Preparation of a biphasic scaffold for osteochondral tissue engineering

Tissue engineering has been developed as a prospective approach for the repair of articular cartilage defects. Engineered osteochondral implants can facilitate the fixation and integration with host tissue, and therefore promote the regeneration of osteochondral defects. A biphasic scaffold with a stratified two-layer structure for osteochondral tissue engineering was developed from biodegradable synthetic and naturally derived polymers. The upper layer of the scaffold for cartilage engineering was collagen sponge; the lower layer for bone engineering was a composite sponge of poly(DL-lactic-co-glycolic acid) (PLGA) and naturally derived collagen. The PLGA–collagen composite sponge layer had a composite structure with collagen microsponge formed in the pores of a skeleton PLGA sponge. The collagen sponge in the two respective layers was connected. Observation of the collagen/PLGA–collagen biphasic scaffold by scanning electron microscopy (SEM) demonstrated the connected stratified structure. The biphasic scaffold was used for culture of canine bone-marrow-derived mesenchymal stem cells. The cell/scaffold construct was implanted in an osteochondral defect in the knee of a one-year old beagle. Osteochondral tissue was regenerated four months after implantation. Cartilage- and bone-like tissues were formed in the respective layers. The collagen/PLGA–collagen biphasic scaffold will be useful for osteochondral tissue engineering.     

Bi-layered constructs based on poly(l-lactic acid) and starch for tissue engineering of osteochondral defects

Articular cartilage has a limited capacity to repair itself, and conventional therapeutic approaches have shown to have limited success as they are deficient and inconsistent in long-term repair. Tissue engineering has shown to be an alternative route to regenerate articular defects. In this work, new bi-layered scaffolds are developed in order to enhance the integration between the engineered cartilage tissue and the corresponding subchondral bone. The concept includes the use of a common polymer in both sides, poly(l-lactic acid), PLLA, to increase the bonding between them, and the use of compression moulding followed by particle leaching to process porous scaffolds with controllable porosities. A compact layer could be observed between the two layers that could be useful for independent cell culturing of the developed osteochondral constructs. A blend of starch and PLLA was used in the cartilage side, which was found to possess adequate hydration capability. For the bone region, where more stiffness and strength was required, PLLA reinforced with hydroxyapatite was used. Preliminary bioactivity tests demonstrated that the bone-layer could induce the formation of a calcium–phosphate layer in vitro, whereas the cartilage layer does not exhibit the ability for calcification.     

Chitosan particles agglomerated scaffolds for cartilage and osteochondral tissue engineering approaches with adipose tissue derived stem cells

It is well accepted that natural tissue regeneration is unlikely to occur if the cells are not supplied with an extracellular matrix (ECM) substitute. With this goal, several different methodologies have been used to produce a variety of 3D scaffolds as artificial ECM substitutes suitable for bone and cartilage tissue engineering. Furthermore, osteochondral tissue engineering presents new challenges since the combination of scaffolding and co-culture requirements from both bone and cartilage applications is required in order to achieve a successful osteochondral construct. In this paper, an innovative processing route based on a chitosan particles aggregation methodology for the production of cartilage and osteochondral tissue engineering scaffolds is reported. An extensive characterization is presented including a morphological evaluation using Micro-Computed Tomography (μCT) and 3D virtual models built with an image processing software. Mechanical and water uptake characterizations were also carried out, evidencing the potential of the developed scaffolds for the proposed applications. Cytotoxicity tests show that the developed chitosan particles agglomerated scaffolds do not exert toxic effects on cells. Furthermore, osteochondral bilayered scaffolds could also be developed. Preliminary seeding of mesenchymal stem cells isolated from human adipose tissue was performed aiming at developing solutions for chondrogenic and osteogenic differentiation for osteochondral tissue engineering applications. An erratum to this article is available at .     

A novel osteochondral scaffold of ceramic–gelatin assembly for articular cartilage repair

A novel ceramic–gelatin assembly (CGA) has been designed as an osteochondral scaffold for articular cartilage repair. The CGA scaffold consists of four layers, that is, a porous ceramic layer as osseous component and also as anchor, a dense ceramic layer to prevent blood vessel penetration and also to stand shear stress, a porous ceramic layer for fixation of bone to cartilage, i.e. for joining the ceramic part to the porous gelatin layer, the latter being used as cartilaginous component. The joining was done by the infiltration of gelatin solution into the porous ceramic layer, gelling and crosslinking. This CGA scaffold can offer solutions to the so-far not satisfactorily resolved issues of the osteochondral scaffold, i.e. anchoring, blood vessel penetration, shear stress distribution during articular joint motion, and enough strength to join the cartilaginous component to the osseous component to prevent delamination. This novel scaffold was tested by in vitro cell culture with Wistar rat's joint chondrocytes. DNA assay, GAGs assay, RT-PCR, and histological evaluations with hematoxylin–eosin and Safranin-O staining were carried out to show that cartilage tissue can be developed in four weeks.     

11.2% All‐Polymer Tandem Solar Cells with Simultaneously Improved Efficiency and Stability

All‐polymer solar cells (all‐PSCs) that contain both p‐type and n‐type polymeric materials blended together as light‐absorption layers have attracted much attention, since the blend of a polymeric donor and acceptor should present superior photochemical, thermal, and mechanical stability to those of small molecular‐based organic solar cells. In this work, the interfacial stability is studied by using highly stable all‐polymer solar cell as a platform. It is found that the thermally deposited metal electrode atoms can diffuse into the active layer during device storage, which consequently greatly decreases the power conversion efficiency. Fortunately, the diffusion of metal atoms can be slowed down and even blocked by using thicker interlayer materials, high‐glass‐transition‐temperature interlayer materials, or a tandem device structure. Learning from this, homojunction tandem all‐PSCs are successfully developed that simultaneously exhibit a record power conversion efficiency over 11% and remarkable stability with efficiency retaining 93% of the initial value after thermally aging at 80 °C for 1000 h.     

Fabrication and development of artificial osteochondral constructs based on cancellous bone/hydrogel hybrid scaffold

Using tissue engineering techniques, an artificial osteochondral construct was successfully fabricated to treat large osteochondral defects. In this study, porcine cancellous bones and chitosan/gelatin hydrogel scaffolds were used as substitutes to mimic bone and cartilage, respectively. The porosity and distribution of pore size in porcine bone was measured and the degradation ratio and swelling ratio for chitosan/gelatin hydrogel scaffolds was also determined in vitro. Surface morphology was analyzed with the scanning electron microscope (SEM). The physicochemical properties and the composition were tested by using an infrared instrument. A double layer composite scaffold was constructed via seeding adipose-derived stem cells (ADSCs) induced to chondrocytes and osteoblasts, followed by inoculation in cancellous bones and hydrogel scaffolds. Cell proliferation was assessed through Dead/Live staining and cellular activity was analyzed with IpWin5 software. Cell growth, adhesion and formation of extracellular matrix in composite scaffolds blank cancellous bones or hydrogel scaffolds were also analyzed. SEM analysis revealed a super porous internal structure of cancellous bone scaffolds and pore size was measured at an average of 410 ± 59 μm while porosity was recorded at 70.6 ± 1.7 %. In the hydrogel scaffold, the average pore size was measured at 117 ± 21 μm and the porosity and swelling rate were recorded at 83.4 ± 0.8 % and 362.0 ± 2.4 %, respectively. Furthermore, the remaining hydrogel weighed 80.76 ± 1.6 % of the original dry weight after hydration in PBS for 6 weeks. In summary, the cancellous bone and hydrogel composite scaffold is a promising biomaterial which shows an essential physical performance and strength with excellent osteochondral tissue interaction in situ. ADSCs are a suitable cell source for osteochondral composite reconstruction. Moreover, the bi-layered scaffold significantly enhanced cell proliferation compared to the cells seeded on either single scaffold. Therefore, a bi-layered composite scaffold is an appropriate candidate for fabrication of osteochondral tissue.     

Hierarchical and Heterogeneous Bioinspired Composites—Merging Molecular Self‐Assembly with Additive Manufacturing

Biological composites display exceptional mechanical properties owing to a highly organized, heterogeneous architecture spanning several length scales. It is challenging to translate this ordered and multiscale structural organization in synthetic, bulk composites. Herein, a combination of top‐down and bottom‐up approach is demonstrated, to form a polymer‐ceramic composite by macroscopically aligning the self‐assembled nanostructure of polymerizable lyotropic liquid crystals via 3D printing. The polymer matrix is then uniformly reinforced with bone‐like apatite via in situ biomimetic mineralization. The combinatorial method enables the formation of macrosized, heterogeneous composites where the nanostructure and chemical composition is locally tuned over microscopic distances. This enables precise control over the mechanics in specific directions and regions, with a unique intrinsic–extrinsic toughening mechanism. As a proof‐of‐concept, the method is used to form large‐scale composites mimicking the local nanostructure, compositional gradients and directional mechanical properties of heterogeneous tissues like the bone‐cartilage interface, for mechanically stable osteochondral plugs. This work demonstrates the possibility to create hierarchical and complex structured composites using weak starting components, thus opening new routes for efficient synthesis of high‐performance materials ranging from biomaterials to structural nanocomposites.     

Evaluation of a press-fit osteochondral poly(ester-urethane) scaffold in a rabbit defect model

The purpose of this study was to evaluate the impact on osteochondral healing of press-fitted multiphasic osteochondral scaffolds consisting of poly(ester-urethane) (PUR) and hydroxyapatite into a cylindric osteochondral defect in the distal non-weight bearing femoral trochlear ridge of the rabbit. Two scaffolds were investigated, one with and one without an intermediate microporous membrane between the cartilage and the bone compartment of the scaffold. A control group without a scaffold placed into the defect was included. After 12 weeks macroscopic and histomorphological analyses were performed. The scaffold was easily press-fitted and provided a stable matrix for tissue repair. The membrane did not demonstrate a detrimental effect on tissue healing compared with the scaffold without membrane. However, the control group had statistically superior healing as reflected by histological differences in the cartilage and subchondral bone compartment between control group and each scaffold group. A more detailed analysis revealed that the difference was localized in the bone compartment healing. The present study demonstrates that an elastomeric PUR scaffold can easily be press-fitted into an osteochondral defect and provides a stable matrix for tissue repair. However, the multi-phasic scaffold did not provide a clear advantage for tissue healing. Future investigations should refine especially the bone phase of the implant to increase its stiffness, biocompatibility and osteoconductive activity. A more precise fabrication technique would be necessary for the matching of tissue organisation.     

Novel alginate biphasic scaffold for osteochondral regeneration: an in vivo evaluation in rabbit and sheep models

Current therapeutic strategies for osteochondral restoration showed a limited regenerative potential. In fact, to promote the growth of articular cartilage and subchondral bone is a real challenge, due to the different functional and anatomical properties. To this purpose, alginate is a promising biomaterial for a scaffold-based approach, claiming optimal biocompatibility and good chondrogenic potential. A previously developed mineralized alginate scaffold was investigated in terms of the ability to support osteochondral regeneration both in a large and medium size animal model. The results were evaluated macroscopically and by microtomography, histology, histomorphometry, and immunohistochemical analysis. No evidence of adverse or inflammatory reactions was observed in both models, but limited subchondral bone formation was present, together with a slow scaffold resorption time.The implantation of this biphasic alginate scaffold provided partial osteochondral regeneration in the animal model. Further studies are needed to evaluate possible improvement in terms of osteochondral tissue regeneration for this biomaterial.     

骨软骨组织工程仿生梯度支架研究进展

骨软骨缺损是导致关节发病和残疾的重要原因,骨软骨组织工程是修复骨软骨缺损的方法之一。骨软骨组织工程方法涉及仿生梯度支架的制造,该支架需模仿天然骨软骨组织的生理特性（例如从软骨表面到软骨下骨之间的梯度过渡）。在许多研究中骨软骨仿生梯度支架表现为离散梯度或连续梯度,用于模仿骨软骨组织的特性,例如生物化学组成、结构和力学性能。连续型骨软骨梯度支架的优点是其每层之间没有明显的界面,因此更相似地模拟天然骨软骨组织。到目前为止,骨软骨仿生梯度支架在骨软骨缺损修复研究中已经取得了良好的实验结果,但是骨软骨仿生梯度支架与天然骨软骨组织之间仍然存在差异,其临床应用还需要进一步研究。本文首先从骨软骨缺损的背景、微尺度结构与力学性能、骨软骨仿生梯度支架制造相关的材料与方法等方面概述了离散和连续梯度支架的研究进展。其次,由于3D打印骨软骨仿生梯度支架的方法能够精确控制支架孔的几何形状和力学性能,因此进一步介绍了计算仿真模型在骨软骨组织工程中的应用,例如采用仿真模型优化支架结构和力学性能以预测组织再生。最后,提出了骨软骨缺损修复相关的挑战以及骨软骨组织再生未来研究的展望。例如,连续型骨软骨仿生梯度支架需要更相似地模拟天然骨软骨组织单元的结构,即力学性能和生化性能的过渡更加自然地平滑。同时,虽然大多数骨软骨仿生梯度支架在体内外实验中均取得了良好的效果,但临床研究和应用仍然需要进行进一步深入研究。     

3D bioprinting of multicellular scaffolds for osteochondral regeneration

Regeneration of osteochondral tissue is of great potentialities in repairing severe osteochondral defects. However, anisotropic physiological characteristics and tissue linage difference make the regeneration of osteochondral tissue remain a huge challenge. Herein, a multicellular system based on a bilayered co-culture scaffold mimicking osteochondral tissues was successfully developed for an alternative of osteochondral regeneration via a 3D bioprinting strategy. The dual function of integrally repairing both cartilage and bone could be achieved by designing multiple-cells distribution and a cell-induced bioink containing bioceramic particles. As an important bioactive agent, the Li-Mg-Si bioceramics-containing bioink exhibited the function of simultaneously stimulating multiple cells for differentiation towards specific directions. The 3D bioprinted co-culture scaffolds showed the capacity for osteochondral tissue regeneration by inducing osteogenic and chondrogenic differentiation in vitro and accelerating the repair of severe osteochondral defects in vivo. This study offers a potential strategy for complex tissue reconstruction through bioprinting multiple tissue cells in combination of bioceramics-stimulating bioinks.     

Processing of novel bioactive polymeric matrixes for tissue engineering using supercritical fluid technology

The aim of this study was to develop a new process for the production of bioactive 3D scaffolds using a clean and environmentally friendly technology. The possibility of preparing composite scaffolds of Bioglass^® and a polymeric blend of starch and poly(l-lactic acid) (SPLA50) was evaluated. Supercritical phase-inversion technique was used to prepare inorganic particles loaded starch-based porous composite matrixes in a one-step process for bone tissue engineering purposes.Due to their osteoconductive properties some glasses and ceramics are interesting materials to be used for bone tissue engineering purposes; however their poor mechanical properties create the need of a polymeric support where the inorganic fraction can be dispersed. Samples impregnated with different concentrations of Bioglass^® (10 and 15% wt/wt polymer) were prepared at 200 bar and 55 °C. The presence of Bioglass^® did not affect the porosity or interconnectivity of the polymeric matrixes. Dynamic mechanical analysis has proven that the modulus of the SPLA50 scaffolds increases when glass particles are impregnated within the matrix.In vitro bioactivity studies were carried out using simulated body fluid and the results show that a calcium-phosphate layer started to be formed after only 1 day of immersion. Chemical analysis of the apatite layer formed on the surface of the scaffold was performed by different techniques, namely EDS and FTIR spectroscopy and X-ray diffraction (XRD). The ion concentration in the simulated body fluid was also carried out by ICP analysis. Results suggest that a bone-like apatite layer was formed.This study reports the feasibility of using supercritical fluid technology to process, in one step, a porous matrix loaded with a bioactive material for tissue engineering purposes.     

聚氨酯软骨-骨双层复合材料的构建及性能研究

关节软骨损伤是临床上的常见病,由于其组织再生能力差,可能导致骨性关节炎的发生,因此,研究开发骨-软骨移植替代材料非常重要。目的就是设计一体化软骨-骨双层复合材料,以解决软骨与骨的整合问题。该双层复合体上层软骨材料为聚氨酯,软骨下骨为羟基磷灰石/聚氨酯复合支架材料,两层结构中引用了同一种材料——聚氨酯,将双层结构有机黏合在一起,使黏合更牢固。下层多孔HA/PU复合支架材料的孔与孔之间相互贯通,孔隙率约为83%,孔径范围分布在200～600μm。体外细胞相容性实验表明,该一体化双层复合材料为细胞的黏附、增殖以及生存活力的维持提供了有利环境。上述结果表明该双层复合材料有望用于软骨组织工程修复。     

Long term results after implantation of tissue engineered cartilage for the treatment of osteochondral lesions in a minipig model

In present study we determined the long term in vivo integration and histological modeling of an in vitro engineered cartilage construct. Tissue engineered autologous cartilagenous tissue was cultured on calcium phosphate cylinders and implanted into osteochondral defects into the femoral condyles in minipigs. Radiological follow-up was performed at 2, 8, 26 and 52 weeks, condyles were harvested 26 and 52 weeks post-implantation. Thickness of cultivated tissue (1.10 ± 0.55 mm) was comparable to in situ cartilage and cells produced in vitro cartilage specific proteins. In vivo, 26 and 52 weeks post-implantation defects were resurfaced with hyaline-like tissue, the implants were well integrated with no gap at the interface between the engineered neocartilage and the adjacent articular cartilage. Synthesis of type II collagen was detected 26 and 52 weeks after implantation. The modified ICRS score increased from 26 to 52 weeks. Histomorphometric evaluation revealed a decrease in cellularity in tissue engineered cartilage from 2.2-fold of native cartilage after 26 weeks to 1.5-fold after 52 weeks. In conclusion, these findings demonstrate the integration and maturation of tissue engineered cartilage pellets attached on a bone substitute carrier implanted in osteochondral defects over a long time. J. P. Petersen, P. Ueblacker, C. Goepfert have contributed equally to this study.     

Long-term in vivo response to citric acid-based nanocomposites for orthopaedic tissue engineering

The disadvantages of current bone grafts have triggered the development of a variety of natural and synthetic bone substitutes. Previously, we have described the fabrication, characterization, and short-term tissue response of poly(1,8-octanediol-co-citrate) (POC) with 60 weight % hydroxyapatite nanocrystals (POC-HA) at 6 weeks. In order to better understand the clinical potential, longer term effects, and the biodegradation, biocompatibility, and bone regenerative properties of these novel nanocomposites, POC-HA, POC, and poly-L-lactide (PLL) were implanted in osteochondral defects in a rabbit model and assessed at 26 weeks. Explants were stained with Masson Goldner Trichrome and the fibrous capsule and tissue ingrowth measured. In addition, the bone-implant and bone-cartilage response of POC-HA, POC, and PLL were assessed through histomorphometry and histological scoring. Upon histological evaluation, both POC-HA and POC implants were biocompatible, but PLL implants were surrounded by a layer of leukocytes at 26 weeks. In addition, due to the degradation properties of POC-HA, tissue grew into the implant and had the highest area of tissue ingrowth although not statistically significant. Histomorphometric analyses supported a similar osteoid, osteoblast, and trabecular bone surface area among all implants although the fibrous capsule thickness was the largest for POC. Moreover, histological scoring demonstrated comparable scores among all three groups of the articular cartilage and subchondral bone. This study provides the long-term bone and cartilage response of novel, citric acid-based nanocomposites and their equivalence to FDA-approved biomaterials. Furthermore, we provide new insights and further discussion of these nanocomposites for orthopaedic applications.     

Identifying chemical changes in subchondral bone taken from murine knee joints using Raman spectroscopy

Application of Raman spectroscopy to analysis of subchondral bone is described. The effect of cartilage health on subchondral bone has been widely studied using radiological and histological methods; however, there is no method to directly assay mineral components. We present Raman spectra of femur condyles and observe mineral bands that arise from the subchondral bone. In two separate experiments, transgenic mouse models of early-onset osteoarthritis (OA) and lipoatrophy were compared to tissue from wild-type mice. Raman spectroscopy was used to identify chemical changes in the mineral of subchondral bone that may accompany or precede morphological changes that can be observed by histology. The transgenic mice were compared to age-matched wild-type mice. Subtle alterations in the mineral or collagen matrix were observed by Raman spectroscopy using established Raman markers such as the carbonate-to-phosphate ratio, mineral-to-matrix ratio (MTMR), and amide I ratio. The Raman microscope configuration enabled rapid collection of Raman spectra from the mineralized layer that lies under an intact layer of non-mineralized articular cartilage. The effect of the cartilage layer on collection of spectra is discussed. The technique proposed is capable of providing insight into the chemical changes that occur in subchondral bone on a molecular level.     

Nanocrystalline apatites in biological systems: characterisation,structure and properties

Nanocrystalline apatitic calcium phosphates play a crucial role in calcified tissues and biomaterials. One of the most interesting characteristics of biomimetic apatite nanocrystals is the existence of a surface hydrated layer essentially related to their formation process in solution. This hydrated layer shows specific spectroscopic characteristics. It seems to exist in its nascent state only in wet samples and is altered on drying. This surface‐hydrated layer progressively disappears as the stable apatite domains develop. The surface ions can be rapidly and reversibly exchanged in solution, mainly with selected bivalent species. The exchange reactions clearly reveal the existence of two domains: the relatively inert apatite core and the very reactive surface‐hydrated domains. The structure of the hydrated layer has been shown to be reversibly affected by the constituting ions. Such a surface layer in bone apatite nanocrystals could participate actively in homeostasis and probably other regulation processes. The specificity of biomimetic apatite nanocrystals also opens interesting possibilities in materials science. The mobility of the mineral ions on the crystal surface, for example, allows strong bonding and interactions either with other crystals or with different substrates. Inter‐crystalline interactions have been described as a “crystal fusion” process in vivo and they could be involved in the setting reaction of biomimetic calcium phosphate cements. Ceramic‐like materials using the surface interaction capabilities of the nanocrystals can be produced at very low temperature (below 200 °C). The surface‐hydrated layer could also be involved in interactions with macromolecules and polymeric materials or in the coating of implants. The ion exchange and adsorption capabilities of the nanocrystals could probably be used for drug release, offering a range of possible behaviours.     

Repair of large osteochondral defects in a beagle model with a novel type I collagen/glycosaminoglycan-porous titanium biphasic scaffold

The limited repair potential of articular cartilage, which hardly heals after injury or debilitating osteoarthritis, is a clinical challenge. The aim of this work was to develop a novel type I collagen (Col)/glycosaminoglycan (GAGs)-porous titanium biphasic scaffold (CGT) and verify its ability to repair osteochondral defects in an animal model with bone marrow stem cells (bMSCs) in the chondral phase. The biphasic scaffold was composed of Col/GAGs as chondral phasic and porous titanium as subchondral phasic. Twenty-four full-thickness defects through the articular cartilage and into the subchondral bone were prepared by drilling into the surface of the femoral patellar groove. Animals were assigned to one of the three groups: 1) CGT with bMSCs (CGTM), 2) only CGT, and 3) no implantation (control). The defect areas were examined grossly, histologically and by micro-CT. The most satisfied cartilage repairing result was in the CGTM group, while CGT alone was better than the control group. Abundant subchondral bone formation was observed in the CGTM and CGT groups but not the control group. Our findings demonstrate that a composite based on a novel biphasic scaffold combined with bMSCs shows a high potential to repair large osteochondral defects in a canine model.     

Cellulose Acetate and Polyetherimide Blend Ultrafiltration Membranes, I: Preparation, Characterization, and Application

Ultrafiltration (UF) techniques have particular advantages for simultaneous purification, concentration, and fractionation of macromolecules. In this study, polymeric blend ultrafiltration membranes based on cellulose acetate and polyetherimide were prepared by phase inversion technique and characterized in terms of compaction time, pure water flux (PWF), water content, membrane hydraulic resistance, and scanning electron microscopy (SEM). The blend membranes prepared were subjected to the separation of macromolecular proteins such as bovine serum albumin (BSA), egg albumin (EA), pepsin, and trypsin. The molecular weight cut-off (MWCO) of blend membranes obtained from the protein separation studies is also presented. Toxic heavy metal ions such as copper, nickel, zinc, and cadmium were subjected to separation by the blend membranes by complexing them with the polymeric ligand polyethyleneimine. The separation and permeate flux efficiencies of the blend membranes are compared with those of pure cellulose acetate membranes.