Cartilage Regeneration Applications Research Articles

Activating UCHL1 through the CRISPR activation system promotes cartilage differentiation mediated by HIF-1α/SOX9.

Developing strategies to enhance cartilage differentiation in mesenchymal stem cells and preserve the extracellular matrix is crucial for successful cartilage tissue reconstruction. Hypoxia-inducible factor-1α (HIF-1α) plays a pivotal role in maintaining the extracellular matrix and chondrocyte phenotype, thus serving as a key regulator in chondral tissue engineering strategies. Recent studies have shown that Ubiquitin C-terminal hydrolase L1 (UCHL1) is involved in the deubiquitylation of HIF-1α. However, the regulatory role of UCHL1 in chondrogenic differentiation has not been investigated. In the present study, we initially validated the promotive effect of UCHL1 expression on chondrogenesis in adipose-derived stem cells (ADSCs). Subsequently, a hybrid baculovirus system was designed and employed to utilize three CRISPR activation (CRISPRa) systems, employing dead Cas9 (dCas9) from three distinct bacterial sources to target UCHL1. Then UCHL1 and HIF-1α inhibitor and siRNA targeting SRY-box transcription factor 9 (SOX9) were used to block UCHL1, HIF-1α and SOX9, respectively. Cartilage differentiation and chondrogenesis were measured by qRT-PCR, immunofluorescence and histological staining. We observed that the CRISPRa system derived from Staphylococcus aureus exhibited superior efficiency in activating UCHL1 compared to the commonly used the CRISPRa system derived from Streptococcus pyogenes. Furthermore, the duration of activation was extended by utilizing the Cre/loxP-based hybrid baculovirus. Moreover, our findings show that UCHL1 enhances SOX9 expression by regulating the stability and localization of HIF-1α, which promotes cartilage production in ADSCs. These findings suggest that activating UCHL1 using the CRISPRa system holds significant potential for applications in cartilage regeneration.

Polysaccharide-Based Composite Systems in Bone Tissue Engineering: A Review.

In recent years, a growing demand for biomaterials has been observed, particularly for applications in bone regenerative medicine. Bone tissue engineering (BTE) aims to develop innovative materials and strategies for repairing and regenerating bone defects and injuries. Polysaccharides, due to their biocompatibility, biodegradability as well as bioactivity, have emerged as promising candidates for scaffolds or composite systems in BTE. Polymers combined with bioactive ceramics can support osteointegration. Calcium phosphate (CaP) ceramics can be a broad choice as an inorganic phase that stimulates the formation of new apatite layers. This review provides a comprehensive analysis of composite systems based on selected polysaccharides used in bone tissue engineering, highlighting their synthesis, properties and applications. Moreover, the applicability of the produced biocomposites has been analyzed, as well as new trends in modifying biomaterials and endowing them with new functionalizations. The effects of these composites on the mechanical properties, biocompatibility and osteoconductivity were critically analyzed. This article summarizes the latest manufacturing methods as well as new developments in polysaccharide-based biomaterials for bone and cartilage regeneration applications.

Construction of ultrasonically treated collagen/silk fibroin composite scaffolds to induce cartilage regeneration

A novel tissue-specific functional tissue engineering scaffold for cartilage repair should have a three-dimensional structure, good biosafety and biological activity, and should be able to promote cartilage tissue regeneration. This study aimed to determine the effect of ultrasound-treated collagen/silk fibroin (Col/SF) composite scaffolds with good mechanical properties and high biological activity on cartilage repair. The characteristics of the scaffolds with different Col/SF ratios (7:3, 8:2, and 9:1) were determined by scanning electron microscopy, Fourier-transform infrared spectroscopy, and porosity, water absorption, and compression tests. In vitro evaluations revealed the biocompatibility of the Col/SF scaffolds. Results suggested that the optimal ratio of Col/SF composite scaffolds was 7:3. The Col/SF scaffolds induced adipose-derived stem cells to undergo chondrogenic differentiation under chondrogenic culture conditions. The efficiency of Col/SF scaffolds for cartilage regeneration applications was further evaluated using an in vivo model of full-thickness articular cartilage defects in New Zealand rabbits. The Col/SF scaffolds effectively promoted osteochondral regeneration as evidenced by macroscopic, histological, and immunohistochemical evaluation. The study demonstrates that ultrasound-treated Col/SF scaffolds show great potential for repairing cartilage defects.

The Mechanical Properties of a Water Hyacinth/Rice Husk Powders Composite for Tissue Engineering Applications

In this study, composites made from water hyacinth powder (WPH) and rice husk powder (RH) were created using the hot press method, and the composites were characterized to determine their suitability for biomedical applications such as tissue engineering. The mixing ratio of WPH/RH was investigated. Fourier transmission infrared spectroscopy (FTIR) revealed the presence of chemical bonds in the composites under investigation. Tensile tests were used to investigate the mechanical properties of the composite, which revealed that adding water WPH to the rice husk composite reduced the composite's strength. A composite with a 5% WPH content had the highest tensile strength of 32.72 MPa. Meanwhile, the mechanical strength of the other composites studied ranged from 25,537 MPa to 29.43 MPa. However, the elastic modulus of the composite increased with the addition of WPH. The SEM image shows that the powder distribution is less even, the interface between WPH-RH and polyester is quite tight, and the composite contains a number of voids. Characterization of the developed composite demonstrates that the WPH/RH addition ratio can be adjusted to achieve the desired composite properties for tissue engineering and cartilage regeneration applications.

Revisiting matrix hydrogel composed of gelatin and hyaluronic acid and its application in cartilage regeneration

With the increasing incidence of knee osteoarthritis (KOA), the reparation of cartilage defects is gaining more attention. Given that tissue integration plays a critical role in repairing cartilage defects, tissue adhesive hydrogels are highly needed in clinics. We constructed a biomacromolecule-based bioadhesive matrix hydrogel and applied it to promote cartilage regeneration. The hydrogel was composed of methacrylate gelatin and N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitroso) butyl amide modified hyaluronic acid (HANB). The methacrylate gelatin provided a stable hydrogel network as a scaffold, and the HANB served as a tissue-adhesive agent and could be favorable for the chondrogenesis of stem cells. Additionally, the chemically modified HA increased the swelling ratio and compressive modulus of the hydrogels. The results of our in vitro study revealed that the hydrogel was compatible with bone marrow stromal cells. In vivo, the hyaluronic-acid-containing hydrogels were found to promote articular cartilage regeneration in the defect site. Therefore, this biomaterial provides promising potential for cartilage repair.

Managing the Heterogeneity of Mesenchymal Stem Cells for Cartilage Regenerative Therapy: A Review.

Articular cartilage defects commonly result from trauma and are associated with significant morbidity. Since cartilage is an avascular, aneural, and alymphatic tissue with a poor intrinsic healing ability, the regeneration of functional hyaline cartilage remains a difficult clinical problem. Mesenchymal stem cells (MSCs) are multipotent cells with multilineage differentiation potential, including the ability to differentiate into chondrocytes. Due to their availability and ease of ex vivo expansion, clinicians are increasingly applying MSCs in the treatment of cartilage lesions. However, despite encouraging pre-clinical and clinical data, inconsistencies in MSC proliferative and chondrogenic potential depending on donor, tissue source, cell subset, culture conditions, and handling techniques remain a key barrier to widespread clinical application of MSC therapy in cartilage regeneration. In this review, we highlight the strategies to manage the heterogeneity of MSCs ex vivo for more effective cartilage repair, including reducing the MSC culture expansion period, and selecting MSCs with higher chondrogenic potential through specific genetic markers, surface markers, and biophysical attributes. The accomplishment of a less heterogeneous population of culture-expanded MSCs may improve the scalability, reproducibility, and standardisation of MSC therapy for clinical application in cartilage regeneration.

Mesenchymal Stem Cell Secreted-Extracellular Vesicles are Involved in Chondrocyte Production and Reduce Adipogenesis during Stem Cell Differentiation

Background:Extracellular vesicles (EVs) are derived from internal cellular compartments, and have potential as a diagnostic and therapeutic tool in degenerative disease associated with aging. Mesenchymal stem cells (MSCs) have become a promising tool for functional EVs production. This study investigated the efficacy of EVs and its effect on differentiation capacity.Methods:The characteristics of MSCs were evaluated by flow cytometry and stem cell differentiation analysis, and a production mode of functional EVs was scaled from MSCs. The concentration and size of EVs were quantitated by Nanoparticle Tracking Analysis (NTA). Western blot analysis was used to assess the protein expression of exosome-specific markers. The effects of MSC-derived EVs were assessed by chondrogenic and adipogenic differentiation analyses and histological observation.Results:The range of the particle size of adipose-derived stem cells (ADSCs)- and Wharton’s jelly -MSCs-derived EVs were from 130 to 150 nm as measured by NTA, which showed positive expression of exosomal markers. The chondrogenic induction ability was weakened in the absence of EVs in vitro. Interestingly, after EV administration, type II collagen, a major component in the cartilage extracellular matrix, was upregulated compared to the EV-free condition. Moreover, EVs decreased the lipid accumulation rate during adipogenic induction.Conclusion:The results indicated that the production model could facilitate production of effective EVs and further demonstrated the role of MSC-derived EVs in cell differentiation. MSC-derived EVs could be successfully used in cell-free therapy to guide chondrogenic differentiation of ADSC for future clinical applications in cartilage regeneration.

Tribological and Rheological Properties of Poly(vinyl alcohol)-Gellan Gum Composite Hydrogels

Polymeric poly(vinyl alcohol) (PVA)-based composite hydrogels are promising materials with various biomedical applications. However, their mechanical and tribological properties should be tailored for such applications. In this study, we report the fabrication of PVA-gellan gum (GG) composite hydrogels and determine the effect of GG content on their rheological and tribological properties. The rheology tests revealed an enhanced storage (elastic) modulus with increased gellan gum (GG) concentration. The results showed up to 89% enhancement of the elastic modulus of PVA by adding 0.5 wt% gellan gum. This elastic modulus (12.1 ± 0.8 kPa) was very close to that of chondrocyte and its surrounding pericellular matrix (12 ± 1 kPa), rendering them ideal for cartilage regeneration applications. Furthermore, the friction coefficient was reduced by up to 80% by adding GG to PVA, demonstrating the increased elastic modulus improved chance of survival under mechanical shear stresses. Examining PVA/GG at different concentrations of 0.1, 0.3, and 0.5 wt% of GG, we demonstrate that at a load of 5 N, the friction coefficient decreases by increasing the GG concentration. However, at higher loads of 10 and 15 N, a 0.3 wt% concentration was sufficient to significantly reduce the friction coefficient. For PVA and PVA/GG composites, we observed a reduction in friction coefficient by increasing the load from 5 to 15 N. We also found the friction to be independent of the sliding velocity. Possible mechanisms of achieving a reduced friction coefficient are discussed.

Fabrication of chitosan/alginate/hydroxyapatite hybrid scaffolds using 3D printing and impregnating techniques for potential cartilage regeneration

Three-dimensional (3D) printed hydrogel scaffolds enhanced with ceramics have shown potential applications for cartilage regeneration, but leaving biological and mechanical properties to be desired. This paper presents our study on the development of chitosan /alginate scaffolds with nano hydroxyapatite (nHA) by combining 3D printing and impregnating techniques, forming a hybrid, yet novel, structure of scaffolds for potential cartilage regeneration. First, we incorporated nHA into chitosan scaffold printing and studied the printability by examining the difference between the printed scaffolds and their designs. Then, we impregnated alginate with nHA into the printed chitosan scaffolds to forming a hybrid structure of scaffolds; and then characterized the scaffolds mechanically and biologically, with a focus on identifying the influence of nHA and alginate for potential cartilage regeneration. The results of compression tests on the scaffolds showed that the inclusion of nHA increased the elastic moduli of scaffolds; while the live/dead assay illustrated that nHA had a great effect on improving attachment and viability of ATCD5 cells on the scaffolds. Also, our results illustrated scaffolds with nHA impregnated in alginate hydrogel enhanced the cell viability and attachment. Furthermore, antibacterial activity of hybrid scaffolds was characterized with results indicating that the chitosan scaffolds had favourable antibacterial ability, which was further enhanced with the impregnated nHA. Taken together, our study has illustrated that chitosan/HA/alginate hybrid scaffolds are promising for cartilage regeneration and the methods developed to create hybrid scaffolds based on 3D printing and impregnating techniques, which can also be extended to fabricating scaffolds for other tissue engineering applications.

Evaluation of novel biomaterials for cartilage regeneration based on gelatin methacryloyl interpenetrated with extractive chondroitin sulfate or unsulfated biotechnological chondroitin.

Gelatin is widely proposed as scaffold for cartilage tissue regeneration due to its high similarities to the extracellular matrix. However, poor mechanical properties and high sensitivity to enzymatic degradation encouraged the scientific community to develop strategies to obtain better performing hydrogels. Gelatin networks, specifically gelatin‐methacryloyl (GM), have been coupled to hyaluronan or chondroitin sulfate (CS). In this study, we evaluated the biophysical properties of an innovative photocross‐linked hydrogel based on GM with the addition of CS or a new unsulfated biotechnological chondroitin (BC). Biophysical, mechanical, and biochemical characterization have been assessed to compare GM hydrogels to the chondroitin containing networks. Moreover, mesenchymal stem cells (MSCs) were seeded on these biomaterials in order to evaluate the differentiation toward the chondrocyte phenotype in 21 days. Rheological characterization showed that both CS and BC increased the stiffness (G' was about 2‐fold), providing a stronger rigid matrix, with respect to GM alone. The biological tests confirmed the onset of MSCs differentiation process starting from 14 days of in vitro culture. In particular, the combination GM + BC resulted to be more effective than GM + CS in the up‐regulation of key genes such as collagen type 2A1 (COLII), SOX‐9, and aggrecan). In addition, the scanning microscope analyses revealed the cellular adhesion on materials and production of extracellular vesicles. Immunofluorescence staining confirmed an increase of COLII in presence of both chondroitins. Finally, the outcomes suggest that BC entangled within cross‐linked GM matrix may represent a promising new biomaterial with potential applications in cartilage regeneration.

Enhancing chondrogenic potential via mesenchymal stem cell sheet multilayering.

Advanced tissue engineering approaches for direct articular cartilage replacement in vivo employ mesenchymal stem cell (MSC) sources, exploiting innate chondrogenic potential to fabricate hyaline-like constructs in vitro within three-dimensional (3D) culture conditions. Cell sheet technology represents one such advanced 3D scaffold-free cell culture platform, and previous work has shown that 3D MSC sheets are capable of in vitro hyaline-like chondrogenic differentiation. The present study aims to build upon this understanding and elucidate the effects of an established cell sheet manipulation technique, cell sheet multilayering, on fabrication of MSC-derived hyaline-like cartilage 3D layered constructs in vitro. To achieve this goal, multilayered MSC sheets are prepared and assessed for structural and biochemical transitions throughout chondrogenesis. Results support MSC multilayering as a means of increasing construct thickness and 3D cellular interactions related to in vitro chondrogenesis, including N-cadherin, connexin 43, and integrin β-1. Data indicate that increasing construct thickness from 14 μm (1-layer construct) to 25 μm (2-layer construct) increases these cellular interactions and subsequent in vitro MSC chondrogenesis. However, a clear initial thickness threshold (33 μm - 3-layer construct) is evident that decreases the rate and extent of in vitro chondrogenesis, specifically chondrogenic gene expressions (Sox9, aggrecan, type II collagen) and sulfated proteoglycan accumulation in deposited extracellular matrix (ECM). Together, these data support the utility of cell sheet multilayering as a platform for tailoring construct thickness and subsequent MSC chondrogenesis for future articular cartilage regeneration applications.

3D printed gelatin-genipin scaffolds for temporomandibular joint cartilage regeneration

Gelatin has emerged as a biocompatible polymer with high printability in scaffold-based tissue engineering. The aim of the current study was to investigate the potential of genipin-crosslinked 3D printed gelatin scaffolds for temporomandibular joint (TMJ) cartilage regeneration. Crosslinking with genipin increased the stability and mechanical properties, without any cytotoxic effects. Chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSC) on the scaffolds were compared to cell pellets and spheres. Although hBMSC seeded scaffolds showed a lower expression of chondrogenesis-related genes compared to cell pellets and spheres, they demonstrated a significantly reduced expression of collagen (COL) 10, suggesting a decreased hypertrophic tendency. After 21 days, staining with Alcian blue and immunofluorescence for SOX9 and COL1 confirmed the chondrogenic differentiation of hBMSC on genipin-crosslinked gelatin scaffolds. In summary, 3D printed gelatin-genipin scaffolds supported the viability, attachment and chondrogenic differentiation of hBMSC, thus, demonstrating potential for TMJ cartilage regeneration applications.

MicroRNA-210-3p Promotes Chondrogenic Differentiation and Inhibits Adipogenic Differentiation Correlated with HIF-3α Signalling in Bone Marrow Mesenchymal Stem Cells.

Cartilage injury of the knee joint is very common. Due to the limited self-healing ability of articular cartilage, osteoarthritis is very likely to occur if left untreated. Bone marrow mesenchymal stem cells (BMMSCs) are widely used in the study of cartilage injury due to their low immunity and good amplification ability, but they still have disadvantages, such as heterogeneous undifferentiated cells. MicroRNAs can regulate the chondrogenic differentiation ability of MSCs by inhibiting or promoting mRNA translation and degradation. In this research, we primarily investigated the effect of microRNA-210-3p (miR-210-3p) on chondrogenic and adipogenic differentiation of BMMSCs in vitro. Our results demonstrate that miR-210-3p promoted chondrogenic differentiation and inhibited adipogenic differentiation of rat BMMSCs, which was related to the HIF-3α signalling pathway. Additionally, miR-210-3p promotes mRNA and protein levels of the chondrogenic expression genes COLII and SOX9 and inhibits mRNA and protein levels of the adipogenic expression genes PPARγ and LPL. Thus, miR-210-3p combined with BMMSCs is a candidate for future clinical applications in cartilage regeneration and could represent a promising new therapeutic target for OA.

Spherowell, a novel system inducing autonomous spheroid formation of cell monolayers

Purpose: Cell spheroids are the most frequently used system for in vitro chondrogenic differentiation and are gaining increasingly importance in clinical applications. However, drawbacks of currently used systems are the lack of early-readout parameters and time as well as reagent intensive procedures. While this can be partially alleviated by process automation, it is often not feasible due to high cost of necessary equipment. Herein we present a completely new concept of spheroid formation (SpheroWell) allowing for mass-production of chondrogenic spheroids from monolayer via growth surface compartmentation with promising applications in cartilage regeneration and in vitro screening applications. Methods: Standard 60 mm cell culture dishes were compartmentalized with a 1 mm or 3 mm raster by laser engraving. Telomerase immortalized adipose derived stromal/stem cells (ASC/TERT1) were seeded in mono-culture and in co-culture with primary human articular chondrocytes (hAC) at a total cell number of 1 x 106 cells/dish on compartmentalized dishes. Co-cultures were conducted at 1:4, 1:1 and 4:1 hAC:ASC ratios. Cells were cultured in differentiation media containing low concentrations of TGFβ-3 and BMP-6 (1 ng/ml). The spheroid formation process was analysed using time-laps imaging and bright field-/confocal microscopy and the resulting spheroid sizes were measured. Chondrogenic differentiation and internal structure were compared to standard pellet culture using qRT-PCR (collagen type 1/2 (Col1/2) and aggrecan (Agg)) and (immuno-) histochemistry (Alcian blue and collagen type II). As a model for tissue regeneration spheroids were embedded into fibrin hydrogel and the cellular outgrowth and matrix deposition were analysed. Finally the system was assessed for its usability as an in vitro testing system using the spheroid formation process as an early readout parameter by assessing the effect of interleukin-1β (IL-1β) on the formation kinetics. Results: After initial seeding, spheroids self-assembled in a multi-phase process. After a short proliferation phase, edge regions (especially in compartment corners) began to retract, followed by roll-up of the monolayer and final condensation into a spheroid (Figure 1). For ASC/TERT1 mono-cultures this process took about 3 weeks, while addition of hAC accelerated the formation time to 1-2 weeks in a cell-ratio dependent manner. However, while ASC and co-cultures up to 1:1 ratio formed pellets of constant size, hAC ratios over 50% lead to variable and smaller spheroids due to faster and incomplete monolayer formation. Apart from cell ratios, spheroid sizes could be controlled by varying the raster size, yielding highly reproducible mean diameters of 340.9 and 134.4 (SD < 10%) for 3 mm and 1 mm raster respectively (with ASC mono-cultures). When comparing spheroids from raster plates with standard pellet culture comparable differentiation capacity was visible despite the 2D-phase of the initial monolayer in SpheroWells. Internal structures revealed differences with spheroids showing strands of matrix homogenously distributed over the cross-section, while pellets generally showed a pronounced aligned cortex region. When embedding spheroids from raster plates into fibrin hydrogels cellular outgrowth into the hydrogel could be observed, with outgrowth speed being controllable via hydrogel density. Matrix deposition could be observed around the outgrowing cells, which was especially pronounced in co-cultures. Finally, first results using the system as an in vitro testing system were obtained. Treatment of raster plate cultures with IL-1β showed a strong reduction in spheroid formation time serving as early readout parameters, while as expected showing abolishment of Col2 expression in endpoint analysis. Conclusions: The new culture system, SpheroWell, is highly efficient in spheroid generation, saving time and culture medium and allowing for additional early read outs via tracking the spheroid formation process. The ability to produce and maintain hundreds of spheroids within one dish, which can be easily integrated into pre-existing production and research workflows makes it attractive for high throughput testing and clinical spheroid application.

Preparation and characterization of a soluble eggshell membrane/agarose composite scaffold with possible applications in cartilage regeneration.

Articular hyaline cartilage is an extremely hydrated, not vascularized tissue with a low-cell density. The damage of this tissue can occur after injuries or gradual stress and tears (osteoarthritis), minor damages can be self-healed in several weeks, but major injuries may eventually require surgery. In fact, in this case, because of nature of the cartilage (the absence of cells and vascularization) it is difficult to expect its natural regeneration in a reasonable amount of time. In recent years, cell therapy, in which cells are directly transplanted, has attracted attention. In this study, a scaffold for implanting chondrocytes was prepared. The scaffold was made as a sponge using the eggshell membrane and agarose. The eggshell membrane is structurally similar to the extracellular matrix and nontoxic due to its many collagen components and has good biocompatibility and biodegradability. However, scaffolds made of collagen only has poor mechanical properties. For this reason, the disulfide bond of collagen extracted from the insoluble eggshell membrane was cut, converted into water-soluble, and then mixed with agarose to prepare a scaffold. Agarose is capable of controlling mechanical properties, has excellent biocompatibility, and is suitable for forming a hydrogel having a three-dimensional porosity. The scaffold was examined for Fourier-transforminfrared, mechanical properties, biodegradability, and biocompatibility. In in vitro experiment, cytotoxicity, cell proliferation, and messenger RNA expression were investigated. The study demonstrated that the agarose/eggshell membrane scaffold can be used for chondrocyte transplantation.

Hyaluronic acid-coated gold nanorods enhancing BMP-2 peptide delivery for chondrogenesis

Bone morphogenic protein-2 (BMP-2) knuckle epitope peptide has been recently discovered and known to activate chondrogenesis. However, the applications of this soluble peptide remain very limited due to rapid diffusion resulting in poor cellular uptake into target cells. We herein designed nanoparticles made from hyaluronic acid functionalized gold nanorods (GNRs) to conjugate with thiolated BMP-2 knuckle epitope peptide via a two-step reaction. Hyaluronic acid was modified to have thiol functional groups to replace the cetyl trimethylammonium bromide ligands on the surface of GNRs. The thiolated peptides were subsequently reacted with hyaluronic acid on the surface on GNRs via a maleimide-hydrazide crosslinker. The conjugation was confirmed by the change of surface charge of GNRs and the plasmon shift. A colorimetric peptide assay suggested more than 69% of the thiolated peptides were conjugated with the hyaluronic acid coated gold nanorods. Moreover, in vitro cell viability showed that BMP-2 conjugated hyaluronic acid functionalized gold nanorods (B2HGR) were cytocompatible and did not cause cytotoxicity to fibroblast cells. The B2HGRs also significantly promote cellular uptake of the BMP-2 peptides in both human mesenchymal stem cells and porcine chondrocytes due to multivalent ligand binding to the BMP receptors on the cell surface resulting in receptor-mediated endocytosis. The enhanced cellular uptake was clearly observed under a confocal microscope resulting in the significant activation of type II collagen gene expression and glucosaminoglycan secretion in those cells. Furthermore, our delivery system is a proof-of-concept of using scaffolds in combination with nanodelivery platform to enhance cartilaginous repair. The peptide loading capacity and the release is not limited by the scaffolds. Therefore, our delivery platform has potential applications for cartilage regeneration in a preclinical and clinical setting in the future.

Injectable gellan gum/lignocellulose nanofibrils hydrogels enriched with melatonin loaded forsterite nanoparticles for cartilage tissue engineering: Fabrication, characterization and cell culture studies

Injectable hydrogels based on natural polysaccharides have attracted considerable attention in cartilage tissue engineering, especially those reinforced with mineral nanofilers carrying drug molecules. Here, a novel injectable hydrogel based on gellan gum (GG)/lignocellulose nanofibrils (LGNF) composite enriched with melatonin (MEL) loaded forsterite (FS) nanoparticles (FS-MEL) was developed to yield enhanced mechanical and biological properties of the hydrogels. Gelation time and temperature were determined for different hydrogel formulation containing 1-5w/v% LGNF and 0.1-0.3w/v% FS-MEL. The injectability test proved the ease of injection of the developed hydrogels. Degradation rate and swelling degree of developed hydrogel were evaluated to determine the effect of LGNF and FS on hydrogel behaviour. Results of mechanical characterization showed that the compressive modulus and strength of GG hydrogels were improved by incorporation of LGNF and FS. The results of MEL release study in PBS revealed that MEL showed more sustained release from the hydrogel compared to FS nanoparticles. Cell-hydrogels interaction was evaluated by culturing chondrocyte cells. Results exhibited higher cell adhesion, proliferation and gene expression on GG/LGNF/FS-MEL hydrogel compared to GG/LGNF and GG/LGNF/FS, which can be attributed to the synergic effect of FS and MEL. Overall results demonstrated that the developed GG/LGNF/FS-MEL hydrogels can be offered as promising materials for cartilage regeneration applications.

Stem Cell Treatment for Knee Articular Cartilage Defects and Osteoarthritis

To review the current basic science and clinical literature on mesenchymal stem cell (MSC) therapy for articular cartilage defects and osteoarthritis of the knee. MSCs derived from bone marrow, adipose, and umbilical tissue have the capacity for self-renewal and differentiation into the chondrocyte lineage. In theory, MSC therapy may help restore cartilage focally or diffusely where nascent regenerative potential in the intra-articular environment is limited. Over the last several years, in vitro and animal studies have elucidated the use of MSCs in isolation as injectables, in combination with biological delivery media and scaffolding, and as surgical adjuvants for cartilage regeneration and treatment of knee degenerative conditions. More recently, clinical and translational literature has grown more convincing from early descriptive case series to randomized controlled trials showing promise in efficacy and safety. Studies describing MSC for knee cartilage regeneration applications are numerous and varied in quality. Future research directions should include work on elucidating optimal cell concentration and dosing, as well as standardization in methodology and reporting in prospective trials. Backed by promise from in vitro and animal studies, preliminary clinical evidence on MSC therapy shows promise as a nonoperative therapeutic option or an adjuvant to existing surgical cartilage restoration techniques. While higher quality evidence to support MSC therapy has emerged over the last several years, further refinement of methodology will be necessary to support its routine clinical use.

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds.

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 µm). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 °C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.

The Potential of Menstrual Blood-Derived Mesenchymal Stem Cells for Cartilage Repair and Regeneration: Novel Aspects.

Menstrual blood is a unique body fluid that contains mesenchymal stem cells (MSCs). These cells have attracted a great deal of attention due to their exceptional advantages including easy access and frequently accessible sample source and no need for complex ethical and surgical interventions, as compared to other tissues. Menstrual blood-derived MSCs possess all the major stem cell properties and even have a greater proliferation and differentiation potential as compared to bone marrow-derived MSCs, making them a perspective tool in a further clinical practice. Although the potential of menstrual blood stem cells to differentiate into a large variety of tissue cells has been studied in many studies, their chondrogenic properties have not been extensively explored and investigated. Articular cartilage is susceptible to traumas and degenerative diseases, such as osteoarthritis, and has poor self-regeneration capacity and therefore requires more effective therapeutic technique. MSCs seem promising candidates for cartilage regeneration; however, no clinically effective stem cell-based repair method has yet emerged. This chapter focuses on studies in the field of menstrual blood-derived MSCs and their chondrogenic differentiation potential and suitability for application in cartilage regeneration. Although a very limited number of studies have been made in this field thus far, these cells might emerge as an efficient and easily accessible source of multipotent cells for cartilage engineering and cell-based chondroprotective therapy.