Novel chitin/nanosilica composite scaffolds for bone tissue engineering applications

Biopolymers like chitin are widely investigated as scaffolds in bone tissue engineering. Its properties like biocompatibility, biodegradability, non-toxicity, wound healing ability, antibacterial activity, hemostatic property, etc., are widely known. However, these materials are not much bioactive. Addition of material like silica can improve the bioactivity and biocompatibility of chitin. In this work, chitin composite scaffolds containing nanosilica were prepared using chitin hydrogel and their bioactivity, swelling ability and cytotoxicity was analyzed in vitro. These scaffolds were found to be bioactive in simulated body fluid (SBF) and biocompatible when tested with MG 63 cell line. These results suggest that chitin/nanosilica composite scaffolds can be useful for bone tissue engineering applications.     

Osteogenic Properties of PBLG-g-HA/PLLA Nanocomposites

New development of biomaterial scaffolds remains a prominent issue for the regeneration of lost or fractured bone. Of these scaffolds, a number of bioactive polymers have been synthesized and fabricated for diverse biological roles. Although recent evidence has demonstrated that composite scaffolds such as HA/PLLA have improved properties when compared to either HA or PLLA alone, recent investigations have demonstrated that the phase compatibility between HA and PLLA layers is weak preventing optimal enhancement of the mechanical properties and making the composites prone to breakdown. In the present study, poly (γ-benzyl-L-glutamate) modified hydroxyapatite/(poly (L-lactic acid)) (PBLG-g-HA/PLLA) composite scaffolds were fabricated with improved phase compatibility and tested for their osteogenic properties in 18 Wistar female rats by analyzing new bone formation in 3 mm bilateral femur defects in vivo. At time points, 2, 4 and 8 weeks post surgery, bone formation was evaluated by µ-CT and histological analysis by comparing 4 treatment groups; 1) blank defect, 2) PLLA, 3) HA/PLLA and 4) PBLG-g-HA/PLLA scaffolds. The in vivo analysis demonstrated that new bone formation was much more prominent in HA/PLLA and PBLG-g-HA/PLLA groups as depicted by µ-CT, H&E staining and immunohistochemistry for collagen I. TRAP staining was also utilized to determine the influence of osteoclast cell number and staining intensity to the various scaffolds. No significant differences in either staining intensity or osteoclast numbers between all treatment modalities was observed, however blank defects did contain a higher number of osteoclast-like cells. The results from the present study illustrate the potential of PBLG-g-HA/PLLA scaffolds for bone tissue engineering applications by demonstrating favorable osteogenic properties.     

Magnesium substitution effect on porous scaffolds for bone repair

Of great interest in developing artificial bone is the incorporation of magnesium (Mg) ions into the ceramic lattice in order to improve the physico-chemical and structural properties of the material and to increase its morphological affinity towards newly formed osseous tissue. In the present study, we evaluated the morphological and biological properties of composite scaffolds fabricated by mixing a nanopowder of Mg-substituted beta-tricalcium phosphate with collagen type I in two dry weight ratios (variant I and II). We used biochemical methods, and electron and light microscopy to investigate their porosity, biodegradability and morphology. Osteoblast cell culture behavior in the presence of nanocomposite variants was also examined. Variant I scaffold presented a higher percentage of cross-links and a better resistance to collagenase degradation compared to variant II scaffold. Their porosity did not vary significantly. Osteoblasts cultivated in the presence of nanocomposite scaffolds for 72 h exhibited good cell viability and a normal morphology. When osteoblasts were injected into the scaffolds, a slightly higher proportion of adhered cells were observed for Mg-substituted samples after 7 days of cultivation. All these results showed that Mg-containing porous composite scaffolds had controlled degradation, allowed osteoblast proliferation and adhesion and are good candidates for bone repair.     

Osteochondral tissue engineering: scaffolds, stem cells and applications

Osteochondral tissue engineering has shown an increasing development to provide suitable strategies for the regeneration of damaged cartilage and underlying subchondral bone tissue. For reasons of the limitation in the capacity of articular cartilage to self-repair, it is essential to develop approaches based on suitable scaffolds made of appropriate engineered biomaterials. The combination of biodegradable polymers and bioactive ceramics in a variety of composite structures is promising in this area, whereby the fabrication methods, associated cells and signalling factors determine the success of the strategies. The objective of this review is to present and discuss approaches being proposed in osteochondral tissue engineering, which are focused on the application of various materials forming bilayered composite scaffolds, including polymers and ceramics, discussing the variety of scaffold designs and fabrication methods being developed. Additionally, cell sources and biological protein incorporation methods are discussed, addressing their interaction with scaffolds and highlighting the potential for creating a new generation of bilayered composite scaffolds that can mimic the native interfacial tissue properties, and are able to adapt to the biological environment.     

Biodegradable zinc oxide composite scaffolds promote osteochondral differentiation of mesenchymal stem cells

Osteoarthritis (OA) involves the degeneration of articular cartilage and subchondral bone. The capacity of articular cartilage to repair and regenerate is limited. A biodegradable, fibrous scaffold containing zinc oxide (ZnO) was fabricated and evaluated for osteochondral tissue engineering applications. ZnO has shown promise for a variety of biomedical applications but has had limited use in tissue engineering. Composite scaffolds consisted of ZnO nanoparticles embedded in slow degrading, polycaprolactone to allow for dissolution of zinc ions over time. Zinc has well-known insulin-mimetic properties and can be beneficial for cartilage and bone regeneration. Fibrous ZnO composite scaffolds, having varying concentrations of 1–10 wt.% ZnO, were fabricated using the electrospinning technique and evaluated for human mesenchymal stem cell (MSC) differentiation along chondrocyte and osteoblast lineages. Slow release of the zinc was observed for all ZnO composite scaffolds. MSC chondrogenic differentiation was promoted on low percentage ZnO composite scaffolds as indicated by the highest collagen type II production and expression of cartilage-specific genes, while osteogenic differentiation was promoted on high percentage ZnO composite scaffolds as indicated by the highest alkaline phosphatase activity, collagen production, and expression of bone-specific genes. This study demonstrates the feasibility of ZnO-containing composites as a potential scaffold for osteochondral tissue engineering.     

In Vitro and In Vivo Evaluations of Nano-Hydroxyapatite/Polyamide 66/Glass Fibre (n-HA/PA66/GF) as a Novel Bioactive Bone Screw

In this study, we prepared nano-hydroxyapatite/polyamide 66/glass fibre (n-HA/PA66/GF) bioactive bone screws. The microstructure, morphology and coating of the screws were characterised, and the adhesion, proliferation and viability of MC3T3-E1 cells on n-HA/PA66/GF scaffolds were determined using scanning electron microscope, CCK-8 assays and cellular immunofluorescence analysis. The results confirmed that n-HA/PA66/GF scaffolds were biocompatible and had no negative effect on MC3T3-E1 cells in vitro. To investigate the in vivo biocompatibility, internal fixation properties and osteogenesis of the bioactive screws, both n-HA/PA66/GF screws and metallic screws were used to repair intercondylar femur fractures in dogs. General photography, CT examination, micro-CT examination, histological staining and biomechanical assays were performed at 4, 8, 12 and 24 weeks after operation. The n-HA/PA66/GF screws exhibited good biocompatibility, high mechanical strength and extensive osteogenesis in the host bone. Moreover, 24 weeks after implantation, the maximum push-out load of the bioactive screws was greater than that of the metallic screws. As shown by their good cytocompatibility, excellent biomechanical strength and fast formation and ingrowth of new bone, n-HA/PA66/GF screws are thus suitable for orthopaedic clinical applications.     

Development of novel α-chitin/nanobioactive glass ceramic composite scaffolds for tissue engineering applications

Bioactive glass ceramic nanoparticles (nBGC) were prepared by sol–gel technique. The novel chitin/nBGC composite scaffolds were prepared using chitin gel with nBGC by lyophilization technique. The prepared nBGC and composite scaffolds were characterized using Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), Fourier Transformed Infrared Spectroscopy (FT-IR) and X-ray diffraction (XRD). The composite scaffolds showed adequate porosity where the nBGC nanoparticles were homogenously distributed on the pore walls. The swelling, density, degradation and in vitro biomineralization capability of the composite scaffolds were also evaluated. The developed composite scaffolds showed adequate swelling and degradation properties along with its ability to become bioactive. Cytocompatability of the scaffolds was assessed using MTT assay, direct contact test and cell attachment studies. Results indicated no sign of toxicity and cells found to be attached to the pore walls offered by the scaffolds. These results suggested that the developed composite scaffold possess the prerequisites for tissue engineering scaffolds and it can be used for tissue engineering applications.     

Composite scaffolds for cartilage tissue engineering

Tissue engineering remains a promising therapeutic strategy for the repair or regeneration of diseased or damaged tissues. Previous approaches have typically focused on combining cells and bioactive molecules (e.g., growth factors, cytokines and DNA fragments) with a biomaterial scaffold that functions as a template to control the geometry of the newly formed tissue, while facilitating the attachment, proliferation, and differentiation of embedded cells. Biomaterial scaffolds also play a crucial role in determining the functional properties of engineered tissues, including biomechanical characteristics such as inhomogeneity, anisotropy, nonlinearity or viscoelasticity. While single-phase, homogeneous materials have been used extensively to create numerous types of tissue constructs, there continue to be significant challenges in the development of scaffolds that can provide the functional properties of load-bearing tissues such as articular cartilage. In an attempt to create more complex scaffolds that promote the regeneration of functional engineered tissues, composite scaffolds comprising two or more distinct materials have been developed. This paper reviews various studies on the development and testing of composite scaffolds for the tissue engineering of articular cartilage, using techniques such as embedded fibers and textiles for reinforcement, embedded solid structures, multi-layered designs, or three-dimensionally woven composite materials. In many cases, the use of composite scaffolds can provide unique biomechanical and biological properties for the development of functional tissue engineering scaffolds.     

牙源性干细胞复合微渠多孔羟基磷灰石支架成骨性能研究*

目的: 探讨牙源性干细胞复合微渠多孔羟基磷灰石支架(grooved porous hydroxyapatite scaffolds, HAG支架)的成骨性能,为骨缺损修复治疗提供新手段。方法: 从健康成人第三磨牙中提取牙周膜干细胞(periodontal ligament stem cells, PDLSCs)及牙髓干细胞(dental pulp stem cells, DPSCs)分别接种于HAG支架上,进行多向分化鉴定及碱性磷酸酶(alkaline phosphatase,ALP)活性测定;并通过CCK-8检测细胞增殖能力;逆转录聚合酶链反应(qRT-PCR)检测骨形态发生蛋白2(bone morphogenetic protein 2, BMP-2)、骨钙素(osteocalcin, OCN)和骨桥蛋白(osteopontin, OPN)等成骨相关基因的表达。体内研究中将搭载PDLSCs和DPSCs的HAG支架移植到裸鼠的背部皮下,8周后取材,组织切片后采用苏木精-伊红(HE)染色观察新骨形成,提取组织蛋白采用Western blot检测ALP、OCN等成骨相关蛋白的表达。结果: 体外研究中DPSCs复合HAG支架组的细胞增殖能力、ALP活性,以及成骨相关基因ALP、BMP2、OCN等的表达均高于PDLSCs复合HAG支架组。体内研究中HE染色显示,PDLSCs复合HAG支架组及DPSCs复合HAG支架组均较空白HAG支架组有更多细胞生长区、纤维细胞增生及骨基质形成,且DPSCs复合HAG支架组的骨基质面积更大,成纤维细胞数量更多;PDLSCs复合HAG支架组及DPSCs复合HAG支架组成骨相关蛋白的表达量均高于空白HAG组,且DPSCs复合HAG支架组中ALP蛋白表达量显著高于PDLSCs复合HAG支架组。结论: PDLSCs、DPSCs复合HAG支架在体内外均表现出良好的成骨性能,其中DPSCs复合HAG支架的成骨性能更为优异。     

A new method of fabricating robust freeform 3D ceramic scaffolds for bone tissue regeneration

Fabrication of three‐dimensional (3D) scaffolds with appropriate mechanical properties and desired architecture for promoting cell growth and new tissue formation is one of the most important efforts in tissue engineering field. Scaffolds fabricated from bioactive ceramic materials such as hydroxyapatite and tricalcium phosphate show promise because of their biological ability to support bone tissue regeneration. However, the use of ceramics as scaffold materials is limited because of their inherent brittleness and difficult processability. The aim of this study was to create robust ceramic scaffolds, which have a desired architecture. Such scaffolds were successfully fabricated by projection‐based microstereolithography, and dilatometric analysis was conducted to study the sintering behavior of the ceramic materials. The mechanical properties of the scaffolds were improved by infiltrating them with a polycaprolactone solution. The toughness and compressive strength of these ceramic/polymer scaffolds were about twice those of ceramic scaffolds. Furthermore, the osteogenic gene expression on ceramic/polymer scaffolds was better than that on ceramic scaffolds. Through this study, we overcame the limitations of previous research on fabricating ceramic scaffolds and these new robust ceramic scaffolds may provide a much improved 3D substrate for bone tissue regeneration. Biotechnol. Bioeng. 2013; 110: 1444–1455. © 2012 Wiley Periodicals, Inc.     

Optimized bone regeneration based on sustained release from three-dimensional fibrous PLGA/HAp composite scaffolds loaded with BMP-2

Contemporary treatment of critical bone defect remains a significant challenge in the field of orthopedic surgery. Engineered biomaterials combined with growth factors have emerged as a new treatment alternative in bone repair and regeneration. Our approach is to encapsulate bone morphogenetic protein-2 (BMP-2) into a polymeric matrix in different ways and characterize their individual performance in a nude mouse model. The main objective of this study is to examine whether the PLGA/HAp composite fibrous scaffolds loaded with BMP-2 through electrospinning can improve bone regeneration. The hypothesis is that different loading methods of BMP-2 and different HAp contents in scaffolds can alternate the release profiles of BMP-2 in vivo, therefore modify the performance of scaffolds in bone regeneration. Firstly, mechanical strength of scaffolds and HAp nanoparticles distribution in scaffolds were investigated. Secondly, nude mice experiments extended to 6 weeks were carried out to test the in vivo performance of these scaffolds, in which measurements, like serum BMP-2 concentration, ALP activity, X-ray qualification, and H&E/IHC tissue staining were utilized to monitor the growth of new bone and the changes of the corresponding biochemical parameters. The results showed that the PLGA/HAp composite scaffolds developed in this study exhibited good morphology/mechanical strength and HAp nanoparticles were homogeneously dispersed inside PLGA matrix. Results from the animal experiments indicate that the bioactivity of BMP-2 released from the fibrous PLGA/HAp composite scaffolds is well maintained, which further improves the formation of new bone and the healing of segmental defects in vivo. It is concluded that BMP-2 loaded PLGA/HAp composite scaffolds are promising for bone healing.     

Fabrication of individual scaffolds based on a patient-specific alveolar bone defect model

Fabricating individualized tissue engineering scaffolds based on the three-dimensional shape of patient bone defects is required for the successful clinical application of bone tissue engineering. However, there are currently no reported studies of individualized bone tissue engineering scaffolds that truly reproduce a patient-specific bone defect. We fabricated individualized tissue engineering scaffolds based on alveolar bone defects. The individualized poly(lactide-co-glycolide) and tricalcium phosphate composite scaffolds were custom-made by acquiring the three-dimensional model through computed tomography, which was input into the computer-aided low-temperature deposition manufacturing system. The three-dimensional shape of the fabricated scaffold was identical to the patient-specific alveolar bone defects, with an average macropore diameter of 380 μm, micropore diameters ranging from 3 to 5 μm, and an average porosity of 87.4%. The mechanical properties of the scaffold were similar to adult cancellous bone. Scaffold biocompatibility was confirmed by attachment and proliferation of human bone marrow mesenchymal stem cells. Successful realization of individualized scaffold fabrication will enable clinical application of tissue-engineered bone at an early date.     

Promotion of human mesenchymal stem cell differentiation on bioresorbable polycaprolactone/biphasic calcium phosphate composite scaffolds for bone tissue engineering

An artificial construct mimicking the intrinsic properties of the natural extracellular matrix in bones has been considered an ideal platform for bone tissue engineering, as it can present an appropriate microenvironment and regulate cell behaviours. In this report, we introduce biodegradable composite scaffolds consisting of polycaprolactone (PCL) and biphasic calcium phosphate (BCP). The scaffolds were fabricated by a salt-leaching process, and the ability of the scaffolds to facilitate osteogenic differentiation was investigated using human mesenchymal stem cells (hMSCs). The scaffolds had an inter-connected porous structure with quadrilateral pores of approximately 200 ～ 500 μm in width. The mechanical properties of the scaffolds changed as the BCP content was increased in the starting mixture. In the hMSC experiment, although we found that hMSCs adhered to the surface, as well as the inside, of the scaffolds, the incorporated BCP did not increase the proliferation of the hMSCs over 7 days in culture. Interestingly, the alkaline phosphatase (ALP) activity was 4 times higher on the PCL/BCP composite scaffold (0.12 ± 0.03 nmol/min/μg protein) thanon the PCL scaffold (0.03 ± 0.01 nmol/min/μg protein), suggesting that BCP can aid in generating a local environment that promotes bone regeneration. Therefore, a strategy combining polymers and ceramics can be considered a useful platform for bone tissue engineering.     

Recent advances in bone tissue engineering scaffolds

Bone disorders are of significant concern due to increase in the median age of our population. Traditionally, bone grafts have been used to restore damaged bone. Synthetic biomaterials are now being used as bone graft substitutes. These biomaterials were initially selected for structural restoration based on their biomechanical properties. Later scaffolds were engineered to be bioactive or bioresorbable to enhance tissue growth. Now scaffolds are designed to induce bone formation and vascularization. These scaffolds are often porous, made of biodegradable materials that harbor different growth factors, drugs, genes, or stem cells. In this review, we highlight recent advances in bone scaffolds and discuss aspects that still need to be improved.     

Polyhydroxyalkanoate (PHA)/inorganic phase composites for tissue engineering applications

Polyhydroxyalkanoates are emerging as a class of biodegradable polymers for applications in tissue engineering. Members of the polyhydroxyalkanoates family encompass a wide variety of materials, from hard and brittle materials to soft and elastomeric. Over the years, efforts have been made to extend the group of polyhydroxyalkanoates and to investigate their use in numerous biomedical applications, such as sutures, cardiovascular patches, wound dressings, guided tissue repair/regeneration devices, and tissue engineering scaffolds. Along with the development of polyhydroxyalkanoates, researchers have looked into the possibility of designing composites in combination with inorganic phases to further improve the mechanical properties, rate of degradation, and also impart bioactivity. Poly(3-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) are some of the polymers which have been studied extensively to fabricate composites in combination with hydroxyapatite, bioactive glass, and glass-ceramic fillers or coatings. This paper reviews international research carried out toward development of polyhydroxyalkanoates/inorganic phase composites in terms of systems investigated, microstructures, properties achieved, and applications, with special focus on tissue engineering scaffolds. A comparison between different composite systems developed in the past few years is presented. The paper also addresses the prospect of potential further development of polyhydroxyalkanoates/inorganic phase composites with optimized microstructure and properties for improved tissue engineering scaffolds.     

骨软骨复合支架的研究进展

软骨的修复是当前医学界十分棘手的难题,人们采取若干手段均收效甚微。由于软骨缺损时,其下的软骨下骨常出现硬化、退变,而新生软骨是无法与病变的软骨下骨进行整合的,所以在修复软骨的同时,必须重视软骨下骨的修复。近十几年来,人们开始发明和利用各种骨软骨复合支架,进行同时修复软骨与软骨下骨的动物实验研究。在正常骨软骨组织中,软骨与软骨下骨被钙化层所相连,此外钙化层也将软骨与软骨下骨分隔在不同的生存环境中。根据仿生学原理,人们又设计出一种带有隔离层的新型骨软骨复合支架,并取得了较为理想的实验结果。本文就国内外骨软骨复合支架的研完进展作一综述。     

Regenerating articular tissue by converging technologies

Scaffolds for osteochondral tissue engineering should provide mechanical stability, while offering specific signals for chondral and bone regeneration with a completely interconnected porous network for cell migration, attachment, and proliferation. Composites of polymers and ceramics are often considered to satisfy these requirements. As such methods largely rely on interfacial bonding between the ceramic and polymer phase, they may often compromise the use of the interface as an instrument to direct cell fate. Alternatively, here, we have designed hybrid 3D scaffolds using a novel concept based on biomaterial assembly, thereby omitting the drawbacks of interfacial bonding. Rapid prototyped ceramic particles were integrated into the pores of polymeric 3D fiber-deposited (3DF) matrices and infused with demineralized bone matrix (DBM) to obtain constructs that display the mechanical robustness of ceramics and the flexibility of polymers, mimicking bone tissue properties. Ostechondral scaffolds were then fabricated by directly depositing a 3DF structure optimized for cartilage regeneration adjacent to the bone scaffold. Stem cell seeded scaffolds regenerated both cartilage and bone in vivo.     

Tissue engineering scaffolds based on photocured dimethacrylate polymers for in vitro optical imaging

Model tissue engineering scaffolds based on photocurable resin mixtures with sodium chloride have been prepared for optical imaging studies of cell attachment. A photoactivated ethoxylated bisphenol A dimethacrylate was mixed with sieved sodium chloride (NaCl) crystals and photocured to form a cross-linked composite. Upon soaking in water, the NaCl dissolved to leave a porous scaffold with desirable optical properties, mechanical integrity, and controlled porosity. Scaffolds were prepared with salt crystals that had been sieved to average diameters of 390, 300, 200, and 100 microm, yielding porosities of approximately 75 vol %. Scanning electron microscopy and X-ray microcomputed tomography confirmed that the pore size distribution of the scaffolds could be controlled using this photocuring technique. Compression tests showed that for scaffolds with 84% (by mass fraction) salt, the larger pore size scaffolds were more rigid, while the smaller pore size scaffolds were softer and more readily compressible. The prepared scaffolds were seeded with osteoblasts, cultured between 3 and 18 d, and examined using confocal microscopy. Because the cross-linked polymer in the scaffolds is an amorphous glass, it was possible to optically image cells that were over 400 microm beneath the surface of the sample.     

Calcium salts bone regeneration scaffolds: a review article

A growing array of synthetic bone regeneration scaffolds has been in use over the last century. These scaffolds aim to provide a three dimensional substrate for bone cells to populate on and to function appropriately. The majority of commercially-available scaffolds are based on calcium sulphate salts, calcium phosphate salts, or composites of the two. The mechanical and biological properties of these scaffolds are a result of the properties of the constituent materials and their ratio in the composite. This review addresses the mechanical and biological characteristics and provides an up-to-date summary of the clinical data available about the use of these calcium-based scaffolds. It will also assess the benefit of using stem cell technology along with this type of scaffolds. This article links between the basic science evidence and the clinical outcomes and details the commercially-available options.     

Effect of osteonectin-derived peptide on the viscoelasticity of hydrogel/apatite nanocomposite scaffolds

Hydrogel/apatite nanocomposites are the ideal biomaterial to mimic the physio-chemical and biologic properties of the bone and to fabricate scaffolds for bone regeneration. The objective of this work was to investigate the effect of an osteonectin derived glutamic acid sequence on the viscoelastic properties of poly(lactide-ethylene oxide-fumarate) (PLEOF)/apatite composite, as a model degradable material in bone regeneration. Osteonectin is an extracellular acidic glycoprotein of the bone matrix, which is believed to be involved in linking the collagen network to hydroxyapatite (HA), the mineral phase of the bone. We synthesized a 6-glutamic acid sequence in solid phase with affinity to HA crystals via ionic interactions. One end of the synthesized peptide was functionalized with an acrylate group to covalently attach the peptide (Ac-Glu6) to the aqueous-based biodegradable and in situ crosslinkable PLEOF hydrogel matrix. To determine the effect of energetic interactions between the fillers and hydrogel matrix, HA nanoparticles were also treated with an acrylate functionalized 6-glycine amino acid peptide (Ac-Gly6) that interacts with the fillers only by van der Waals and polar interactions (without ionic interactions). Crosslinked PLEOF/apatite scaffolds were prepared using PLEOF as the degradable macromer, HA nanofillers treated with Ac-Glu6 peptide linker, and a neutral redox initiation system. The viscoelastic properties were studied by dynamic time sweep, strain sweep, and small amplitude oscillatory rheometry. Composites without surface treatment, treated with Ac-Gly6, and treated with Ac-Glu6 at different volume fractions and various particle sizes were examined. The results showed that the 6-mer glutamic acid sequence significantly affects the shear modulus of the scaffold because of ionic interactions between the peptide and HA crystals.