GAG Deposition Research Articles

Donor ethnicity, sex, and age impact chondrogenic re-differentiation capacity: a multi-demographic study of human articular chondrocytes in vitro

ABSTRACT To unravel causes of disparities in osteoarthritis (OA) prevalence and to improve cell-based cartilage repair treatments, there is need to investigate the multifactorial impact of patient demographics on a biophysiological level. In this study, we systematically analyse single- and multi-demographic impact on in vitro chondrogenic re-differentiation capacity of human articular chondrocytes (hACs), specifically of an Aotearoa-New Zealand (NZ) patient cohort which displays unique demographic diversity. HACs were isolated from 14 NZ donors with distinct demographics (ethnicity: indigenous Māori vs European descendant Pākehā; sex; age 18–24 vs 25–30 yrs). In vitro chondrogenic re-differentiation capacity of donor chondrocytes was assessed through quantifications of cartilage matrix deposition (GAG, DNA, GAG/DNA) and by histological visualisation. Isolated chondrocytes ranged from low chondrogenic re-differentiation capacity, characterised by fibrocartilage tissue deposition, to high re-differentiation capacity to deposit GAG-rich tissue. Age-related reduction in GAG/DNA content was detected while no impact of ethnicity or sex was observed. Multi-demographic analysis revealed reduced GAG deposition in Māori male (compared to Māori female), and Māori (18–24 yrs) compared to Māori (25–30 yrs) and Pākehā (18–24 yrs) within our cohort. Multi-demographic analysis is a promising strategy to understand disparities in OA prevalence in patient cohorts and can help guide development of cell-based strategies for diverse patient populations.

Investigating the Interplay Between Matrix Compliance and Passaging History on Chondrogenic Differentiation of Mesenchymal Stem Cells Encapsulated Within Alginate-Gelatin Hybrid Hydrogels

Mesenchymal stem cells (MSCs) are used widely in tissue engineering and regenerative medicine because of their ease of isolation and their pluripotency. The low survival and retention rate of MSCs at the target site upon implantation can be addressed via encapsulation within hydrogels capable of directing their fate. In this study, the interplay between matrix mechanics and the passage number of MSCs on their chondrogenic differentiation was assessed. Human bone marrow-derived MSCs between passages 4 and 6 were encapsulated within alginate-gelatin hybrid gels. The stiffness of the gels was varied by varying alginate concentrations while maintaining the concentration of gelatin and consequently, the cell adhesion sites, constant. The study revealed that within 4.8 kPa gels, GAG deposition was higher by P4 MSCs compared to P6 MSCs. However, an opposite trend was observed with collagen type 2 deposition. Further, we observed enhanced chondrogenic differentiation upon encapsulation of MSCs within 6.7 kPa hydrogel irrespective of passaging history. However, the effect of matrix compliance was more prominent in the case of higher passaged MSCs suggesting that matrix stiffness can help rescue the reduced differentiation capability of these cells.

Performance of Colombian Silk Fibroin Hydrogels for Hyaline Cartilage Tissue Engineering.

The development and evaluation of scaffolds play a crucial role in the engineering of hyaline cartilage tissue. This work aims to evaluate the performance of silk fibroin hydrogels fabricated from the cocoons of the Colombian hybrid in the in vitro regeneration of hyaline cartilage. The scaffolds were physicochemically characterized, and their performance was evaluated in a cellular model. The results showed that the scaffolds were rich in random coils and β-sheets in their structure and susceptible to various serine proteases with different degradation profiles. Furthermore, they showed a significant increase in ACAN, COL10A1, and COL2A1 expression compared to pellet culture alone and allowed GAG deposition. The soluble portion of the scaffold did not affect chondrogenesis. Furthermore, they promoted the increase in COL1A2, showing a slight tendency to differentiate towards fibrous cartilage. The results also showed that Colombian silk could be used as a source of biomedical devices, paving the way for sericulture to become a more diverse economic activity in emerging countries.

Silk fibroin based interpenetrating network hydrogel for corneal stromal regeneration

There is a need to develop tissue engineering based approaches to address the shortage of donor corneas worldwide for transplantation. To do this a novel approach to fabricate three-dimensional hydrogels using free-radical polymerization was investigated to generate constructs for corneal stromal tissue regeneration. Different ratios of silk fibroin (SF) to polyacrylamide (PA) were used to fabricate semi-interpenetrating hydrogels. Scanning electron micrograph displayed the interconnectivity of pores within the fabricated hydrogels. Pore sizes ranged from 25 to 66 μm. Scaffolds with increasing concentration of SF had enhanced β-sheet structure (verified by Fourier transform infrared spectroscopy). The biological response of human corneal stromal cells to these hydrogels was examined using cellular adhesion, proliferation, cytoskeleton organization, gene expression and immunocytochemical analysis. The fabricated hydrogels possess rapid gelation (∼3 min) at 37 °C, 84 % porosity facilitating keratocyte migration during healing, improved cellular adhesion and no cytotoxicity, indicating their efficiency for in-situ corneal tissue regeneration. Presence of SF in semi-interpenetrating network hydrogel enhanced cellular proliferation, elevated GAG deposition, and increased expression of keratocyte genes, normally associated with healthy corneal stromal tissue. This study acts as an initial step towards fabricating SF based semi-interpenetrating network hydrogels for developing clinically applicable ocular implants.

Genipin-crosslinked collagen scaffolds inducing chondrogenesis: a mechanical and biological characterization.

Articular cartilage degeneration is still an unsolved issue owing to its weak repairing capabilities, which usually result in fibrocartilage tissue formation. This fibrous tissue lacks of structural and bio-mechanical properties, degrading over time. Currently, arthroscopic techniques and autologous transplantation are the most used clinical procedures. However, rather than restoring cartilage integrity, these methods only postpone further cartilage deterioration. Therefore, tissue engineering strategies aimed at selecting scaffolds that remarkably support the chondrogenic differentiation of human mesenchymal stem cells (hMSCs) could represent a promising solution, but they are still challenging for researchers. In this study, the influence of two different genipin (Gp) crosslinking routes on collagen (Coll)-based scaffolds in terms of hMSCs chondrogenic differentiation and biomechanical performances was investigated. Three-dimensional (3D) porous Coll scaffolds were fabricated by freeze-drying techniques and were crosslinked with Gp following a "two-step" and an in "bulk" procedure, in order to increase the physico-mechanical stability of the structure. Chondrogenic differentiation efficacy of hMSCs and biomechanical behavior under compression forces through unconfined stress-strain tests were assessed. Coll/Gp scaffolds revealed an isotropic and highly homogeneous pore distribution along with an increase in the stiffness, also supported by the increase in the Coll denaturation temperature (Td =57-63°C) and a significant amount of Coll and GAG deposition during the 3weeks of chondrogenic culture. In particular, the presence of Gp in "bulk" led to a more uniform and homogenous chondral-like matrix deposition by hMSCs if compared to the results obtained from the Gp "two-step" functionalization procedure.

Influence of Medical Marijuana on Interleukin-1Β Treated Cartilage: An in Vitro Study

Category:Basic Sciences/BiologicsIntroduction/Purpose:Medical marijuana is reported to have potent analgesic, immunomodulatory and anti-inflammatory properties. Some recent studies have shown that cannabinoids may attenuate joint damage in animal models of arthritis. However, the underlying mechanism has not been completely elucidated. Interleukin-1β (IL-1β), a proinflammatory cytokine that can result in the degradation of cartilage, is known to be associated with the pathogenesis of osteoarthritis. We hypothesize that cannabinoids can mitigate the detrimental effect of IL-1β on cartilage, thus slowing the progression of osteoarthritis. We exposed human chondrocyte-derived engineered cartilage pellet cultures to IL-1 β for 48 hours or not and then treated the samples with a synthetic cannabinoid agonist, Win-55,212-2(Win). The tissue phenotypes were thereafter assessed by real time polymerase chain reaction (RT-PCR), histology and western blotting.Methods:Medical marijuana is reported to have potent analgesic, immunomodulatory and anti-inflammatory properties. Some recent studies have shown that cannabinoids may attenuate joint damage in animal models of arthritis. However, the underlying mechanism has not been completely elucidated. Interleukin-1β (IL-1β), a proinflammatory cytokine that can result in the degradation of cartilage, is known to be associated with the pathogenesis of osteoarthritis. We hypothesize that cannabinoids can mitigate the detrimental effect of IL-1β on cartilage, thus slowing the progression of osteoarthritis. We exposed human chondrocyte-derived engineered cartilage pellet cultures to IL-1 β for 48 hours or not and then treated the samples with a synthetic cannabinoid agonist, Win-55,212-2(Win). The tissue phenotypes were thereafter assessed by real time polymerase chain reaction (RT-PCR), histology and western blotting.Results:RT-PCR revealed persistent increase in the expression of inflammatory mediators IL-1beta, TNF-alpha and COL2 in IL1beta+ cultures over IL1beta- controls at all levels of WIN exposure. In contrast there was clear decrease in IL-6 expression at high WIN doses coincident with a downward trend the other mediators. WIN was unable to rescue IL-1beta-induced reductions in COL2 and ACN, while inducing ALP, a marker of osteogenesis and cartilage hypertrophy. Safranin O/Fast Green histology revealed progressive loss in GAG deposition with increasing doses of WIN (Figure 2). Western blots confirmed reductions in COL2 and COMP protein, persistent COX2 production, and increasing translation of the osteogenic transcription factor RUNX2 and p-SMAD1/5/8 (Figure 3).Conclusion:At doses below 1µM, Win does not impact IL-1beta-induced chondrocyte inflammation. while inhibiting chondrogenesis and stimulating osteogenesis through RUNX2 as compared to IL-1β controls. In proper context, BMP2 stimulates osteogenesis through SMAD1/5/8 and the induction of RUNX2. We did observe an apparent anti-inflammatory role of WIN at 1µM, but this dose did not rescue a loss in cartilage pellet GAG loss. Our data suggests that cannabinoids are anti-inflammatory, but are unable to rescue cartilage degeneration by IL-1β and promote an osteogenic phenotype. However, the data are not conclusive with regarding the beneficial or detrimental to arthritic bones and cartilage

An In Vitro System to Study the Effect of Subchondral Bone Health on Articular Cartilage Repair in Humans.

Chondrocyte-based cartilage repair strategies, such as articular chondrocyte implantation, are widely used, but few studies addressed the communication between native subchondral bone cells and the transplanted chondrocytes. An indirect co-culture model was developed, representing a chondrocyte/scaffold-construct repair of a cartilage defect adjoining bone, where the bone could have varying degrees of degeneration. Human BM-MSCs were isolated from two areas of subchondral bone in each of five osteochondral tissue specimens from five patients undergoing knee arthroplasty. These two areas underlaid the macroscopically and histologically best and worst cartilage, representing early and late-stage OA, respectively. BM-MSCs were co-cultured with normal chondrocytes suspended in agarose, with the two cell types separated by a porous membrane. After 0, 7, 14 and 21 days, chondrocyte–agarose scaffolds were assessed by gene expression and biochemical analyses, and the abundance of selected proteins in conditioned media was assessed by ELISA. Co-culture with late-OA BM-MSCs resulted in a reduction in GAG deposition and a decreased expression of genes encoding matrix-specific proteins (COL2A1 and ACAN), compared to culturing with early OA BM-MSCs. The concentration of TGF-β1 was significantly higher in the early OA conditioned media. The results of this study have clinical implications for cartilage repair, suggesting that the health of the subchondral bone may influence the outcomes of chondrocyte-based repair strategies.

MiR-218 affects hypertrophic differentiation of human mesenchymal stromal cells during chondrogenesis via targeting RUNX2, MEF2C, and COL10A1

BackgroundHuman mesenchymal stromal cells (MSC) hold hopes for cartilage regenerative therapy due to their chondrogenic differentiation potential. However, undesirable occurrence of calcification after ectopic transplantation, known as hypertrophic degeneration, remains the major obstacle limiting application of MSC in cartilage tissue regeneration approaches. There is growing evidence that microRNAs (miRs) play essential roles in post-transcriptional regulation of hypertrophic differentiation during chondrogenesis. Aim of the study was to identify new miR candidates involved in repression of hypertrophy-related targets.MethodsThe miR expression profile in human articular chondrocytes (AC) was compared to that in hypertrophic chondrocytes derived from human MSC by microarray analysis, and miR expression was validated by qPCR. Putative targets were searched by in silico analysis and validated by miR reporter assay in HEK293T, by functional assays (western blotting and ALP-activity) in transiently transfected SaOS-2 cells, and by a miR pulldown assay in human MSC. The expression profile of miR-218 was assessed by qPCR during in vitro chondrogenesis of MSC and re-differentiation of AC. MSC were transfected with miR-218 mimic, and differentiation outcome was assessed over 28 days. MiR-218 expression was quantified in healthy and osteoarthritic cartilage of patients.ResultsWithin the top 15 miRs differentially expressed between chondral AC versus endochondral MSC differentiation, miR-218 was selected as a candidate miR predicted to target hypertrophy-related genes. MiR-218 was downregulated during chondrogenesis of MSC and showed a negative correlation to hypertrophic markers, such as COL10A1 and MEF2C. It was confirmed in SaOS-2 cells that miR-218 directly targets hypertrophy-related COL10A1, MEF2C, and RUNX2, as a gain of ectopic miR-218 mimic caused drop in MEF2C and RUNX2 protein accumulation, with attenuation of COL10A1 expression and significant concomitant reduction of ALP activity. A miR pulldown assay confirmed that miR-218 directly targets RUNX2, MEF2C in human MSC. Additionally, the gain of miR-218 in human MSC attenuated hypertrophic markers (MEF2C, RUNX2, COL10A1, ALPL), although with no boost of chondrogenic markers (GAG deposition, COL2A1) due to activation of WNT/β-catenin signaling. Moreover, no correlation between miR-218 expression and a pathologic phenotype in the cartilage of osteoarthritis (OA) patients was found.ConclusionsAlthough miR-218 was shown to target pro-hypertrophic markers MEF2C, COL10A1, and RUNX2 in human MSC during chondrogenic differentiation, overall, it could not significantly reduce the hypertrophic phenotype or boost chondrogenesis. This could be explained by a concomitant activation of WNT/β-catenin signaling counteracting the anti-hypertrophic effects of miR-218. Therefore, to achieve a full inhibition of the endochondral pathway, a whole class of anti-hypertrophic miRs, including miR-218, needs to be taken into consideration.

Inhibition of hypertrophy and improving chondrocyte differentiation by MMP-13 inhibitor small molecule encapsulated in alginate-chondroitin sulfate-platelet lysate hydrogel

BackgroundMesenchymal stem cells are a promising cell source for chondrogenic differentiation and have been widely used in several preclinical and clinical studies. However, they are prone to an unwanted differentiation process towards hypertrophy that limits their therapeutic efficacy. Matrix metallopeptidase 13 (MMP-13) is a well-known factor regulated during this undesirable event. MMP-13 is a collagen degrading enzyme, which is also highly expressed in the hypertrophic zone of the growth plate and in OA cartilage. Accordingly, we investigated the effect of MMP-13 inhibition on MSC hypertrophy.MethodsIn this study, 5-bromoindole-2-carboxylic acid (BICA) was used as an inhibitory agent for MMP-13 expression. After identifying its optimal concentration, BICA was mixed into a hydrogel and the release rate was studied. To prepare the ideal hydrogel, chondroitin sulfate (CS) and platelet lysate (PL) were mixed with sodium alginate (Alg) at concentrations selected based on synergistic mechanical and rheometric properties. Then, four hydrogels were prepared by combining alginate (1.5%w/v) and/or CS (1%w/v) and/or PL (20%v/v). The chondrogenic potential and progression to hypertrophy of human bone marrow-derived mesenchymal stem cell (hBM-MSC)-loaded hydrogels were investigated under free swelling and mechanical loading conditions, in the presence and absence of BICA.ResultsViability of hBM-MSCs seeded in the four hydrogels was similar. qRT-PCR revealed that BICA could successfully inhibit MMP-13 expression, which led to an inhibition of Coll X and induction of Coll-II, in both free swelling and loading conditions. The GAG deposition was higher in the group combining BICA and mechanical stimulation.ConclusionsIt is concluded that BICA inhibition of MMP-13 reduces MSC hypertrophy during chondrogenesis.Graphical abstract

Aspergillus-Derived Galactosaminogalactan Triggers Complement Activation on Human Platelets.

Invasive fungal infections caused by Aspergillus (A.) and Mucorales species still represent life-threatening diseases in immunocompromised individuals, and deeper knowledge about fungal interactions with elements of innate immunity, such as complement and platelets, appears essential for optimized therapy. Previous studies showed that galactosaminogalactan secreted by A. fumigatus and A. flavus is deposited on platelets, thereby inducing their activation. Since the altered platelet surface is a putative trigger for complement activation, we aimed to study the interplay of platelets with complement in the presence of fungal GAG. Culture supernatants (SN) of A. fumigatus and A. flavus both induced not only GAG deposition but also subsequent deposition of complement C3 fragments on the platelet surface. The SN of a Δuge3 mutant of A. fumigatus, which is unable to synthesize GAG, did not induce complement deposition on platelets, nor did the SN of other Aspergillus species and all tested Mucorales. Detailed analysis revealed that GAG deposition itself triggered the complement cascade rather than the GAG-induced phosphatidylserine exposure. The lectin pathway of complement could be shown to be crucially involved in this process. GAG-induced complement activation on the platelet surface was revealed to trigger processes that might contribute to the pathogenesis of invasive aspergillosis by A. fumigatus or A. flavus. Both pro-inflammatory anaphylatoxins C3a and C5a arose when platelets were incubated with SN of these fungal species; these processes might favor excessive inflammation after fungal infection. Furthermore, platelets were stimulated to shed microparticles, which are also known to harbor pro-inflammatory and pro-coagulant properties. Not only did early processes of the complement cascade proceed on platelets, but also the formation of the terminal complement C5b-9 complex was detected on platelets after incubation with fungal SN. Subsequently, reduced viability of the platelets could be shown, which might contribute to the lowered platelet numbers found in infected patients. In summary, fungal GAG initiates an interplay between complement and platelets that can be supposed to contribute to excessive inflammation, thrombocytopenia, and thrombosis, which are important hallmarks of fatal invasive mycoses.

Microwave assisted methacrylation of Kappa carrageenan: A bioink for cartilage tissue engineering

In this study, we aimed to obtain stable Kappa carrageenan (κCar) hydrogel that could be used as a bioink for cartilage regeneration. For this purpose, we described an effective and considerably faster methacrylation process by using microwave energy. Thus, microwave-methacrylated κCar (Mw-κCar-MA) with ≥85% degree of methacrylation (DM) was synthesized despite the use of a low concentration of methacrylic anhydride (MA) at 1000 W in 5 min. Then, Mw-κCar-MA was photo-crosslinked by only using UV irradiation for 40 s. Characterization studies proved that Mw-κCar-MA hydrogels were stronger and have lower weight loss (~20% at 30 days) than that of conventionally synthesized κCar-MA hydrogels. Viscosities of the Mw-κCar-MA hydrogels were found to be sufficient to use in 3D bioprinters. Furthermore, Mw-κCar-MA hydrogels enhanced the viability, proliferation, and GAG deposition of ATDC5 chondrogenic cells. Therefore, we proposed that Mw-κCar-MA can be considered as a suitable bioink for cartilage tissue engineering.

Potential for combined delivery of riboflavin and all-trans retinoic acid, from silk fibroin for corneal bioengineering.

Millions of people worldwide suffer from vision impairing conditions resulting from corneal injury or disease. Silk fibroin (SF) is an emerging biopolymer that has been used for several applications including the fabrication of bioengineered corneas and ocular prostheses. To improve the cell response to SF, riboflavin (RF) and all-trans retinoic acid (RA) were coupled onto SF matrices. RF is a photo-initiator that has previously been combined with ultraviolet light to crosslink corneal collagen while RA has been used to regulate the phenotype of corneal stromal cells and their extracellular matrix deposition. Different concentrations of RF and RA were respectively photo-crosslinked and covalently bound through carbodiimide coupling onto 2% SF matrices. The effect of incorporating these molecules on the physical, chemical and mechanical properties of the matrices was evaluated. The biological response of human corneal stromal cells to the matrices was examined using cellular adhesion assays, proliferation assays, cytoskeleton staining, gene expression analysis and immunocytochemical staining. RF and RA both led to changes in the surface nanostructure and hydrophilicity while just RF increased the material stiffness. Cells cultured on the matrices containing both biomolecules displayed improved cellular proliferation, increased GAG deposition and increased expression of keratocyte genes that are normally associated with healthy corneal stromal tissue. These in vitro studies serve as a starting point for the optimization of loading bioactive molecules on SF based matrices for formulating clinically relevant ocular implants.

Biodegradable water-based polyurethane scaffolds with a sequential release function for cell-free cartilage tissue engineering

Three-dimensional (3D) printing technology has rapidly developed as a promising technology for manufacturing tissue engineering scaffolds. Cells used in tissue engineering are subjected to the quality management and risk of contamination, while cell-free scaffolds may not have sufficient therapeutic efficacy. In this study, water-based 3D printing ink containing biodegradable polyurethane (PU), chemokine SDF-1, and Y27632 drug-embedding PU microspheres was printed at low temperature (−40 °C) to fabricate tissue engineering scaffolds with sequential drug release function. The scaffolds containing 200 ng/ml SDF-1 and 22 wt% Y27632-encapsulated microspheres (55 µg/ml Y27632 in microspheres) (abbreviated PU/SDF-1/MS_Y scaffolds) had the optimal performance. The structural design of the scaffolds allowed each of SDF-1 and Y27632 to be released sequentially in vitro and reach the effective concentration (∼100 ng/ml and 3.38 µg/ml, respectively) after the appropriate time (24 h and 62 h, respectively). Human mesenchymal stem cells (hMSCs) seeded in the scaffolds showed significant GAG deposition in 7 days. Besides, the gradual release of SDF-1 from the PU/SDF-1/MS_Y scaffolds could induce the migration of hMSCs. Implantation of the cell-free PU/SDF-1/MS_Y scaffolds in rabbit articular cartilage defects supported the potential of the scaffolds to promote cartilage regeneration. The 3D printed scaffolds with sequential releases of SDF-1 and Y27632 may have potential in cartilage tissue engineering. Statement of SignificanceThe clinical success of tissue engineering depends highly on the quality of externally supplied cells, while cell-free scaffolds may not have sufficient therapeutic efficacy. In this manuscript, water-based 3D printing ink containing biodegradable polyurethane (PU), chemokine SDF-1, and Y27632 drug-embedding PU microspheres was printed at low temperature to fabricate tissue engineering scaffolds with sequential drug release function. The structural design of the scaffolds allowed each of SDF-1 and Y27632 to be released sequentially in vitro. SDF-1 was released earlier from the scaffolds to promote cell migration. The drug Y27632 was released later from the microspheres into the matrix of the scaffolds to induce the chondrogenic differentiation of the attracted cells. Implantation of the cell-free PU/SDF-1/MS_Y scaffolds in rabbit articular cartilage defects supported the potential of the scaffolds to promote cartilage regeneration. We hypothesized that the cell-free scaffolds may improve the clinical applicability and convenience without the use of exogenous cells or growth factor.

Enzymatically Cross-Linked Silk Fibroin-Based Hierarchical Scaffolds for Osteochondral Regeneration.

Osteochondral (OC) regeneration faces several limitations in orthopedic surgery, owing to the complexity of the OC tissue that simultaneously entails the restoration of articular cartilage and subchondral bone diseases. In this study, novel biofunctional hierarchical scaffolds composed of a horseradish peroxidase (HRP)-cross-linked silk fibroin (SF) cartilage-like layer (HRP-SF layer) fully integrated into a HRP-SF/ZnSr-doped β-tricalcium phosphate (β-TCP) subchondral bone-like layer (HRP-SF/dTCP layer) were proposed as a promising strategy for OC tissue regeneration. For comparative purposes, a similar bilayered structure produced with no ion incorporation (HRP-SF/TCP layer) was used. A homogeneous porosity distribution was achieved throughout the scaffolds, as shown by micro-computed tomography analysis. The ion-doped bilayered scaffolds presented a wet compressive modulus (226.56 ± 60.34 kPa) and dynamic mechanical properties (ranging from 403.56 ± 111.62 to 593.56 ± 206.90 kPa) superior to that of the control bilayered scaffolds (189.18 ± 90.80 kPa and ranging from 262.72 ± 59.92 to 347.68 ± 93.37 kPa, respectively). Apatite crystal formation, after immersion in simulated body fluid (SBF), was observed in the subchondral bone-like layers for the scaffolds incorporating TCP powders. Human osteoblasts (hOBs) and human articular chondrocytes (hACs) were co-cultured onto the bilayered structures and monocultured in the respective cartilage and subchondral bone half of the partitioned scaffolds. Both cell types showed good adhesion and proliferation in the scaffold compartments, as well as adequate integration of the interface regions. Osteoblasts produced a mineralized extracellular matrix (ECM) in the subchondral bone-like layers, and chondrocytes showed GAG deposition. The gene expression profile was different in the distinct zones of the bilayered constructs, and the intermediate regions showed pre-hypertrophic chondrocyte gene expression, especially on the BdTCP constructs. Immunofluorescence analysis supported these observations. This study showed that the proposed bilayered scaffolds allowed a specific stimulation of the chondrogenic and osteogenic cells in the co-culture system together with the formation of an osteochondral-like tissue interface. Hence, the structural adaptability, suitable mechanical properties, and biological performance of the hierarchical scaffolds make these constructs a desired strategy for OC defect regeneration.

Hypoxic condition enhances chondrogenesis in synovium-derived mesenchymal stem cells

BackgroundThe chondrogenic differentiation of mesenchymal stem cells (MSCs) is regulated by many factors, including oxygen tensions, growth factors, and cytokines. Evidences have suggested that low oxygen tension seems to be an important regulatory factor in the proliferation and chondrogenic differentiation in various MSCs. Recent studies report that synovium-derived mesenchymal stem cells (SDSCs) are a potential source of stem cells for the repair of articular cartilage defects. But, the effect of low oxygen tension on the proliferation and chondrogenic differentiation in SDSCs has not characterized. In this study, we investigated the effects of hypoxia on proliferation and chondrogenesis in SDSCs.MethodSDSCs were isolated from patients with osteoarthritis at total knee replacement. To determine the effect of oxygen tension on proliferation and colony-forming characteristics of SDSCs, A colony-forming unit (CFU) assay and cell counting-based proliferation assay were performed under normoxic (21% oxygen) or hypoxic (5% oxygen). For in vitro chondrogenic differentiation, SDSCs were concentrated to form pellets and subjected to conditions appropriate for chondrogenic differentiation under normoxia and hypoxia, followed by the analysis for the expression of genes and proteins of chondrogenesis. qRT-PCR, histological assay, and glycosoaminoglycan assays were determined to assess chondrogenesis.ResultsLow oxygen condition significantly increased proliferation and colony-forming characteristics of SDSCs compared to that of SDSCs under normoxic culture. Similar pellet size and weight were found for chondrogensis period under hypoxia and normoxia condition. The mRNA expression of types II collagen, aggrecan, and the transcription factor SOX9 was increased under hypoxia condition. Histological sections stained with Safranin-O demonstrated that hypoxic conditions had increased proteoglycan synthesis. Immunohistochemistry for types II collagen demonstrated that hypoxic culture of SDSCs increased type II collagen expression. In addition, GAG deposition was significantly higher in hypoxia compared with normoxia at 21 days of differentiation.ConclusionThese findings show that hypoxia condition has an important role in regulating the synthesis ECM matrix by SDSCs as they undergo chondrogenesis. This has important implications for cartilage tissue engineering applications of SDSCs.

Notochordal cell matrix as a bioactive lubricant for the osteoarthritic joint

Notochordal cell derived matrix (NCM) can induce regenerative effects on nucleus pulposus cells and may exert such effects on chondrocytes as well. Furthermore, when dissolved at low concentrations, NCM forms a viscous fluid with potential lubricating properties. Therefore, this study tests the feasibility of the use of NCM as a regenerative lubricant for the osteoarthritic joint. Chondrocyte-seeded alginate beads were cultured in base medium (BM), BM with NCM (NCM), or BM with TGF-β1 (TGF), as well as BM and NCM treated with IL-1β. NCM increased GAG deposition and cell proliferation (stronger than TGF), and GAG/DNA ratio and hydroxyproline content (similar to TGF). These effects were maintained in the presence of IL-1β. Moreover, NCM mitigated expression of IL-1β-induced IL-6, IL-8, ADAMTS-5 and MMP-13. Reciprocating sliding friction tests of cartilage on glass were performed to test NCM’s lubricating properties relative to hyaluronic acid (HA), and showed a dose-dependent reduction in coefficient of friction with NCM, similar to HA. NCM has anabolic and anti-inflammatory effects on chondrocytes, as well as lubricating properties. Therefore, intra-articular NCM injection may have potential as a treatment to minimize pain while restoring the affected cartilage tissue in the osteoarthritic joint.

In vitro culture increases mechanical stability of human tissue engineered cartilage constructs by prevention of microscale scaffold buckling

Many studies have measured the global compressive properties of tissue engineered (TE) cartilage grown on porous scaffolds. Such scaffolds are known to exhibit strain softening due to local buckling under loading. As matrix is deposited onto these scaffolds, the global compressive properties increase. However the relationship between the amount and distribution of matrix in the scaffold and local buckling is unknown. To address this knowledge gap, we studied how local strain and construct buckling in human TE constructs changes over culture times and GAG content. Confocal elastography techniques and digital image correlation (DIC) were used to measure and record buckling modes and local strains. Receiver operating characteristic (ROC) curves were used to quantify construct buckling. The results from the ROC analysis were placed into Kaplan-Meier survival function curves to establish the probability that any point in a construct buckled. These analysis techniques revealed the presence of buckling at early time points, but bending at later time points. An inverse correlation was observed between the probability of buckling and the total GAG content of each construct. This data suggests that increased GAG content prevents the onset of construct buckling and improves the microscale compressive tissue properties. This increase in GAG deposition leads to enhanced global compressive properties by prevention of microscale buckling.

TGF-β1 presenting enzymatically cross-linked injectable hydrogels for improved chondrogenesis

In this work, we developed a novel enzymatically cross-linked injectable hydrogel composed of carboxymethyl cellulose (CMC), sulfated carboxymethyl cellulose (sCMC) and gelatin for delivery of infrapatellar fat pad derived MSCs and articular chondrocytes to a cartilage defect site while enabling TGF-β1 mediated chondrogenesis. The sCMC component in the hydrogel served the purpose of mimicking heparan sulfate and thus enabled strong binding with TGF-β1 and its consequential long term presentation to the encapsulated cells. We demonstrated that amongst CMC/sCMC/gelatin hydrogels cross-linked with 1 and 2mM H2O2, the latter demonstrated significantly higher compressive modulus and supported better in vitro cartilage formation. Thereafter, we explored the utility of this system to present TGF-β1 to encapsulated cells for prolonged time period. It was observed that these hydrogels could sequester >90% of encapsulated TGF-β1 for at least 4 weeks. The encapsulated TGF-β1 was shown to be bioactive and supported significantly better cell survival over control hydrogels. Further, the TGF loaded hydrogels demonstrated good sulfated GAG and collagen deposition which was higher than control hydrogels and comparable to those treated with soluble TGF-β1 through media. Interestingly, incorporation of TGF-β1 in hydrogels not only enhanced the expression and deposition of hyaline cartilage markers, but it also significantly reduced the deposition of fibrocartilage and hypertrophy markers. Overall, it was concluded that TGF-β1 immobilized CMC/sCMC/gelatin injectable hydrogels encapsulated with IFP MSCs and ACs present a promising, cost effective and easily translatable strategy for cartilage tissue engineering.

Structural and Biochemical Modification of a Collagen Scaffold to Selectively Enhance MSC Tenogenic, Chondrogenic, and Osteogenic Differentiation

Biomaterial approaches for engineering orthopedic interfaces such as the tendon-bone junction (TBJ) are limited by a lack of understanding of how insoluble (microstructure, composition) and soluble regulators of stem cell fate work in concert to promote bioactivity and differentiation. One strategy for regenerating the interface is to design biomaterials containing spatially graded structural properties sufficient to induce divergent mesenchymal stem cell (MSC) differentiation into multiple interface-specific phenotypes. This work explores the hypothesis that selective structural modification to a 3D collagen-glycosaminoglycan (CG) scaffold combined with biochemical supplementation can drive human bone-marrow-derived MSC differentiation down tenogenic, osteogenic, and chondrogenic lineages. Tenogenic differentiation is enhanced in geometrically anisotropic scaffolds versus a standard isotropic control. Notably, blebbistatin treatment abrogates this microstructurally driven effect. Further, enhanced osteogenic differentiation and new mineral synthesis are achieved by incorporation of a calcium phosphate mineral phase within the CG scaffold along with the use of osteogenic induction media. Finally, chondrogenic differentiation is optimally driven by combining chondrogenic induction media with a reduced density scaffold that promotes increased cellular condensation, significantly higher expression of chondrogenic genes, and increased GAG deposition. Together these data provide critical insight regarding design rules for elements of an integrated biomaterial platform for orthopedic interface regeneration.

Evaluation of insulin medium or chondrogenic medium on proliferation and chondrogenesis of ATDC5 cells.

Background. The ATDC5 cell line is regarded as an excellent cell model for chondrogenesis. In most studies with ATDC5 cells, insulin medium (IM) was used to induce chondrogenesis while chondrogenic medium (CM), which was usually applied in chondrogenesis of mesenchymal stem cells (MSCs), was rarely used for ATDC5 cells. This study was mainly designed to investigate the effect of IM, CM, and growth medium (GM) on chondrogenesis of ATDC5 cells. Methods. ATDC5 cells were, respectively, cultured in IM, CM, and GM for a certain time. Then the proliferation and the chondrogenesis progress of cells in these groups were analyzed. Results. Compared with CM and GM, IM promoted the proliferation of cells significantly. CM was effective for enhancement of cartilage specific markers, while IM induced the cells to express endochondral ossification related genes. Although GAG deposition per cell in CM group was significantly higher than that in IM and GM groups, the total GAG contents in IM group were the most. Conclusion. This study demonstrated that CM focused on induction of chondrogenic differentiation while IM was in favor of promoting proliferation and expression of endochondral ossification related genes. Combinational use of these two media would be more beneficial to bone/cartilage repair.