Engineered Osteochondral Grafts Using Biphasic Composite Solid Free-Form Fabricated Scaffolds (original) (raw)
Related papers
Journal of Tissue Engineering and Regenerative Medicine, 2014
Sufficient treatment of chondral and osteochondral defects to restore function of the respective tissue remains challenging in regenerative medicine. Biphasic scaffolds that mimic properties of bone and cartilage are appropriate to regenerate both tissues at the same time. The present study describes the development of biphasic, but monolithic scaffolds based on alginate, which are suitable for embedding of living cells in the chondral part. Scaffolds are fabricated under sterile and cellcompatible conditions according to the principle of diffusion-controlled, directed ionotropic gelation, which leads to the formation of channel-like, parallel aligned pores, running through the whole length of the biphasic constructs. The synthesis process leads to an anisotropic structure, as it is found in many natural tissues. The two different layers of the scaffolds are characterized by different microstructure and mechanical properties which provide a suitable environment for cells to form the respective tissue. Human chondrocytes and human mesenchymal stem cells were embedded within the chondral layer of the biphasic scaffolds during hydrogel formation and their chondrogenic (re)differentiation was successfully induced. Whereas viability of non-induced human mesenchymal stem cells decreased during culture, cell viability of human chondrocytes and chondrogenically induced human mesenchymal stem cells remained high within the scaffolds over the whole culture period of 3 weeks, demonstrating successful fabrication of cell-laden centimetre-scaled constructs for potential application in regenerative treatment of osteochondral defects.
Design and characterization of a tissue-engineered bilayer scaffold for osteochondral tissue repair
Journal of Tissue Engineering and Regenerative Medicine, 2012
Treatment of full-thickness cartilage defects relies on osteochondral bilayer grafts, which mimic the microenvironment and structure of the two affected tissues: articular cartilage and subchondral bone. However, the integrity and stability of the grafts are hampered by the presence of a weak interphase, generated by the layering processes of scaffold manufacturing. We describe here the design and development of a bilayer monolithic osteochondral graft, avoiding delamination of the two distinct layers but preserving the cues for selective generation of cartilage and bone. A highly porous polycaprolactone-based graft was obtained by combining solvent casting/particulate leaching techniques. Pore structure and interconnections were designed to favour in vivo vascularization only at the bony layer. Hydroxyapatite granules were added as bioactive signals at the site of bone regeneration. Unconfined compressive tests displayed optimal elastic properties and low residual deformation of the graft after unloading (< 3%). The structural integrity of the graft was successfully validated by tension fracture tests, revealing high resistance to delamination, since fractures were never displayed at the interface of the layers (n = 8). Ectopic implantation of grafts in nude mice, after seeding with bovine trabecular bone-derived mesenchymal stem cells and bovine articular chondrocytes, resulted in thick areas of mature bone surrounding ceramic granules within the bony layer, and a cartilaginous alcianophilic matrix in the chondral layer. Vascularization was mostly observed in the bony layer, with a statistically significant higher blood vessel density and mean area. Thus, the easily generated osteochondral scaffolds, since they are mechanically and biologically functional, are suitable for tissue-engineering applications for cartilage repair.
The goal of this study was to determine the efficacy of the bioactive scaffold system to initiate bone marrow stromal cell (BMSC) differentiation into osteogenic and chondrogenic lineages in various culture media compositions. In the biphasic polymeric scaffolds, the chondrogenic layer contained aligned polycaprolactone nanofibers embedded with chondroitin sulfate and hyaluronic acid, while osteogenic layer carried nano-hydroxyapatite. Many studies for in vitro testing of osteochondral scaffolds incorporate the use of complicated bioreactors or growth factors for the formation of cartilage and bone tissue, thus true efficacy of the scaffold system cannot be determined. The present study compared the effect of several media compositions consisting of osteogenic, chondrogenic components, and control basal media. Scaffolds seeded with BMSCs following 28 days in vitro culture in different induction and basal media were evaluated for osteogenic and chondrogenic markers such as aggrecan, collagen type II, bone sialoprotein, alkaline phosphatase (ALP), and runt-related transcription factor 2 (Runx-2). Cartilage scaffold layer of the biphasic scaffold resulted in the expression of chondrogenic markers such as aggrecan and collagen type II by BMSCs in control and induction media compositions. The bone scaffold layer supported the expression of osteogenic markers such as ALP and Runx-2 by BMSCs in control and induction media compositions. The cartilage scaffold layer under the osteogenic induction media encouraged the growth of hypertrophic cartilage as marked by the positive expression of Runx-2. Expression of collagen type II and aggrecan on the cartilage layer in basal media was confirmed by immunostaining. These studies suggest that the bioactive scaffolds were able to support the osteogenic and chondrogenic phenotype development in the absence of growth factors and induction media.
Tissue Engineering, 2006
Animal studies in cartilage tissue engineering usually include the transfer of cultured cells into chondral or osteochondral defects. Immediately at implantation, the cells are exposed to a dramatically changed environment. The aim of this study was to determine the viability of two cell types currently considered for cellular therapies of cartilage defects-chondrocytes and progenitor cells-shortly after exposure to an osteochondral defect in rabbit knees. To that end, autogenic chondrocytes and periosteal cells were labeled with CM-DiI fluorochrome, seeded or cultured in PEGT/PBT scaffolds for periods up to 2 weeks, transferred into osteochondral defects, harvested 5 days postimplantation, and analyzed for cell viability. In order to further elucidate factors effecting cell viability within our model system, we investigated the effect of serum, 2) extracellular matrix surrounding implanted cells, 3) scaffold interconnectivity, and 4) hyaluronan, as a known cell protectant. Controls included scaffolds with devitalized cells and scaffolds analyzed at implantation. We found that the viability of periosteum cells (14%), but not of chondrocytes (65-95%), was significantly decreased after implantation. The addition of hyaluronan increased periostium cell viability to 44% (p Ͻ 0.05). Surprisingly, cell viability in less interconnected compression-molded scaffolds was higher compared to that of fully interconnected scaffolds produced by rapid prototyping. All other factors tested did not affect viability significantly. Our data suggest chondrocytes as a suitable cell source for cartilage repair in line with clinical data on several chondrocyte-based therapies. Although we did not test progenitor cells other the periosteum cells, tissue-engineering approaches using such cell types should take cell viability aspects into consideration.
Current Concepts and Challenges in Osteochondral Tissue Engineering and Regenerative Medicine
ACS Biomaterials Science & Engineering, 2015
In the last few years, great progress has been made to validate tissue engineering strategies in preclinical studies and clinical trials on the regeneration of osteochondral defects. In the preclinical studies, one of the dominant strategies comprises the development of biomimetic/bioactive scaffolds, which are used alone or incorporated with growth factors and/or stem cells. Many new trends are emerging for modulation of stem cell fate towards osteogenic and chondrogenic differentiations, but bone/cartilage interface regeneration and physical stimulus have been showing great promise. Besides the matrix-associated autologous chondrocyte implantation (MACI) procedure, the matrix-associated stem cells implantation (MASI) and layered scaffolds in acellular or cellular strategy are also applied in clinic. This review outlines the progresses at preclinical and clinical levels, and identifies the new challenges in osteochondral tissue engineering. Future perspectives are provided, e.g., the applications of extracellular matrix-like biomaterials, computer-aided design/manufacture of osteochondral implant and reprogrammed cells for osteochondral regeneration.
Journal of Tissue Engineering, 2019
Bone/cartilage interfacial tissue engineering needs to satisfy the differential properties and architectures of the osteochondral region. Therefore, biphasic or multiphasic scaffolds that aim to mimic the gradient hierarchy are widely used. Here, we find that two differently structured (topographically) three-dimensional scaffolds, namely, "dense" and "nanofibrous" surfaces, show differential stimulation in osteo-and chondro-responses of cells. While the nanofibrous scaffolds accelerate the osteogenesis of mesenchymal stem cells, the dense scaffolds are better in preserving the phenotypes of chondrocytes. Two types of porous scaffolds, generated by a salt-leaching method combined with a phase-separation process using the poly(lactic acid) composition, had a similar level of porosity (~90%) and pore size (~150 μm). The major difference in the surface nanostructure led to substantial changes in the surface area and water hydrophilicity (nanofibrous >> dense); as a result, the nanofibrous scaffolds increased the cell-to-matrix adhesion of mesenchymal stem cells significantly while decreasing the cell-to-cell contracts. Importantly, the chondrocytes, when cultured on nanofibrous scaffolds, were prone to lose their phenotype, including reduced chondrogenic expressions (SOX-9, collagen type II, and Aggrecan) and glycosaminoglycan content, which was ascribed to the enhanced cell-matrix adhesion with reduced cell-cell contacts. On the contrary, the osteogenesis of mesenchymal stem cells was significantly accelerated by the improved cell-to-matrix adhesion, as evidenced in the enhanced osteogenic expressions (RUNX2, bone sialoprotein, and osteopontin) and cellular mineralization. Based on these findings, we consider that the dense scaffold is preferentially used for the chondral-part, whereas the nanofibrous structure is suitable for osteo-part, to provide an optimal biphasic matrix environment for osteochondral tissue engineering.
Tissue Engineering, 2006
In order to evaluate the repair potential in large osteochondral defects on high load-bearing sites, a hybrid scaffold system was made of three-dimensional porous Polycaprolactone (PCL) scaffold for the cartilage and tricalcium phosphate-reinforced PCL for the bone portion. Osteochondral defects of 4-mm diameter ؋ 5.5-mm depth were created in the medial femoral condyle of adult New Zealand White rabbits. The defects were treated with hybrid scaffolds without cells (control group) or seeded with allogenic bone marrow-derived mesenchymal stem cells (BMSC) in each part (experimental group) by press-fit implantation. Implanted cells were tracked using Adeno-LacZ labeling. Repair tissues were evaluated at 3 and 6 months after implantation. Overall, the experimental group showed superior repair results as compared to the control group using gross examination, qualitative and quantitative histology, and biomechanical assessment. With BMSC implantation, the hybrid scaffolds provided sufficient support to new osteochondral tissues formation. The bone regeneration was consistently good from 3 to 6 months with firm integration to the host tissue. Cartilage layer resurfacing was more complicated. All of the samples showed cartilage tissues mixed with PCL scaffold filaments at 3 months. Histology at 6 months revealed some degradation phenomenon in several samples whereas others had a good appearance; however, the Young's moduli from the experimental group (0.72 MPa) approached that of normal cartilage (0.81 MPa).
Biomaterials, 2010
In this study, we aimed at developing and validating a technique for the engineering of osteochondral grafts based on the biological bonding of a chondral layer with a bony scaffold by cell-laid extracellular matrix. Osteochondral composites were generated by combining collagen-based matrices (Chondro-Gide Ò ) containing human chondrocytes with devitalized spongiosa cylinders (Tutobone Ò ) using a fibrin gel (Tisseel Ò ). We demonstrate that separate pre-culture of the chondral layer for 3 days prior to the generation of the composite allows for (i) more efficient cartilaginous matrix accumulation than no pre-culture, as assessed histologically and biochemically, and (ii) superior biological bonding to the bony scaffold than 14 days of pre-culture, as assessed using a peel-off mechanical test, developed to measure integration of bilayered materials. The presence of the bony scaffold induced an upregulation in the infiltrated cells of the osteoblast-related gene bone sialoprotein, indicative of the establishment of a gradient of cell phenotypes, but did not affect per se the quality of the cartilaginous matrix in the chondral layer. The described strategy to generate osteochondral plugs is simple to be implemented and -since it is based on clinically compliant cells and materials -is amenable to be readily tested in the clinic.
Tissue Engineering Part C: Methods, 2011
Our long-term goal is to treat osteochondral lesions with bioengineered biphasic constructs. We have previously demonstrated that biphasic constructs, created in vitro with primary chondrocytes harvested from healthy joints and a porous calcium polyphosphate (CPP) substrate bone substitute, could successfully repair a focal defect in sheep joints. However, primary chondrocytes are limited in supply and cannot be used in engineering constructs large enough for clinical use. Thus, we developed a robust protocol to predifferentiate sheep bone marrow-derived stromal cells to chondrocytes on collagen-coated polytetrafluoroethane membrane inserts, and harvest the chondrocytes that develop and subsequently culturing these predifferentiated cells scaffold-free on the intended articulation surface of the CPP. Chondrocytes predifferentiated on membrane culture accumulated similar matrix as those in conventional pellet culture, but expressed less Col1a1 RNA. Membrane culture predifferentiated cells gave rise to a functionally superior hyaline cartilage tissue compared to pellet culture predifferentiated cells. Studies demonstrated that 2 weeks of membrane predifferentiation culture followed by 8 weeks of biphasic construct culture was the optimal culture period at which the compressive mechanical strength and the accumulation of extracellular matrix were maximized while avoiding tissue mineralization. This protocol will be used to generate implants for preclinical study to determine their ability to repair osteochondral lesions.
Veterinary Sciences
Osteochondral defects are a common problem in both human medicine and veterinary practice although with important limits concerning the cartilaginous tissue regeneration. Interest in the subchondral bone has grown, as it is now considered a key element in the osteochondral defect healing. The aim of this work was to generate and to evaluate the architecture of three cell-free scaffolds made of collagen, magnesium/hydroxyapatite and collagen hydroxyapatite/wollastonite to be implanted in a sheep animal model. Scaffolds were designed in a bilayer configuration and a novel “Honey” configuration, where columns of hydroxyapatite were inserted within the collagen matrix. The use of different types of scaffolds allowed us to identify the best scaffold in terms of integration and tissue regeneration. The animals included were divided into four groups: three were treated using different types of scaffold while one was left untreated and represented the control group. Evaluations were made at...