Cartilage Regeneration Applications Research Articles

Natural polymer-based hydrogels for cartilage regeneration

Purpose: Cartilage is a tough, flexible tissue found throughout the body. It can become damaged because of a sudden injury, such as a sports injury, or gradual wear and tear (osteoarthritis). Injured articular cartilage has a poor regenerative capability, for this reason, tissue engineering may provide a fundamental therapeutic solution to repair cartilage injury. Human articular cartilages are composed of dense extracellular matrix and chondrocytes. One of the major components in the extracellular matrix is hyaluronic acid. The combination of hyaluronic acid with other natural polymers to prepare hydrogel scaffolds may be potentially useful in many tissue engineering applications, including cartilage regeneration. We propose the synthesis of hydrogels based on hyaluronic acid and chitosan by a new strategy of synthesis using di-isocyanates to obtain an interpenetrated network of chitosan and hyaluronic acid. Methods: Different kinds of hydrogels were synthesized using hyaluronic acid (Bioibérica, Spain), natural polymers (gelatine, chitosan) and stabilized with diisocianates. The hydrogels were charged with chondroitin sulfate (Bioibérica, Spain). Morphological Characterization: The hydrogels were characterized by scanning electron microscopy and energy dispersive X-ray microscopy in order to study the morphology and the composition of the hydrogels. Hydrogel Swelling: Dynamic swelling experiments were performed by placing discs of stabilized hydrogels in PBS at 37.0 °C, and measuring their weight gain as a function of time. Biocompatibility: Cytotoxicity in vitro assays (MTT) and cell adhesion tests (Alamar Blue) were performed on the hydrogels using different cell lines to corroborate the biocompatibility of the hydrogels. Results: All the synthesized hydrogels present a high interconnected porosity. The studied hydrogels swell around 200%. Aaround five hours equilibrium is reached and this equilibrium depend on the ratio hyaluronic acid/natural polymer increasing with the amount of hyaluronic acid present on the hydrogel. MTT assay indicated that the extracts collected at different times from the hydrogels are not cytotoxic and could be considered biocompatible. Cell adhesion assay indicates that the hydrogels support cell adhesion and proliferation, and this behaviour is better on SC-loaded hydrogels. Conclusions: Hydrogels based on hyaluronic acid and natural polymers are proposed as scaffolds for tissue engineering. The presence of interconnected porous and the good swelling properties and biocompatibility represent a great advantage for application in cartilage regeneration. The addition of drugs or glycosaminoglycan is being studied to improve the cartilage regeneration.

실크 피브로인과 젤란검 하이드로젤에서 연골 재생 효과에 대한 연구

최근 조직 공학에서 새로운 생체 재료로 관심을 받고 있는 젤란검은 포도당, 글루콘산, 람노스로 구성된 다당류로서 연골 조직 재생의 대체재로 각광받고 있다. 실크 피브로인은 생체적합성과 생분해성이 뛰어나며 우수한 기계적 특성을 갖고 있다. 본 실험은 실크 피브로인이 연골 재생에 미치는 영향을 확인하기 위해 2 wt% 젤란검 수용액에 2 wt% 실크 피브로인 수용액을 100/0, 70/50, 50/50 비율로 첨가하여 하이드로젤 지지체를 제조하였으며, 실크 피브로인 수용액이 다양한 함량으로 제작된 젤란검 하이드로젤 지지체의 물리적 특성을 알아보고자 압축강도, 다공도, FTIR 등을 실시하였고 연골 세포의 증식과 유전자 발현을 평가하기 위해 MTT, bio-SEM, RT-PCR을 실시한 결과 50/50 비율의 젤란검/실크 피브로인(GG/SF) 하이드로젤 지지체에서 높은 세포 증식률과 우수한 연골재생 효과를 확인하였다. 결과적으로 50/50 비율의 젤란검/실크 피브로인 하이드로젤 지지체가 연골 조직 재생에 긍정적으로 작용함을 확인하였다.

Bone Marrow- and Adipose Tissue-Derived Mesenchymal Stem Cells: Characterization, Differentiation, and Applications in Cartilage Tissue Engineering.

Articular cartilage defects have very limited self-repair potential, and traditional bone marrow-stimulating therapy is not effective. Cartilage tissue engineering using bone marrow mesenchymal stem cells (BMSCs) and adipose tissue-derived mesenchymal stem cells (ADSCs) is considered an attractive treatment for cartilage lesions and osteoarthritis. However, studies proved that both BMSCs and ADSCs have their own advantages and shortcomings, including their sources, isolation methods, characterizations and differentiation potential. Understanding the properties and differences between ADSCs and BMSCs is important for clinical application in cartilage regeneration. This review provides an overview of BMSCs and ADSCs based on their characterization, isolation. Then, we summarized their differentiation potential in different experimental conditions. Finally, we discuss the applications of BMSCs and ADSCs in scaffold-free and scaffold-based cartilage tissue engineering. Based on different properties of BMSCs and ADSCs, and patient's physical condition, a more suitable therapeutic strategy can be selected.

Enhanced chondrogenic differentiation of human mesenchymal stems cells on citric acid-modified chitosan hydrogel for tracheal cartilage regeneration applications.

Congenital tracheal stenosis in infants and children is a worldwide clinical problem. Tissue engineering is a promising method for correcting long segmental tracheal defects. Nonetheless, the lack of desirable scaffolds always limits the development and applications of tissue engineering in clinical practice. In this study, a citric-acid-functionalized chitosan (CC) hydrogel was fabricated by a freeze–thaw method. Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) confirmed that citric acid was successfully attached to the chitosan hydrogel. Scanning electron microscopy (SEM) images and compression tests showed that the CC hydrogel had an interconnected porous structure and better wet mechanical properties. Using morphological and proliferation analyses, cell biocompatibility of the CC hydrogel was shown by culturing human mesenchymal stem cells (hMSCs) on it. Specific expression of cartilage-related markers was analyzed by real-time polymerase chain reaction and western blotting. The expression of chondrocytic markers was strongly upregulated in the culture on the CC hydrogel. Hematoxylin and eosin staining revealed that the cells had the characteristic shape of chondrocytes and clustered into the CC hydrogel. Both Alcian blue staining and a sulfated glycosaminoglycan (sGAG) assay indicated that the CC hydrogel promoted the expression of glycosaminoglycans (GAGs). In a nutshell, these results suggested that the CC hydrogel enhanced chondrogenic differentiation of hMSCs. Thus, the newly developed CC hydrogel may be a promising tissue-engineered scaffold for tracheal cartilage regeneration.

A novel hybrid multichannel biphasic calcium phosphate granule-based composite scaffold for cartilage tissue regeneration.

The objective of the present study was to develop a novel hybrid multichannel biphasic calcium phosphate granule (MCG)-based composite system for cartilage regeneration. First, hyaluronic acid-gelatin (HG) hydrogel was coated onto MCG matrix (MCG-HG). Poly(lactic-co-glycolic acid) (PLGA) microspheres was separately prepared and modified with polydopamine subsequent to BMP-7 loading (B). The surface-modified microspheres were finally embedded into MCG-HG scaffold to develop the novel hybrid (MCG-HG-PLGA-PD-B) composite system. The newly developed MCG-HG-PLGA-PD-B composite was then subjected to scanning electron microscopy, energy dispersive X-ray spectroscopy, Fourier Transform infrared spectroscopy, porosity, compressive strength, swelling, BMP-7 release and in-vitro biocompatibility studies. Results showed that 60% of BMP-7 retained on the granular surface after 28 days. A hybrid MCG-HG-PLGA-PD-B composite scaffold exhibited higher swelling and compressive strength compared to MCG-HG or MCG. In-vitro studies showed that MCG-HG-PLGA-PD-B had improved cell viability and cell proliferation for both MC3T3-E1 pre-osteoblasts and ATDC5 pre-chondrocytes cell line with respect to MCG-HG or MCG scaffold. Our results suggest that a hybrid MCG-HG-PLGA-PD-B composite scaffold can be a promising candidate for cartilage regeneration applications.

Mesenchymal Stem/Progenitor Cells Derived from Articular Cartilage, Synovial Membrane and Synovial Fluid for Cartilage Regeneration: Current Status and Future Perspectives.

Large articular cartilage defects remain an immense challenge in the field of regenerative medicine because of their poor intrinsic repair capacity. Currently, the available medical interventions can relieve clinical symptoms to some extent, but fail to repair the cartilaginous injuries with authentic hyaline cartilage. There has been a surge of interest in developing cell-based therapies, focused particularly on the use of mesenchymal stem/progenitor cells with or without scaffolds. Mesenchymal stem/progenitor cells are promising graft cells for tissue regeneration, but the most suitable source of cells for cartilage repair remains controversial. The tissue origin of mesenchymal stem/progenitor cells notably influences the biological properties and therapeutic potential. It is well known that mesenchymal stem/progenitor cells derived from synovial joint tissues exhibit superior chondrogenic ability compared with those derived from non-joint tissues; thus, these cell populations are considered ideal sources for cartilage regeneration. In addition to the progress in research and promising preclinical results, many important research questions must be answered before widespread success in cartilage regeneration is achieved. This review outlines the biology of stem/progenitor cells derived from the articular cartilage, the synovial membrane, and the synovial fluid, including their tissue distribution, function and biological characteristics. Furthermore, preclinical and clinical trials focusing on their applications for cartilage regeneration are summarized, and future research perspectives are discussed.

Sol-gel derived lithium-releasing glass for cartilage regeneration.

Wnt-signalling cascade is one of the crucial pathways involved in the development and homeostasis of cartilage. Influencing this pathway can potentially contribute to improved cartilage repair or regeneration. One key molecular regulator of the Wnt pathway is the glycogen synthase kinase-3 enzyme, the inhibition of which allows initiation of the signalling pathway. This study aims to utilise a binary SiO2-Li2O sol-gel derived glass for controlled delivery of lithium, a known glycogen synthase kinase-3 antagonist. The effect of the dissolution products of the glass on chondrogenic differentiation in an invitro 3D pellet culture model is reported. Dissolution products that contained 5 mM lithium and 3.5 mM silicon were capable of inducing chondrogenic differentiation and hyaline cartilaginous matrix formation without the presence of growth factors such as TGF-β3. The results suggest that sol-gel derived glass has the potential to be used as a delivery vehicle for therapeutic lithium ions in cartilage regeneration applications.

Supramolecular polypseudorotaxane gels for controlled delivery of rAAV vectors in human mesenchymal stem cells for regenerative medicine

The aim of this work was to investigate, for the first time, the possibility of using supramolecular polypseudorotaxane gels as scaffolds that can durably deliver rAAV vectors for applications in cartilage regeneration. Dispersions of Pluronic® F68 (PF68) or Tetronic® 908 (T908) containing either hyaluronic acid (HA) or chondroitin sulfate (CS) were prepared in PBS. Then, alpha-cyclodextrin (αCD) was added to some dispersions to form polypseudorotaxane gels. Polysaccharides and αCD reinforced the viscoelasticity of the gels, which could withstand autoclaving without changes. In vitro release of rAAV vectors and subsequent transduction of human mesenchymal stem cells (hMSCs) by rAAV vectors from the release medium and from gels in direct contact with the cells were investigated. Compared with free vectors, the gels provided higher levels of transgene expression. CS (or HA)/PF68/αCD gels rapidly released rAAV vectors while CS (or HA)/T908/αCD gels provided sustained release probably due to different interactions with the viral vectors. Incorporation of αCD into CS (or HA)/PF68 gels resulted on higher rAAV concentrations and sustained levels of transgene expression over time. HA increased the bioactivity and cytocompatibility of the gels, especially those based on T908. Overall, combining rAAV gene transfer with polypseudorotaxane gels may provide new, promising tools for human tissue engineering and regenerative medicine strategies.

Alginate hydrogels modified with low molecular weight hyaluronate for cartilage regeneration

Alginate is a typical biomaterial that forms hydrogels in the presence of calcium ions and has often been utilized in tissue engineering approaches. However, it lacks biofunctionality in the form of interactions with cells and proteins. Hyaluronate, a main component of glycosaminoglycans, provides CD44-specific interactions with chondrocytes but typically requires chemical cross-linking agents to fabricate hydrogels, which may cause unexpected side effects in the body. In this study, we propose the design and fabrication of a hybrid structure of alginate and hyaluronate useful for cartilage regeneration. Alginate was used as a backbone, and hyaluronate with a low molecular weight was introduced to the backbone to fabricate alginate-hyaluronate hybrid coupled by ethylenediamine. We hypothesized that alginate-hyaluronate hybrid (AH) could maintain its ability to form gels in the presence of calcium ions and could be useful for cartilage regeneration as an injectable system. Characteristics of AH hydrogels containing various composition ratios of hyaluronate to alginate were investigated, and the chondrogenic potential of ATDC5 cells encapsulated within AH hydrogels was evaluated in vitro. Consequently, AH hydrogels having a defined polymer composition and mechanical stiffness were useful to successfully regulate chondrogenic differentiation and to maintain the chondrocytic cell phenotype, which may lead to many useful applications in cartilage regeneration.

Multifunctional biomaterials from the sea: Assessing the effects of chitosan incorporation into collagen scaffolds on mechanical and biological functionality

Natural biomaterials such as collagen show promise in tissue engineering applications due to their inherent bioactivity. The main limitation of collagen is its low mechanical strength and somewhat unpredictable and rapid degradation rate; however, combining collagen with another material, such as chitosan, can reinforce the scaffold mechanically and may improve the rate of degradation. Additionally, the high cost and the risk of prion transmission associated with mammal-derived collagen has prompted research into alternative sources such as marine-origin collagen. In this context, the overall goal of this study was to determine if the incorporation of chitosan into collagen scaffolds could improve the mechanical and biological properties of the scaffold. In addition the study assessed if collagen, derived from salmon skin (marine), can provide an alternative to collagen derived from bovine tendon (mammal) for tissue engineering applications. Scaffold architecture and mechanical properties were assessed as well as their ability to support mesenchymal stem cell growth and differentiation. Overall, the addition of chitosan to bovine and salmon skin-derived collagen scaffolds improved the mechanical properties, increasing the compressive strength, swelling ratio and prolonged the degradation rate. Mesenchymal stem cell (MSC) attachment and proliferation was most improved on the bovine-derived collagen scaffold containing a 75:25 ratio of collagen:chitosan, and when MSC osteogenic and chondrogenic potential on the scaffold was assessed, a significant increase in calcium production (p<0.001) and sulfated glycosaminoglycan (sGAG) production (p<0.001) was observed respectively. Regardless of chitosan content, the bovine-derived collagen scaffolds out-performed the salmon skin-derived collagen scaffolds, displaying a larger pore size and higher percentage porosity, more regular architecture, higher compressive modulus, a greater capacity for water uptake and allowed for more MSC proliferation and differentiation. This versatile scaffold incorporating the marine biomaterial chitosan show great potential as appropriate platforms for promoting orthopaedic tissue repair while the use of salmon skin-derived collagen may be more suitable in the repair of soft tissues such as skin. Statement of SignificanceCollagen is commonly used in tissue engineering due to its biocompatibility; however, it has low mechanical strength and an unpredictable degradation rate. In addition, high cost and risk of prion transmission associated with mammalian-derived collagen has prompted research into alternative collagen sources, namely, marine-derived collagen. In this study, scaffolds made from salmon-skin collagen were compared to the more commonly used bovine-derived collagen with a focus on orthopaedic applications. To improve the mechanical properties of these scaffolds, another marine biomaterial, chitosan, was added to produce scaffolds with increased mechanical stability. The collagen-chitosan composites were also shown to support mesenchymal stem cell differentiation towards both bone and cartilage tissue. This multi-functional scaffold therefore has potential in both bone and cartilage regeneration applications.

Cruciate ligament-derived mesenchymal stem cells: a potential cell source for cartilage regeneration

Background: Mesenchymal stem cells (MSCs) have been widely investigated for potential in tissue regeneration. The aim of this study was to compare the proliferation and induced differentiation potentials of MSCs isolated from bone marrow (BMSCs), adipose (AMSCs) and cruciate ligament (CLMSCs). Methods: BMSCs, AMSCs and CLMSCs were isolated and identified by flow cytometry with CD markers. Their clonogenicity, proliferation and multi-differentiation capacities were also analyzed. The mRNA expression of adipogenic, osteogenic, and chondrogenic markers at basal state and after multi-lineage inductions was examined using quantitative real-time polymerase chain reaction (qRT-PCR). Results: BMSCs, AMSCs and CLMSCs showed similar positive expression for CD29, CD90, and negative for CD45. Comparative analyses suggested the superior colony forming and proliferation capacity in CLMSCs. When induced toward adipo-, osteo- and chondro-lineages, BMSCs, AMSCs and CLMSCs could successfully differentiate toward target lineages. At basal state and after multi-lineage inductions, AMSCs expressed the highest level of adipogenic markers [peroxisome proliferator-activated receptor gamma 2 (PPARγ2) and CCAAT enhancer binding protein alpha (c/EBPα)]; BMSCs expressed the highest level of osteogenic markers [Alpl, Bglap, Runx2, bone morphogenetic protein 2 (Bmp2), and Spp1]; CLMSCs expressed the highest level of chondrogenic markers (Col2A1, Acan, and Sox9). Conclusions: This study demonstrates that CLMSCs and AMSCs exhibit superiority in cell proliferation compared to BMSCs. CLMSCs can be a potential cell source for clinical application in cartilage regeneration.

Polycaprolactone foam functionalized with chitosan microparticles - a suitable scaffold for cartilage regeneration.

For biodegradable porous scaffolds to have a potential application in cartilage regeneration, they should enable cell growth and differentiation and should have adequate mechanical properties. In this study, our aim was to prepare biocompatible scaffolds with improved biomechanical properties. To this end, we have developed foam scaffolds from poly-epsilon-caprolactone (PCL) with incorporated chitosan microparticles. The scaffolds were prepared by a salt leaching technique from either 10 or 15 wt% PCL solutions containing 0, 10 and 20 wt% chitosan microparticles, where the same amount and size of NaCl was used as a porogen in all the cases. PCL scaffolds without and with low amounts of chitosan (0 and 10 wt% chitosan) showed higher DNA content than scaffolds with high amounts of chitosan during a 22-day experiment. 10 wt% PCL with 10 and 20 wt% chitosan showed significantly increased viscoelastic properties compared to 15 wt% PCL scaffolds with 0 and 10 wt% chitosan. Thus, 10 wt% PCL scaffolds with 0 wt% and 10 wt% chitosan are potential scaffolds for cartilage regeneration.

Combinatory approach for developing silk fibroin-based scaffolds with hierarchical porosity and enhanced performance for cartilage tissue engineering applications

Event Abstract Back to Event Combinatory approach for developing silk fibroin-based scaffolds with hierarchical porosity and enhanced performance for cartilage tissue engineering applications Viviana P. Ribeiro1, 2*, Le-Ping Yan1, 2, Ana L. Oliveira1, 2, 3*, Joaquim M. Oliveira1, 2* and Rui L. Reis1, 2* 1 University of Minho, 3B’s Research Group – Biomaterials, Biodegradables and Biomimetics, Portugal 2 University of Minho, ICVS/3B’s - PT Government Associate Laboratory, Portugal 3 Portuguese Catholic University, CBQF–Center for Biotechnology and Fine Chemistry, Portugal Introduction: The combination of several processing technologies can open the possibility for producing scaffolds with superior performance for tissue engineering (TE) applications. Hydrogels are structurally similar to the natural extracellular matrix microenvironment presenting high elasticity and resistance to compression forces. They have been extensively used in biomedical devices fabrication and for TE applications, including for cartilage defects repair[1]. Recently, it was found that proteins like silk fibroin (SF), presenting tyrosine groups can be used to prepare fast formed hydrogels with controlled gelation properties, via an enzyme-mediated cross-linking reaction using horseradish peroxidase (HRP) and hydrogen peroxide (H2O2)[2],[3]. Moreover, the high versatility, processability and tailored mechanical properties of SF, make this natural polymer attractive for the development of innovative scaffolding strategies for cartilage TE applications[4],[5]. Materials and Methods: The present work proposes a novel route for developing SF-based scaffolds derived from high-concentrated SF (16wt%) enzymatically cross-linked by a HRP/H2O2 complex. The combination of salt-leaching and freeze-drying methodologies was used to prepare macro/microporous SF scaffolds with an interconnected structure and specific features regarding biodegradation and mechanical properties (Fig. 1a). The scaffolds morphology and porosity were analyzed by SEM and micro-CT. The mechanical properties (Instron) and protein conformation (FTIR, XRD) were also assessed. In order to evaluate the scaffolds structural integrity, swelling ratio and degradation profile studies were performed for a period of 30 day. This work also aims to evaluate the in vitro chondrogenic differentiation response by culturing human adipose derived stem cells (hASCs) over 21 days in basal and chondrogenic conditions. Cell behaviour in the presence of the macro/microporous structures will be evaluated through different quantitative (Live/Dead, DNA, GAGs, RT PCR) and qualitative (SEM, histology, immunocytochemistry) assays. Results and Discussion: The macro/microporous SF scaffolds showed high porosity and interconnectivity with the trabecular structures evenly distributed (Fig. 1b,c). A dramatic decrease of compressive modulus was observed for samples in hydrated state. Chemical analysis revealed that SF scaffolds displayed the characteristic peaks for β-sheet conformation. Swelling ratio data demonstrated a large swelling capacity, maintaining their structural integrity for 30 days. As expected, when immersed in protease XIV the degradation rate of SF scaffolds increased. Based on the promising morphology and physicochemical properties of the developed SF scaffolds, in vitro chondrogenic differentiation studies with hASCs are envisioned in order to validate their performance for cartilage regeneration applications. Figure 1- (a) Macroscopic image, (b)Three-dimensional micro-CT image and (c) SEM micrograph (cross-section) of the SF scaffolds. Conclusion: This study proposes an innovative approach to produce fast-formed porous SF scaffolds using enzymatically cross-linked SF hydrogels structured by the combination of salt-leaching and freeze-drying methodologies. The obtained results can provide a valuable reference of SF as a tunable and versatile biomaterial with great potential for applications in cartilage TE scaffolding. Portuguese Foundation for Science and Technology (FCT) project PEst-C/SAU/LA0026/201; ERDP funding through POCTEP Project 0687_NOVOMAR_1_P; Investigator FCT program IF/00423/2012 and IF/00411/2013

Transforming Growth Factor-β-Induced KDM4B Promotes Chondrogenic Differentiation of Human Mesenchymal Stem Cells.

The high prevalence of cartilage diseases and limited treatment options create a significant biomedical burden. Due to the inability of cartilage to regenerate itself, introducing chondrocyte progenitor cells to the affected site is of significant interest in cartilage regenerative therapies. Tissue engineering approaches using human mesenchymal stem cells (MSCs) are promising due to their chondrogenic potential, but a comprehensive understanding of the mechanisms governing the fate of MSCs is required for precise therapeutic applications in cartilage regeneration. TGF-β is known to induce chondrogenesis by activating SMAD signaling pathway and upregulating chondrogenic genes such as SOX9; however, the epigenetic regulation of TGF-β-mediated chondrogenesis is not understood. In this report, we found that TGF-β dramatically induced the expression of KDM4B in MSCs. When KDM4B was overexpressed, chondrogenic differentiation was significantly enhanced while KDM4B depletion by shRNA led to a significant reduction in chondrogenic potential. Mechanistically, upon TGF-β stimulation, KDM4B was recruited to the SOX9 promoter, removed the silencing H3K9me3 marks, and activated the transcription of SOX9. Furthermore, KDM4B depletion reduced the occupancy of SMAD3 in the SOX9 promoter, suggesting that KDM4B is required for SMAD-dependent coactivation of SOX9. Our results demonstrate the critical role of KDM4B in the epigenetic regulation of TGF-β-mediated chondrogenic differentiation of MSCs. Since histone demethylases are chemically modifiable, KDM4B may be a novel therapeutic target in cartilage regenerative therapy.

Three-dimensional dynamic fabrication of engineered cartilage based on chitosan/gelatin hybrid hydrogel scaffold in a spinner flask with a special designed steel frame.

Cartilage transplantation using in vitro tissue engineered cartilage is considered a promising treatment for articular cartilage defects. In this study, we assessed the advantages of adipose derived stem cells (ADSCs) combined with chitosan/gelatin hybrid hydrogel scaffolds, which acted as a cartilage biomimetic scaffold, to fabricate a tissue engineered cartilage dynamically in vitro and compared this with traditional static culture. Physical properties of the hydrogel scaffolds were evaluated and ADSCs were inoculated into the hydrogel at a density of 1×10(7) cells/mL and cultured in a spinner flask with a special designed steel framework and feed with chondrogenic inductive media for two weeks. The results showed that the average pore size, porosity, swelling rate and elasticity modulus of hybrid scaffolds with good biocompatibility were 118.25±19.51 μm, 82.60±2.34%, 361.28±0.47% and 61.2±0.16 kPa, respectively. ADSCs grew well in chitosan/gelatin hybrid scaffold and successfully differentiated into chondrocytes, showing that the scaffolds were suitable for tissue engineering applications in cartilage regeneration. Induced cells cultivated in a dynamic spinner flask with a special designed steel frame expressed more proteoglycans and the cell distribution was much more uniform with the scaffold being filled mostly with extracellular matrix produced by cells. A spinner flask with framework promoted proliferation and chondrogenic differentiation of ADSCs within chitosan/gelatin hybrid scaffolds and accelerated dynamic fabrication of cell-hydrogel constructs, which could be a selective and good method to construct tissue engineered cartilage in vitro.

Natural assembly of platelet lysate-loaded nanocarriers into enriched 3D hydrogels for cartilage regeneration

The role of Platelet Lysates (PLs) as a source of growth factors (GFs) and as main element of three-dimensional (3D) hydrogels has been previously described. However, the resulting hydrogels usually suffer from high degree of contraction, limiting their usefulness. This work describes the development of a stable biomimetic 3D hydrogel structure based on PLs, through the spontaneous assembling of a high concentration of chitosan-chondroitin sulfate nanoparticles (CH/CS NPs) with PLs loaded by adsorption. The interactions between the NPs and the lysates resemble the ones observed in the extracellular matrix (ECM) native environment between glycosaminoglycans and ECM proteins. In vitro release studies were carried out focusing on the quantification of PDGF-BB and TGF-β1 GFs. Human adipose derived stem cells (hASCs) were entrapped in these 3D hydrogels and cultured in vitro under chondrogenic stimulus, in order to assess their potential use for cartilage regeneration. Histological, immunohistological and gene expression analysis demonstrated that the PL-assembled constructs entrapping hASCs exhibited results similar to the positive control (hASCS cultured in pellets), concerning the levels of collagen II expression and immunolocalization of collagen type I and II and aggrecan. Moreover, the deposition of new cartilage ECM was detected by alcian blue and safranin-O positive stainings. This work demonstrates the potential of PLs to act simultaneously as a source/carrier of GFs and as a 3D structure of support, through the application of a “bottom-up” approach involving the assembly of NPs, resulting in an enriched construct for cartilage regeneration applications.

Jellyfish collagen scaffolds for cartilage tissue engineering

Porous scaffolds were engineered from refibrillized collagen of the jellyfish Rhopilema esculentum for potential application in cartilage regeneration. The influence of collagen concentration, salinity and temperature on fibril formation was evaluated by turbidity measurements and quantification of fibrillized collagen. The formation of collagen fibrils with a typical banding pattern was confirmed by atomic force microscopy and transmission electron microscopy analysis. Porous scaffolds from jellyfish collagen, refibrillized under optimized conditions, were fabricated by freeze-drying and subsequent chemical cross-linking. Scaffolds possessed an open porosity of 98.2%. The samples were stable under cyclic compression and displayed an elastic behavior. Cytotoxicity tests with human mesenchymal stem cells (hMSCs) did not reveal any cytotoxic effects of the material. Chondrogenic markers SOX9, collagen II and aggrecan were upregulated in direct cultures of hMSCs upon chondrogenic stimulation. The formation of typical extracellular matrix components was further confirmed by quantification of sulfated glycosaminoglycans.

Editorial [Hot Topic: Stem Cell Therapy and Tissue Engineering Applications for Cartilage Regeneration

Editorial [Hot Topic: Stem Cell Therapy and Tissue Engineering Applications for Cartilage Regeneration

Adult human articular chondrocytes in a microcarrier‐based culture system: expansion and redifferentiation

Expanding human chondrocytes in vitro while maintaining their ability to form cartilage remains a key challenge in cartilage tissue engineering. One promising approach to address this is to use microcarriers as substrates for chondrocyte expansion. While microcarriers have shown beneficial effects for expansion of animal and ectopic human chondrocytes, their utility has not been determined for freshly isolated adult human articular chondrocytes. Thus, we investigated the proliferation and subsequent chondrogenic differentiation of these clinically relevant cells on porous gelatin microcarriers and compared them to those expanded using traditional monolayers. Chondrocytes attached to microcarriers within 2 days and remained viable over 4 weeks of culture in spinner flasks. Cells on microcarriers exhibited a spread morphology and initially proliferated faster than cells in monolayer culture, however, with prolonged expansion they were less proliferative. Cells expanded for 1 month and enzymatically released from microcarriers formed cartilaginous tissue in micromass pellet cultures, which was similar to tissue formed by monolayer-expanded cells. Cells left attached to microcarriers did not exhibit chondrogenic capacity. Culture conditions, such as microcarrier material, oxygen tension, and mechanical stimulation require further investigation to facilitate the efficient expansion of clinically relevant human articular chondrocytes that maintain chondrogenic potential for cartilage regeneration applications. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:539-546, 2011.

Behavior of human chondrocytes in engineered porous bacterial cellulose scaffolds

Regeneration of articular cartilage damage is an area of great interest due to the limited ability of cartilage to self-repair. The latest cartilage repair strategies are dependent on access to biomaterials to which chondrocytes can attach and in which they can migrate and proliferate, producing their own extracellular matrix. In the present study, engineered porous bacterial cellulose (BC) scaffolds were prepared by fermentation of Acetobacter xylinum (A. xylinum) in the presence of slightly fused wax particles with a diameter of 150-300 microm, which were then removed by extrusion. This porous material was evaluated as a scaffold for cartilage regeneration. Articular chondrocytes from young adult patients as well as neonatal articular chondrocytes were seeded with various seeding techniques onto the porous BC scaffolds. Scanning electron microscopy (SEM) analysis and confocal microscopy analysis showed that cells entered the pores of the scaffolds and that they increasingly filled out the pores over time. Furthermore, DNA analysis implied that the chondrocytes proliferated within the porous BC. Alcian blue van Gieson staining revealed glycosaminoglycan (GAG) production by chondrocytes in areas where cells were clustered together. With some further development, this novel biomaterial can be a suitable candidate for cartilage regeneration applications.