Chondrocyte Cultures Research Articles

Efficient engineering of human auricular cartilage through mesenchymal stem cell chaperoning

A major challenge to the clinical translation of tissue-engineered ear scaffolds for ear reconstruction is the limited auricular chondrocyte (hAuC) yield available from patients. Starting with a relatively small number of chondrocytes in culture results in dedifferentiation and loss of phenotype with subsequent expansion. To significantly decrease the number of chondrocytes required for human elastic cartilage engineering, we co-cultured human mesenchymal stem cells (hMSCs) with HAuCs to promote healthy elastic cartilage formation. HAuCs along with human bone marrow-derived hMSCs were encapsulated into 1% Type I collagen at 25 million/mL total cell density with different ratios (HAuCs/hMSCs: 10/90, 25/75, 50/50) and then injected into customized 3D-printed polylactic acid (PLA) ridged external scaffolds, which simulate the shape of the auricular helical rim, and implanted subcutaneously in nude rats for 1, 3 and 6months. The explanted constructs demonstrated near complete volume preservation and topography maintenance of the ridged "helical" feature after 6months with all ratios. Cartilaginous appearing tissue formed within scaffolds by 3months, verified by histologic analysis demonstrating mature elastic cartilage within the constructs with chondrocytes seen in lacunae within a Type II collagen and proteoglycan-enriched matrix, and surrounded by a neoperichondrial external layer. Compressive mechanical properties comparable to human elastic cartilage were achieved after 6months. Co-implantation of hAuCs and hMSCs in collagen within an external scaffold efficiently produced shaped human elastic cartilage without volume loss even when hAuC comprised only 10% of the implanted cell population, marking a crucial step toward the clinical translation of auricular tissue engineering.

Silk-based hydrogel incorporated with metal-organic framework nanozymes for enhanced osteochondral regeneration.

Osteochondral defects (OCD) cannot be efficiently repaired due to the unique physical architecture and the pathological microenvironment including enhanced oxidative stress and inflammation. Conventional strategies, such as the control of implant microstructure or the introduction of growth factors, have limited functions failing to manage these complex environments. Here we developed a multifunctional silk-based hydrogel incorporated with metal-organic framework nanozymes (CuTA@SF) to provide a suitable microenvironment for enhanced OCD regeneration. The incorporation of CuTA nanozymes endowed the SF hydrogel with a uniform microstructure and elevated hydrophilicity. In vitro cultivation of mesenchymal stem cells (MSCs) and chondrocytes showed that CuTA@SF hydrogel accelerated cell proliferation and enhanced cell viability, as well as had antioxidant and antibacterial properties. Under the inflammatory environment with the stimulation of IL-1β, CuTA@SF hydrogel still possessed the potential to promote MSC osteogenesis and deposition of cartilage-specific extracellular matrix (ECM). The proteomics analysis further confirmed that CuTA@SF hydrogel promoted cell proliferation and ECM synthesis. In the full-thickness OCD model of rabbit, CuTA@SF hydrogel displayed successfully in situ OCD regeneration, as evidenced by micro-CT, histology (HE, S/O, and toluidine blue staining) and immunohistochemistry (Col I and aggrecan immunostaining). Therefore, CuTA@SF hydrogel is a promising biomaterial targeted at the regeneration of OCD.

Development of meniscus cartilage using polycaprolactone and decellularized meniscus surface modified by gelatin, hyaluronic acid biomacromolecules: A rabbit model

The lack of vascularization in the white-red and white zone of the meniscus causes these zones of tissue to have low self-healing capacity in case of injury and accelerate osteoarthritis (OA). In this study, we have developed hybrid constructs using polycaprolactone (PCL) and decellularized meniscus extracellular matrix (DMECM) surface modified by gelatin (G), hyaluronic acid (HU) and selenium (Se) nanoparticles (PCL/DMECM/G/HU/Se), following by the cross-linking of the bio-polymeric surface. Material characterization has been performed on the fabricated scaffold using scanning electron microscopy (SEM), Fourier transforms infrared (FTIR) spectroscopy, swelling and degradation analyses, and mechanical tests. In Vitro, investigations have been conducted by C28/I2 human chondrocyte culture into the scaffold and evaluated the cytotoxicity and cell/scaffold interaction. For the in vivo study, the scaffolds were transplanted into the defect sites of female New Zealand white rabbits. Good regeneration was observed after two months. We have concluded that the designed PCL/DMECM/G/HU construct can be a promising candidate as a meniscus tissue engineering scaffold to facilitate healing.

Comparison of engineered cartilage based on BMSCs and chondrocytes seeded on PVA-PPU scaffold in a sheep model.

In this study, polyvinyl alcohol hydrogel chains were crosslinked by polyurethane in order to synthesize a suitable substrate for cartilage lesions. The substrate was fully characterized, and in vitro and in vivo investigations were conducted based on a sheep model. In vitro tests were performed based on the chondrocyte cells with the Alcian Blue and safranin O staining in order to prove the presence of proteoglycan on the surface of the synthesized substrate, which has been secreted by cultures of chondrocytes. Furthermore, the expression of collagen type I, collagen type II, aggrecan, and Sox9 was presented in the chondrocyte cultures on the synthesized substrate through RT-PCR. In addition, the H&E analysis and other related tests demonstrated the formation of neocartilage tissue in a sheep model. The results were found to be promising for cartilage tissue engineering and verified that the isolated chondrocyte cultures on the synthesized substrate retain their original composition.

GRK2, a novel regulator of skeletal development

STUDY OBJECTIVEDuring skeletal development, the cartilaginous growth plate (GP) allows for bone elongation, composed of chondrocytes organized into three histological zones: resting (RZ), proliferative (PZ), and hypertrophic (HZ). Resting chondrocytes proliferate, become prehypertrophic, then hypertrophic, to create room for bone deposition. The G‐protein coupled receptor (GPCR) Parathyroid Hormone Receptor‐1 (PTH1R) expressed on the chondrocytes of the prehypertrophic zone, decelerates the transition to hypertrophy. PTH1R signaling is regulated by GPCR kinase 2 (GRK2); GRK2 phosphorylates PTH1R leading to its internalization and signal termination. Recent studies showed that loss of function mutations in GRK2 result in skeletal deformities, and we demonstrated that GRK2 promotes chondrocyte hypertrophy (CH) in adult arthritic cartilage. However, the role of GRK2 in molecular signaling in GP chondrocytes has yet to be explored.HYPOTHESISWe hypothesized that GRK2 desensitizes PTH1R to promote CH in the GP.METHODSWe utilized the Cre‐Lox system to generate transgenic mice with inducible ‐conditional‐knockout of GRK2 (GRK2‐icKO) in chondrocytes. KO was initiated at postnatal day 3 (PN3) by Tamoxifen ip injections and mice were sacrificed at PN10 to examine the effects on early and late stages of skeletal development. We performed H&E staining to analyze histological changes in GP structure. To determine changes in molecular signaling, we used ISH and IF staining to quantify expression of GRK2, PTH1R, BrdU (proliferation marker), and ColX (CH marker). At the chondro‐osseous junction, we quantified apoptosis (TUNEL staining), bone deposition (Col1 and Runx2 IF), and matrix mineralization (Alizarin Red (AR) staining). We used in vitro experiments with primary chondrocyte culture to confirm in vivoresults. GRK2 knockdown (KD) was achieved with siRNA before inducing CH. Gene and protein expression of the same molecular markers were analyzed with qPCR and WB, and matrix mineralization quantified by AR.RESULTSGKR2‐icKO mice had: (1) Shorter bones (2) Retained PTH1R expression in the HZ, with reduced gene expression (Fig. 1A), as confirmed in vitro (Fig. 1D) (3) Longer PZ and more proliferative chondrocytes confirmed by BrdU, (4) shorter HZ and less hypertrophic chondrocytes, confirmed by ColX IF (Fig. 1B). (5) At the chondro‐osseous junction, GRK2‐icKO mice had reduced chondrocyte apoptosis, bone deposition, and matrix mineralization (Fig. 1C). In vitroexperiments demonstrated increased GRK2 expression with CH, where GRK2 KD reduced CH and matrix mineralization (Fig. 1D).CONCLUSIONSWe show a novel role of GRK2 in skeletal development as a major regulator of PTH1R in GP chondrocytes. GRK2‐icKO increases chondrocyte proliferation and reduces hypertrophy and apoptosis, leading to reduced bone deposition, matrix mineralization, and shorter bones. Altogether, this presents GRK2 as a novel therapeutic target for skeletal abnormalities.

Maternal Hyperthyroidism in Rats Alters the Composition and Gene Expression of the Matrix Produced In Vitro by Chondrocytes from Offspring with Intrauterine Growth Restriction.

Herein, we aimed to evaluate cultures of femoral chondrocytes from offspring of rats with intrauterine growth restriction (IUGR) induced by maternal hyperthyroidism. Fourteen adult female Wistar rats were divided into two groups, a control group and a group treated with daily L-thyroxine administration using an orogastric tube (50 µg/animal/day) during pregnancy. Three days after birth, the offspring were euthanized for chondrocyte extraction. At 7, 14, and 21 days, viability and alkaline-phosphatase (ALP) activity were assessed using the MTT assay and BCIP/NBT method, respectively, in a 2D culture. Pellets (3D cultures) were stained with periodic acid Schiff (PAS) to assess the morphology and percentage of PAS+ areas. The gene transcripts for Col2, Col10, Acan, Sox9, and Runx2 were evaluated by qRT-PCR. The MTT and ALP-assay results showed no significant differences between the groups. Maternal hyperthyroidism did not alter the chondrocyte morphology, but significantly reduced the percentage of PAS+ areas, decreased the expression of the gene transcripts of Col2 and Acan, and increased Sox9 expression. Maternal hyperthyroidism in rats alters the composition and gene expression of the matrix produced by chondrocytes from offspring with IUGR. This may be one of the mechanisms through which excess maternal thyroid hormones reduce offspring bone growth.

Blocking chondrocyte hypertrophy in conditional Evc knockout mice does not modify cartilage damage in osteoarthritis.

Chondrocytes in osteoarthritic (OA) cartilage acquire a hypertrophic-like phenotype, where Hedgehog (Hh) signaling is pivotal. Hh overexpression causes OA-like cartilage lesions, whereas its downregulation prevents articular destruction in mouse models. Mutations in EVC and EVC2genes disrupt Hh signaling, and are responsible for the Ellis-van Creveld syndrome skeletal dysplasia. Since Ellis-van Creveld syndrome protein (Evc) deletion is expected to hamper Hh target gene expression we hypothesized that it would also prevent OA progression avoiding chondrocyte hypertrophy. Our aim was to study Evc as a new therapeutic target in OA, and whether Evc deletion restrains chondrocyte hypertrophy and prevents joint damage in an Evc tamoxifen induced knockout (EvccKO ) model of OA. For this purpose, OA was induced by surgical knee destabilization in wild-type (WT) and EvccKO adult mice, and healthy WT mice were used as controls (n=10knees/group). Hypertrophic markers and Hh genes were measured by qRT-PCR, and metalloproteinases (MMP) levels assessed by western blot. Human OA chondrocytes and cartilage samples were obtained from patients undergoing knee joint replacement surgery. Cyclopamine (CPA) was used for Hh pharmacological inhibition and IL-1 beta as an inflammatory insult. Our results showed that tamoxifen induced inactivation of Evc inhibited Hh overexpression and partially prevented chondrocyte hypertrophy during OA, although it did not ameliorate cartilage damage in DMM-EvccKO mice. Hh pathway inhibition did not modify the expression of proinflammatory mediators induced by IL-1 beta in human OA chondrocytes in culture. We found that hypertrophic-IHH-and inflammatory-COX-2-markers co-localized in OA cartilage samples. We concluded that tamoxifen induced inactivation of Evc partially prevented chondrocyte hypertrophy in DMM-EvccKO mice, but it did not ameliorate cartilage damage. Overall, our results suggest that chondrocyte hypertrophy per se is not a pathogenic event in the progression of OA.

Selected physico-chemical properties of composite scaffolds of sintered submicrocrystalline corundum and bioglass

Presented paper contains description and interpretation of the results of selected physicochemical and structural properties of two types of composite sinters. They were constituted of a mixture of sintered microcrystalline corundum and bioglass CaO-P2O5-SiO2-Na2O system intended for scaffolds to cell culture of human chondrocytes. The composites contained a mixture of both above-mentioned components in the volumetric proportion of 50:50 (W5) and 30:70 (W7). They were obtained using powder metallurgy by free sintering in air atmosphere. Phase analysis of composites and verification of theoretical identification using X-ray diffraction were performed. The same phases were found in both cases (Al2O3 SiO2 CaAl2Si2O8, Ca3 (PO4)2, Ca2Al4O7 and NaAlSiO4). Microscopic tests of composite surfaces were performed and some differences were found. W5 sample was not completely covered with bioglass, whilst W7 sample was completely covered with bioglass with few fine pores. Tests of surface topography confirmed the presence of large and small pores. Composite surfaces immersed for 30 days in artificial blood plasma were tested and then electron microscopy analysis was performed. It was found that no significant changes occurred on the surface of the W5 composite, probably partial corrosion of the glass happened. Spherical forms characteristic of HA-hydroxyapatites were observed on the surface of sample W7. Human articular chondrocyte cells were seeded on both types of sinters and proliferation assay was performed. Results indicate that tested scaffolds support cellular attachment and proliferation of chondrocytes.

Lithium chloride-induced primary cilia recovery enhances biosynthetic response of chondrocytes to mechanical stimulation

Mechanical stimulation is commonly used in cartilage tissue engineering for enhancing tissue formation and improving the mechanical properties of resulting engineered tissues. However, expanded chondrocytes tend to dedifferentiate and lose expression of their primary cilia, which is necessary for chondrocyte mechanotransduction. As treatment with lithium chloride (LiCl) can restore passaged chondrocytes in monolayer, in this study, we investigated whether this approach would be effective in 3D culture and restore chondrocyte mechanosensitivity. Chondrocytes at different passages (P0 to P2) were treated with 0-50mM LiCl for 24h, with different pre-culture durations (0 to 4days). The primary cilia incidence and length were measured in α-tubulin-stained images. Treated chondrocytes were cultured with or without dynamic compression to evaluate the effect of LiCl-induced primary cilia expression on matrix synthesis by mechanically stimulated chondrocytes. LiCl treatment of chondrocytes in 3D agarose culture increased primary cilia incidence and length, with significant increases in incidence and length using 50mM LiCl compared to other concentrations (P < 0.05). This effect was further optimized by including a 4-day pre-culture prior to the 24-h 50mM LiCl treatment. Importantly, LiCl-induced primary cilia expression increased chondrocyte mechanosensitivity. When stimulated with dynamic compression, LiCl-treated P1 chondrocytes increased collagen (1.4-fold, P < 0.1) and proteoglycan (1.5-fold, P < 0.05) synthesis compared to untreated, unstimulated cells. The LiCl treatment method described here can be used to restore primary cilia in passaged chondrocytes, transforming them into a mechanosensitive cell source for cartilage tissue engineering.

Higher Chondrogenic Potential of Extracellular Vesicles Derived from Mesenchymal Stem Cells Compared to Chondrocytes-EVs In Vitro.

The inability of cartilage to self-repair necessitates an effective therapeutic approach to restore damaged tissues. Extracellular vesicles (EVs) are attractive options because of their roles in cellular communication and tissue repair where they regulate the cellular processes of proliferation, differentiation, and recruitment. However, it is a challenge to determine the relevant cell sources for isolation of EVs with high chondrogenic potential. The current study aims to evaluate the chondrogenic potential of EVs derived from chondrocytes (Cho-EV) and mesenchymal stem cells (MSC-EV). The EVs were separately isolated from conditioned media of both rabbit bone marrow MSCs and chondrocyte cultures. The isolated vesicles were assessed in terms of size, morphology, and surface marker expression. The chondrogenic potential of MSCs in the presence of different concentrations of EVs (50, 100, and 150 μg/ml) was evaluated during 21 days, and chondrogenic surface marker expressions were checked by qRT-PCR and histologic assays. The extracted vesicles had a spherical morphology and a size of 44.25 ± 8.89 nm for Cho-EVs and 112.1 ± 10.10 nm for MSC-EVs. Both groups expressed the EV-specific surface markers CD9 and CD81. Higher expression of chondrogenic specified markers, especially collagen type II (COL II), and secretion of glycosaminoglycans (GAGs) and proteoglycans were observed in MSCs treated with 50 and 100 μg/ml MSC-EVs compared to the Cho-EVs. The results from the use of EVs, particularly MSC-EVs, with high chondrogenic ability will provide a basis for developing therapeutic agents for cartilage repair.

Autologous chondrocyte transplantation with bone augmentation for the treatment of osteochodral defects of the knee : Treatment of osteochondral defects of the femoral condyles using autologous cancellous bone from the iliac crest combined with matrix-guided autologous chondrocyte transplantation

OperationszielOffene Therapie osteochondraler Läsionen des Kniegelenks zur vollständigen Auffüllung knöcherner Defekte und Wiederherstellung der Gelenkfläche.IndikationenFokale, symptomatische osteochondrale Defekte des Kniegelenks ab einer Knochendefekttiefe von ≥ 5 mm und Größe von ≥ 1,5 cm2.KontraindikationenArthrose (> KL Grad 2), „kissing lesions“ (ICRS > Grad 2), Alter > 50 Jahre, unbehandelte Bandinstabilitäten oder Beinachsenfehstellungen zum Nachteil des betroffenen Gelenkkompartiments, fehlendes Defektcontainment, entzündliche Gelenkerkrankungen.OperationstechnikZweizeitiger Eingriff: Ersteingriff (arthroskopische Knorpelprobenentnahme): Defektevaluierung, Entnahme von Knorpelstücken für die Chondrozytenkultivierung, bei Bedarf Behandlung von Begleitpathologien. Zweiteingriff (offene Defektbehandlung): Arthrotomie, Präparation des knöchernen Defekts, Auffüllung mit autologen Spongiosazylindern aus dem Beckenkamm, Knorpeldefektpräparation (kann größer als knöcherner Defekt sein) und matrixgestützte autologe Chondrozytentransplantation.WeiterbehandlungErsteingriff: frühfunktionelle Nachbehandlung mit schmerzorientierter Vollbelastung je nach Begleiteingriffen. Zweiteingriff: keine Drainage, funktionelle Kniegelenkorthese in Streckstellung für 1 Woche, danach schrittweise Freigabe der Flexion, Teilbelastung für 6 Wochen, Motorschiene (CPM) ab dem 1. postoperativen Tag.ErgebnisseSeit 2018 sind 8 Patienten (mittleres Alter 29,4 Jahre, Spanne 18 bis 36) mit der beschriebenen Technik behandelt worden. Alle Patienten konnten nach durchschnittlich 12 Monaten nachuntersucht werden. Der Gesamt-KOOS (Knee injury and Osteoarthritis Outcome Score) verbesserte sich im Mittel von 45,8 auf 81,3, und die postoperativen radiologischen Kontrollen zeigten die Einheilung der Spongiosazylinder bei allen Patienten. Der MOCART (Magnetic Resonance Observation of Cartilage Repair Tissue) Score ergab 80,4 Punkte.

Cross-talk between EGFR and BMP signals regulates chondrocyte maturation during endochondral ossification.

Progressive maturation of growth plate chondrocytes drives long bone growth during endochondral ossification. Signals from the epidermal growth factor receptor (EGFR), and from bone morphogenetic protein-2 (BMP2), are required for normal chondrocyte maturation. Here, we investigated cross-talk between EGFR and BMP2 signals in developing and adult growth plates. Using in vivo mouse models of conditional cartilage-targeted EGFR or BMP2 loss, we show that canonical BMP signal activation is increased in the hypertrophic chondrocytes of EGFR-deficient growth plates; whereas EGFR signal activation is increased in the reserve, prehypertrophic and hypertrophic chondrocytes of BMP2-deficient growth plates. EGFR-deficient chondrocytes displayed increased BMP signal activation in vitro, accompanied by increased expression of IHH, COL10A1, and RUNX2. Hypertrophic differentiation and BMP signal activation were suppressed in normal chondrocyte cultures treated with the EGFR ligand betacellulin, effects that were partially blocked by simultaneous treatment with BMP2 or a chemical EGFR antagonist. Cross-talk between EGFR and BMP2 signals occurs during chondrocyte maturation. In the reserve and prehypertrophic zones, BMP2 signals unilaterally suppress EGFR activity; in the hypertrophic zone, EGFR and BMP2 signals repress each other. This cross-talk may play a role in regulating chondrocyte maturation in developing and adult growth plates.

Progenitor Cells in Healthy and Osteoarthritic Human Cartilage Have Extensive Culture Expansion Capacity while Retaining Chondrogenic Properties.

Articular cartilage-derived progenitor cells (ACPCs) are a potential new cell source for cartilage repair. This study aims to characterize endogenous ACPCs from healthy and osteoarthritic (OA) cartilage, evaluate their potential for cartilage regeneration, and compare this to cartilage formation by chondrocytes. ACPCs were isolated from full-thickness healthy and OA human cartilage and separated from the total cell population by clonal growth after differential adhesion to fibronectin. ACPCs were characterized by growth kinetics, multilineage differentiation, and surface marker expression. Chondrogenic redifferentiation of ACPCs was compared with chondrocytes in pellet cultures. Pellets were assessed for cartilage-like matrix production by (immuno)histochemistry, quantitative analyses for glycosaminoglycans and DNA content, and expression of chondrogenic and hypertrophic genes. Healthy and OA ACPCs were successfully differentiated toward the adipogenic and chondrogenic lineage, but failed to produce calcified matrix when exposed to osteogenic induction media. Both ACPC populations met the criteria for cell surface marker expression of mesenchymal stromal cells (MSCs). Healthy ACPCs cultured in pellets deposited extracellular matrix containing proteoglycans and type II collagen, devoid of type I collagen. Gene expression of hypertrophic marker type X collagen was lower in healthy ACPC pellets compared with OA pellets. This study provides further insight into the ACPC population in healthy and OA human articular cartilage. ACPCs show similarities to MSCs, yet do not produce calcified matrix under well-established osteogenic culture conditions. Due to extensive proliferative potential and chondrogenic capacity, ACPCs show potential for cartilage regeneration and possibly for clinical application, as a promising alternative to MSCs or chondrocytes.

Hyaluronic Acid In Vitro Response: Viability and Proliferation Profile of Human Chondrocytes in 3D-Based Culture

This study aimed to evaluate the efficacy of hyaluronic acid in the viability and proliferation profile of human femoral-tibial joint cartilage affected by osteoarthritis using in vitro models of chondrocytes in a 2-dimensional (2D)- and 3-dimensional (3D)-based culture model by spheroids. In vitro study of knee cartilage affected by osteoarthritis that required surgical treatment. Samples were cultured and exposed to hyaluronic acid (100 and 500 μM; intervention group) or vehicle solution. In monolayer or 2D culture, proliferation and cell viability were measured, and nuclear morphometry was analyzed by 4',6'-diamino-2-fenil-indol (DAPI) staining. The 3D-based culture established from the culture of articular cartilage of patients submitted to total knee arthroplasty evaluated the diameter, viability, and fusion ability of the chondrospheres created. Samples from 3 patients resulted in viable cultures, with chondrocyte cells exhibiting a potential for cell proliferation and viability to establish a culture. Hyaluronic acid (100 and 500 μM) improved chondrocyte viability and proliferation up to 72 hours in contact when compared with the control group, and no nuclear irregularities in morphology cell characteristics were observed by DAPI. In the 3D evaluation, hyaluronic acid (500 μM) improved the cellular feedback mechanisms, increasing the survival and maintenance of the chondrospheres after 7 days of analysis, showing the intrinsic capacity of chondrospheres grouped in the attempt to rearrange and reestablish new articular tissue. The 2D- and 3D-based culture models with hyaluronic acid improved chondrocyte viability and proliferation and demonstrated the ability of freshly formed chondrospheres to undergo fusion when placed together in the presence of hyaluronic acid.

AMIC Technique for the Treatment of Chondral Injuries of the Hand and Wrist

AbstractScaffolds, either alone or combined with cultured chondrocyte cells, are an effective treatment for chondral or osteochondral defects of the knee and ankle joints.Scaffolds are a more sophisticated solution and have some advantages compared with the isolated use of the more traditional treatments of microfractures or nanofractures.In addition, scaffolds represent a less complicated technique and a less expensive treatment compared with chondrocyte culture treatments, which are accessible by very few patients.In the present article, we detail the surgical technique and provide advices and tips for the treatment of ostecochondral hand and wrist lesions using the Chondro-Gide (Geistlich Pharma AG, Wolhausen, Switzerland) scaffold and its patented autologous matrix-induced chondrogenesis (AMIC, Geistlich Pharma AG) technique.

Early Efficacy of Type I Collagen-Based Matrix-Assisted Autologous Chondrocyte Transplantation for the Treatment of Articular Cartilage Lesions.

Background: Articular cartilage is a complex structure that allows for low frictional gliding and effective shock absorption. Various sports injuries and inflammatory conditions can lead to lesions in the articular cartilage, which has limited regenerative potential. Type I collagen combined with autologous chondrocytes in a three-dimensional culture were used to induce the regeneration of single-layer autologous expanded chondrocytes without chondrogenic differentiation. Purpose: To assess the clinical, radiological, and histological changes following collagen-based autologous chondrocyte transplantation (MACT) for chondral knee lesions. Methods: The study prospectively enrolled 20 patients with symptomatic knee chondral lesions (mean size lesion was 2.41 ± 0.43 cm2, range: 2.0–3.4 cm2) in the lateral femoral condyle and femoral groove who underwent type I collagen-based MACT between July 2017 and July 2019. knee injury and osteoarthritis outcome score (KOOS) was assessed before the procedure, and periodic clinical follow-up was conducted every 3 months for a maximum of 12 months following the procedure and at 1-year intervals thereafter. Magnetic resonance imaging (MRI) T2 mapping of repaired cartilage was also used for the quantitative analysis of regeneration. In one patient, second-look arthroscopy was performed to assess cartilage regeneration characteristics, and a portion of regenerated cartilage was harvested for histological evaluation 12 months after implantation. Results: At pre-operation and at three, six, 12, and 24 months after the operation, KOOS pain, symptoms, daily life activities, sports and recreation, as well as the quality of life were significantly improved between every two time points. Hematoxylin and eosin (HE) staining indicated that the newly formed cartilage was comprised of naive chondrocytes. Safranin O-fast (S-O) green staining of the regenerated tissue revealed fibroblast-like cells surrounded by glycosaminoglycans. Immunohistochemistry (IHC) analysis indicated that collagen type II was uniformly distributed at the deep zone of articular cartilage and type I collagen mainly depositing in the superficial cartilage layer. The T2 values for repaired tissue gradually decreased, eventually approaching near-average values. Conclusion: The present study demonstrated that type I collagen-based MACT is a clinically effective treatment for improving functionality and pain levels. Histological evidence confirmed hyaline cartilage induction and showed that repaired cartilage tended to emerge from the deep to the superficial layer. The quantitative MRI T2 mapping test indicated that there still was a difference between the transplanted cartilage and the surrounding hyaline cartilage. Taken together, the current method represents an efficient approach for the restoration of knee cartilage lesions.

In Vitro Evaluation of a Composite Gelatin-Hyaluronic Acid-Alginate Porous Scaffold with Different Pore Distributions for Cartilage Regeneration.

Although considerable achievements have been made in the field of regenerative medicine, since self-repair is not an advanced ability of articular cartilage, the regeneration of osteochondral defects is still a challenging problem in musculoskeletal diseases. Cartilage regeneration aims to design a scaffold with appropriate pore structure and biological and mechanical properties for the growth of chondrocytes. In this study, porous scaffolds made of gelatin, hyaluronic acid, alginate, and sucrose in different proportions of 2 g (SL2) and 4 g (SL4) were used as porogens in a leaching process. Sucrose with particle size ranges of 88–177 μm (Hμ) and 44–74 μm (SHμ) was added to the colloid, and the individually cross-linked hydrogel scaffolds with controllable pore size for chondrocyte culture were named Hμ-SL2, Hμ-SL4, SHμ-SL2 and SHμ-SL4. The perforation, porosity, mechanical strength, biocompatibility, and proliferation characteristics of the hydrogel scaffold and its influence on chondrocyte differentiation are discussed. Results show that the addition of porogen increases the porosity of the hydrogel scaffold. Conversely, when porogens with the same particle size are added, the pore size decreases as the amount of porogen increases. The perforation effect of the hydrogel scaffolds formed by the porogen is better at 88–177 μm compared with that at 44–74 μm. Cytotoxicity analysis showed that all the prepared hydrogel scaffolds were non-cytotoxic, indicating that no cross-linking agent residues that could cause cytotoxicity were found. In the proliferation and differentiation of the chondrocytes, the SHμ-SL4 hydrogel scaffold with the highest porosity and strength did not achieve the best performance. However, due to the compromise between perforation pores, pore sizes, and strength, as well as considering cell proliferation and differentiation, Hμ-SL4 scaffold provided a more suitable environment for the chondrocytes than other groups; therefore, it can provide the best chondrocyte growth environment for this study. The development of hydrogels with customized pore properties for defective cartilage is expected to meet the requirements of the ultimate clinical application.

Phenotype and synthesis activity of joint chondrocytes extracted from newborn rats with prenatal ethanol exposure.

Thirteen female Wistar rats were divided into two groups: one treated with ethanol and the other of untreated. Four newborns from each mother were selected and weighed, measured, and evaluated for physical characteristics. From these neonates, chondrocytes were extracted from the articular cartilages of the femur and tibia, and cultivated in a chondrogenic medium at 37oC and 5% CO2. At 7, 14, and 21days of cultivation, alkaline phosphatase activity tests, MTT conversion to formazan, and percentage area covered by cells per field were performed. At 21days, the percentage of PAS+ areas in 3D cultures was performed, as well as the evaluation of gene transcript expression for aggrecan, SOX-9, collagen type II, collagen X, Runx-2, and VEGF by real-time RT-PCR. The means were compared by Student's t-test. The weight of the ethanol group neonates was significantly lower than that of the controls. Chondrocyte cultures from the ethanol group showed significantly higher AP activity, MTT conversion, and cell percentage. There was higher expression of collagen type II and lower expression of SOX-9 in the ethanol group. There was no difference in the percentage of PAS+ areas in pellets and in expression of aggrecan, collagen X, Runx-2, or VEGF between groups. In conclusion, prenatal exposure to ethanol alters the phenotype and activity of offspring chondrocytes, which may be mechanisms by which endochondral bone formation is compromised by maternal ethanol consumption.

Ex Vivo and In Vivo Properties of an Injectable Hydrogel Derived From Acellular Ear Cartilage Extracellular Matrix.

Extracellular matrix (ECM) hydrogels provide advantages such as injectability, the ability to fill an irregularly shaped space, and the adequate bioactivity of native matrix. In this study, we developed decellularized cartilage ECM (dcECM) hydrogels from porcine ears innovatively via the main method of enzymatic digestion and verified good biocompatible properties of dcECM hydrogels to deliver chondrocytes and form subcutaneous cartilage in vivo. The scanning electron microscopy and turbidimetric gelation kinetics were used to characterize the material properties and gelation kinetics of the dcECM hydrogels. Then we evaluated the biocompatibility of hydrogels via the culture of chondrocytes in vitro. To further explore the dcECM hydrogels in vivo, grafts made from the mixture of dcECM hydrogels and chondrocytes were injected subcutaneously in nude mice for the gross and histological analysis. The structural and gelation kinetics of the dcECM hydrogels altered according to the variation in the ECM concentrations. The 10 mg/ml dcECM hydrogels could support the adhesion and proliferation of chondrocytes in vitro. In vivo, at 4 weeks after transplantation, cartilage-like tissues were detected in all groups with positive staining of toluidine blue, Safranin O, and collagen II, indicating the good gelation of dcECM hydrogels. While with the increasing concentration, the tissue engineering cartilages formed by 10 mg/ml dcECM hydrogel grafts were superior in weights, volumes, collagen, and glycosaminoglycan (GAG) content compared to the dcECM hydrogels of 1 mg/ml and 5 mg/ml. At 8 weeks after grafting, dcECM hydrogel grafts at 10 mg/ml showed very similar qualities to the control, collagen I grafts. After 12 weeks of in vivo culture, the histological analysis indicated that 10 mg/ml dcECM hydrogel grafts were similar to the normal cartilage from pig ears, which was the source tissue. In conclusion, dcECM hydrogel showed the promising potential as a tissue engineering biomaterial to improve the regeneration and heal injuries of ear cartilage.

Inhibitory Effects of IL-6-Mediated Matrix Metalloproteinase-3 and -13 by Achyranthes japonica Nakai Root in Osteoarthritis and Rheumatoid Arthritis Mice Models

Achyranthes japonica Nakai root (AJNR) is used to treat osteoarthritis (OA) and rheumatoid arthritis (RA) owing to its anti-inflammatory and antioxidant effects. This study investigated the inhibitory effects of AJNR on arthritis. AJNR was extracted using supercritical carbon dioxide (CO2), and its main compounds, pimaric and kaurenoic acid, were identified. ANJR’s inhibitory effects against arthritis were evaluated using primary cultures of articular chondrocytes and two in vivo arthritis models: destabilization of the medial meniscus (DMM) as an OA model, and collagenase-induced arthritis (CIA) as an RA model. AJNR did not affect pro-inflammatory cytokine (IL-1β, TNF-α, IL-6)-mediated cytotoxicity, but attenuated pro-inflammatory cytokine-mediated increases in catabolic factors, and recovered pro-inflammatory cytokine-mediated decreases in related anabolic factors related to in vitro. The effect of AJNR is particularly specific to IL-6-mediated catabolic or anabolic alteration. In a DMM model, AJNR decreased cartilage erosion, subchondral plate thickness, osteophyte size, and osteophyte maturity. In a CIA model, AJNR effectively inhibited cartilage degeneration and synovium inflammation in either the ankle or knee and reduced pannus formation in both the knee and ankle. Immunohistochemistry analysis revealed that AJNR mainly acted via the inhibitory effects of IL-6-mediated matrix metalloproteinase-3 and -13 in both arthritis models. Therefore, AJNR is a potential therapeutic agent for relieving arthritis symptoms.