam-bending technique and compared with the composite response under indentation and unconfined compression. Cellular solids models, using the measured strut properties, overestimated the overall mechanical properties for the scaffolds; the discrepancy arises from defects such as disconnected pore walls within the scaffold. As the scaffold stiffness and strength decreased with increasing overall mineral content and were less than that of natural, mineralized collagen scaffolds, these microstructural/mechanical relations will be used to further improve scaffold design for bone regeneration applications.     

Collagen-silica xerogel nanohybrid membrane for guided bone regeneration

A collagen-silica xerogel hybrid membrane was fabricated by a sol-gel process for guided bone regeneration (GBR). The silica xerogel synthesized by the sol-gel method was distributed uniformly within the collagen matrix in the form of nanoparticles. The hybridization of the silica xerogel with collagen improved the biological properties of the membrane significantly. Preosteoblast cells were observed to adhere well and grow much more actively on the hybrid membrane than on the pure collagen membrane. In particular, the hybrid membrane containing 30% of the silica xerogel showed the highest level of osteoblast differentiation. Moreover, the GBR ability, as assessed by the in vivo animal test, was superior to that of the pure collagen membrane. These findings suggest that the collagen-silica xerogel hybrid can be used as a GBR membrane.     

Optimally porous and biomechanically compatible scaffolds for large-area bone regeneration

Large-area or critical-sized bone defects pose a serious challenge in orthopedic surgery, as all current treatment options present with shortcomings. Bone tissue engineering offers a more promising alternative treatment strategy. However, this approach requires mechanically stable scaffolds that support homogenous bone formation throughout the scaffold thickness. Despite advances in scaffold fabrication, current scaffold-based techniques are unable to support uniform, three-dimensional bone regeneration, and are limited to only the scaffold surface in vitro and in vivo. This is mainly because of inadequate scaffold pore sizes (<200?μm) and accessible pore volume, and the associated limited oxygen diffusion and vascular invasion. In this study, we have adopted a method combining microsphere-sintering and porogen-leaching techniques to fabricate scaffolds with an increased accessible pore volume. Of the scaffolds developed, moderately porous poly(85 lactide-co-15 glycolide) (PLGA) microsphere scaffolds were selected as most advantageous, since they retain mechanical strength in the range of human cancellous bone and display a significantly higher accessible pore volume, which is attributed to an increased percentage of larger pores (i.e., size range 200-600?μm). Unlike control scaffolds with a limited pore size and an accessible pore volume, moderately porous scaffolds displayed increased oxygen diffusion, pre-osteoblast cell infiltration, proliferation, and survival throughout the entire scaffold. Furthermore, moderately porous PLGA microsphere scaffolds displayed enhanced and homogenous mineralization in vitro. Since these newly designed moderately porous scaffolds are weight bearing, are fully osteoconductive, and have the ability to support vascularization, they may serve as effective scaffolds for large-area bone defect repair/regeneration. In addition, this study demonstrates the ability to modulate scaffold porosity and, in turn, to develop oxygen tension-controlled matrices that are effective for large-area bone regeneration.     

Hierarchically mesoporous-macroporous bioactive glasses scaffolds for bone tissue regeneration

Hierarchically 2D/3D mesoporous-macroporous bioactive glasses (MMBG) with good molding capabilities and compressive modulus were synthesized by sol-gel method and evaporation-induced self-assembly process in the presence of both nonionic triblock copolymers, EO(70)PO(20)EO(70) (P123) or EO(100)PO(65)EO(100) (F127), templates and methyl cellulose template. P123 or F127 acts as both a template, inducing the formation of mesopore, and an effective dispersant of MC, which produces macropores. In vitro bioactivity studies were carried out in simulated body fluid and showed superior bone-forming bioactivities of hierarchical MMBG. Human osteoblastlike cells, MG63, were seeded on MMBG and were determined using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyl-tetrazolium bromide] assay to confirm biocompatibilities of MMBG.     

Prefabrication of vascularized bone graft using guided bone regeneration

This article describes the prefabrication of a vascularized bone graft composed of autologous particulate cancellous bone and marrow (PCBM), a vessel bundle, and a biodegradable membrane. The PCBM was placed around the saphenous vessel bundle of rats and rolled with a biodegradable membrane of L-lactide-epsilon-caprolactone copolymer to prepare the prefabricated vascularized bone graft (group A). As controls, combinations of PCBM and membrane (group B), vessel bundle and membrane (group C), and PCBM and vessel bundle (group D) were prepared. A radiographic study revealed radio-opacity in the implantation site of group A 1 week later, in contrast to the other groups. Newly formed bone in the membrane roll was histologically confirmed, and neomicrovasculature circulating from the vessel bundle through the newly formed bone tissue was observed. The increase in alkaline phosphatase activity and osteocalcin content was significant for the group A preparation compared with the other groups. We concluded that the combination of autologous PCBM, a vessel bundle, and a biodegradable membrane was promising in the prefabrication of vascularized bone with good blood circulation.     

应用羟基磷灰石生物陶瓷及口腔膜引导骨再生修复牙周骨缺损

背景：羟基磷灰石生物陶瓷以天然优质海洋珊瑚为原料,在珊瑚骨架上形成羟基磷灰石薄层,保留珊瑚天然孔孔相同的支架结构,为组织生长提供了良好空间。 目的：观察羟基磷灰石生物陶瓷膜引导骨再生修复牙周骨缺损的临床效果。 方法：将42例下颌第一磨牙牙周病致骨缺损患者随机分组：实验组采用羟基磷灰石生物陶瓷结合口腔修复膜充填修复骨缺损,对照组采用单独羟基磷灰石生物陶瓷充填修复。 结果与结论：临床随访观察12个月,两组牙周组织附着丧失、牙周探诊深度较治疗前明显改善(P < 0.05),且实验组牙周组织附着丧失、牙周探诊深度改善优于对照组(P < 0.05);实验组骨缺损区新骨形成密度和骨量均优于对照组(P < 0.05)。表明采用羟基磷灰石生物陶瓷充填骨缺损区同时覆盖生物膜的引导骨再生技术可获得良好的骨引导再生效果,修复骨缺损。     

Porous collagen-apatite nanocomposite foams as bone regeneration scaffolds

We have created a porous bioresorbable nanocomposite bone scaffold that chemically, structurally and mechanically matched natural bone so that it could be recognized and remodeled by natural bone. Containing collagen fibers and synthetic apatite nanocrystals, our scaffold has high strength for supporting the surrounding tissue. The foam-like scaffold has a similar microstructure as trabecular bone, with nanometer-sized and micron-sized pores. The apatitic phase of the scaffold exhibited similar chemical composition, crystalline phase and grain size as the trabecular bone apatite. The nanocomposite scaffold demonstrated excellent bioactivity for promoting cell attachment and proliferation. It was osteoconductive and successfully healed a non-union fracture in rat femur as well as a critical-sized defect in pig tibia.     

Tissue engineering for bone regeneration using differentiated alveolar bone cells in collagen scaffolds

Regeneration of osseous defects by a tissue-engineering approach provides a novel means of treatment utilizing cell biology, materials science, and molecular biology. In this study the concept of tissue engineering was tested with collagen type I matrices seeded with cells with osteogenic potential and implanted into sites where osseous damage had occurred. Explant cultures of cells from human alveolar bone and gingiva were established. When seeded into a three-dimensional type I collagen-based scaffold, the bone-derived cells maintained their osteoblastic phenotype as monitored by mRNA and protein levels of the bone-related proteins including bone sialoprotein, osteocalcin, osteopontin, bone morphogenetic proteins 2 and 4, and alkaline phosphatase. These in vitro-developed matrices were implanted into critical-size bone defects in skulls of immunodeficient (SCID) mice. Wound healing was monitored for up to 4 weeks. When measured by microdensitometry the bone density within defects filled with osteoblast-derived matrix was significantly higher compared with defects filled with either collagen scaffold alone or collagen scaffold impregnated with gingival fibroblasts. New bone formation was found at all the sites treated with the osteoblast-derived matrix at 28 days, whereas no obvious new bone formation was identified at the same time point in the control groups. In situ hybridization for the human-specific Alu gene sequence indicated that the newly formed bone tissue resulted from both transplanted human osteoblasts and endogenous mesenchymal stem cells. The results indicate that cells derived from human alveolar bone can be incorporated into bioengineered scaffolds and synthesize a matrix, which on implantation can induce new bone formation.     

Coating of VEGF-releasing scaffolds with bioactive glass for angiogenesis and bone regeneration

Bioactive glasses are potentially useful as bone defect fillers, and vascular endothelial growth factor (VEGF) has demonstrated benefit in bone regeneration as well. We hypothesized that the specific combination of prolonged localized VEGF presentation from a matrix coated with a bioactive glass may enhance bone regeneration. To test this hypothesis, the capacity of VEGF-releasing polymeric scaffolds with a bioactive glass coating was examined in vitro and in vivo using a rat critical-sized defect model. In the presence of a bioactive glass coating, we did not detect pronounced differences in the differentiation of human mesenchymal stem cells in vitro. However, we observed significantly enhanced mitogenic stimulation of endothelial cells in the presence of the bioactive glass coating, with an additive effect with VEGF release. This trend was maintained in vivo, where coated VEGF-releasing scaffolds demonstrated significant improvements in blood vessel density at 2 weeks versus coated control scaffolds. At 12 weeks, bone mineral density was significantly increased in coated VEGF-releasing scaffolds versus coated controls, while only a slight increase in bone volume fraction was observed. The results of this study suggest that a bioactive glass coating on a polymeric substrate participates in bone healing through indirect processes which enhance angiogenesis and bone maturation and not directly on osteoprogenitor differentiation and bone formation. The mass of bioactive glass used in this study provides a comparable and potentially additive, response to localized VEGF delivery over early time points. These studies demonstrate a materials approach to achieve an angiogenic response formerly limited to the delivery of inductive growth factors.     

New macroporous calcium phosphate glass ceramic for guided bone regeneration

This work describes a method to obtain macroporous resorbable glass and glass ceramic scaffolds with controlled biodegradability for tissue engineering applications. The constructs consisted of glass and glass ceramics in the system P(2)O(5)-CaO-Na(2)O-TiO(2) and they were prepared by foaming a slurry of glass particles by addition of a H(2)O(2) solution, and subsequent sintering of the porous structures obtained. Different thermal treatments were applied to control the degree of devitrification of the glass. The resultant materials showed a porosity percentage between 40% and 55% with a wide variety of pores ranging from 20 to 500 microm in diameter as determined by SEM and Image Analysis. The resulting constructs were predominantly formed by a vitreous phase, although small amounts of calcium metaphosphate and pyrophosphates were detected by X-ray diffraction and Raman spectroscopy after the sintering process. The biological response was also evaluated by means of the MTT test, the material showed a non-cytotoxic effect.     

Unique microstructural design of ceramic scaffolds for bone regeneration under load

During the past two decades, research on ceramic scaffolds for bone regeneration has progressed rapidly; however, currently available porous scaffolds remain unsuitable for load-bearing applications. The key to success is to apply microstructural design strategies to develop ceramic scaffolds with mechanical properties approaching those of bone. Here we report on the development of a unique microstructurally designed ceramic scaffold, strontium–hardystonite–gahnite (Sr–HT–gahnite), with 85% porosity, 500 μm pore size, a competitive compressive strength of 4.1 ± 0.3 MPa and a compressive modulus of 170 ± 20 MPa. The in vitro biocompatibility of the scaffolds was studied using primary human bone-derived cells. The ability of Sr–HT–gahnite scaffolds to repair critical-sized bone defects was also investigated in a rabbit radius under normal load, with β-tricalcium phosphate/hydroxyapatite scaffolds used in the control group. Studies with primary human osteoblast cultures confirmed the bioactivity of these scaffolds, and regeneration of rabbit radial critical defects demonstrated that this material induces new bone defect bridging, with clear evidence of regeneration of original radial architecture and bone marrow environment.     

Three-dimensional printing of strontium-containing mesoporous bioactive glass scaffolds for bone regeneration

In this study, we fabricated strontium-containing mesoporous bioactive glass (Sr-MBG) scaffolds with controlled architecture and enhanced mechanical strength using a three-dimensional (3-D) printing technique. The study showed that Sr-MBG scaffolds had uniform interconnected macropores and high porosity, and their compressive strength was ∼170 times that of polyurethane foam templated MBG scaffolds. The physicochemical and biological properties of Sr-MBG scaffolds were evaluated by ion dissolution, apatite-forming ability and proliferation, alkaline phosphatase activity, osteogenic expression and extracelluar matrix mineralization of osteoblast-like cells MC3T3-E1. The results showed that Sr-MBG scaffolds exhibited a slower ion dissolution rate and more significant potential to stabilize the pH environment with increasing Sr substitution. Importantly, Sr-MBG scaffolds possessed good apatite-forming ability, and stimulated osteoblast cells’ proliferation and differentiation. Using dexamethasone as a model drug, Sr-MBG scaffolds also showed a sustained drug delivery property for use in local drug delivery therapy, due to their mesoporous structure. Therefore, the 3-D printed Sr-MBG scaffolds combined the advantages of Sr-MBG such as good bone-forming bioactivity, controlled ion release and drug delivery and enhanced mechanical strength, and had potential application in bone regeneration.     

Hierarchical mesoporous silica nanofibers as multifunctional scaffolds for bone tissue regeneration

Mesoporous materials with pore sizes between 2 and 50?nm have elicited widespread interest in catalysis, separation, adsorption, sensors, and drug delivery applications due to its highly ordered pore size along with high hydrothermal stability and easily modifiable surface functionalities. Fabricating these mesoporous materials as continuous fibers offers exciting vistas for biomedical applications especially in tissue engineering. The aim of the present study was to fabricate, characterize, and evaluate the cellular and gene expression of mesoporous silica with a long ordered fibrous morphology to support regeneration of bone tissue. Tetraethyl orthosilicate, polyvinyl pyrrolidone, and the tri-block copolymer P-123 were subjected to electrospinning to fabricate continuous ordered mesoporous silica nanofibers by optimizing solution and operation parameters. Mesoporous silica fibers with an average diameter of 470?nm and mesopores of dimension 5.97?nm were obtained. The combination of micropores, mesopores, macropores, and the nanofibrous morphology imparted excellent bioactivity to the mesoporous silica fibrous scaffolds as demonstrated by the proliferation of human osteoblast-like cells (MG63) and by the maintenance of its phenotype. The upregulation of collagen I, alkaline phosphatase, osteocalcin, osteopontin, and bone sialoprotein signifies the maturation of MG63 cells on the silica scaffold. Hence, these novel scaffolds are promising new biomaterials for orthopaedic applications.     

Engineering bone regeneration with bioabsorbable scaffolds with novel microarchitecture

Critical-sized defects (CSDs) were introduced into rat calvaria to test the hypothesis that absorption of surrounding blood, marrow, and fluid from the osseous wound into a bioabsorbable polymer matrix with unique microarchitecture can induce bone formation via hematoma stabilization. Scaffolds with 90% porosity, specific surface areas of approximately 10 m2/g, and median pore sizes of 16 and 32 microm, respectively, were fabricated using an emulsion freeze-drying process. Contact radiography and radiomorphometry revealed the size of the initial defects (50 mm2) were reduced to 27 +/- 11 mm2 and 34 +/- 17 mm2 for CSDs treated with poly(D,L-lactide-co-glycolide). Histology and histomorphometry revealed scaffolds filled with significantly more de novo bone than negative controls (p < 0. 007), more osteoid than both the negative and autograft controls (p < 0.002), and small masses of mineralized tissue (< 15 mm in diameter) observed within the scaffolds. Based on these findings, we propose a change in the current paradigm regarding the microarchitecture of scaffolds for in vivo bone regeneration to include mechanisms based on hematoma stabilization.     

Three-layered membranes of collagen/hydroxyapatite and chitosan for guided bone regeneration

The purpose of this study was to design a novel hybrid membrane with optimized properties for guided bone regeneration (GBR). Both the top and bottom layers of the sandwich-structured membrane were composed of collagen containing 20 wt% hydroxyapatite (HA), while the middle layer was made of chitosan. The above three layers were formulated into an integral membrane from their respective slurries through a layer-by-layer filtration process. The phase and composition of the membrane were confirmed by FT-IR and XRD analyses. The observation of its morphology by SEM showed that the membrane had a porous structure and structural integrity. The chitosan layer ensured the high tensile strength and elastic modulus of the membrane, while the presence of the collagen/HA composite layers endowed it with good flexibility and bioactivity. These results suggest that the integrated membrane prepared in this study would have the potential for use as a GBR material.     

3D printing of composite calcium phosphate and collagen scaffolds for bone regeneration

Low temperature 3D printing of calcium phosphate scaffolds holds great promise for fabricating synthetic bone graft substitutes with enhanced performance over traditional techniques. Many design parameters, such as the binder solution properties, have yet to be optimized to ensure maximal biocompatibility and osteoconductivity with sufficient mechanical properties. This study tailored the phosphoric acid-based binder solution concentration to 8.75 wt% to maximize cytocompatibility and mechanical strength, with a supplementation of Tween 80 to improve printing. To further enhance the formulation, collagen was dissolved into the binder solution to fabricate collagen-calcium phosphate composites. Reducing the viscosity and surface tension through a physiologic heat treatment and Tween 80, respectively, enabled reliable thermal inkjet printing of the collagen solutions. Supplementing the binder solution with 1–2 wt% collagen significantly improved maximum flexural strength and cell viability. To assess the bone healing performance, we implanted 3D printed scaffolds into a critically sized murine femoral defect for 9 weeks. The implants were confirmed to be osteoconductive, with new bone growth incorporating the degrading scaffold materials. In conclusion, this study demonstrates optimization of material parameters for 3D printed calcium phosphate scaffolds and enhancement of material properties by volumetric collagen incorporation via inkjet printing.