From Chondrocytes to Chondrons, Maintenance of Phenotype and Matrix Production in a Composite 3D Hydrogel Scaffold.

Osteoarthritis (OA) is a degenerative disease characterized by articular cartilage (AC) degradation that affects more than 30 million people in the USA. OA is managed with symptom-alleviating medications. Matrix-assisted autologous chondrocyte transplantation (MACT) is a tissue-engineered option, but current products are expensive and lack mechanical tunability or processability to match defect mechanical properties and anatomical shapes. Here, we explore the efficacy of a biocompatible hydrogel-based scaffold composed of sodium alginate, gelatin, and gum Arabic-referred to by SA-GEL-GA-to support bovine articular chondrocyte (bAChs) proliferation, pericellular matrix (PCM), and extracellular matrix (ECM) production. bAChs were grown for 45 days in SA-GEL-GA. Their viability, their live/dead status, histological staining, biochemical assays for glycosaminoglycans (GAGs) and collagen, atomic force microscopy (AFM) imaging, and immunofluorescence staining of collagen I, collagen II, aggrecan, and CD44 were assessed. We found that SA-GEL-GA was not cytotoxic, induced cellular proliferation by 6.1-fold while maintaining a round morphology, and supported ECM deposition by producing 3.9-fold more GAG compared to day 0. bAChs transformed into chondrons and produced a PCM enriched with collagen II (3.4-fold), aggrecan (1.7-fold), and CD44 (1.3-fold) compared to day 0. In summary, SA-GEL-GA supported the proliferation, ECM production, and PCM production of bAChs in vitro.

Enhanced chondrogenesis from chondrocytes co-cultured on mesenchymal stromal cells: Implication for cartilage repair.

Cellular therapy based on chondrocytes implantation is the most widely used procedure for inducing cartilage regeneration. However, the dedifferentiation process that these cells suffer and their limited capacity of proliferation, when they are cultured in vitro, restrict their use in cellular therapy protocols. To investigate the capacity of mesenchymal stromal cells (MSCs) to promote chondrogenesis from chondrocytes or chondrons in 2D and 3D coculture systems. Murine chondrocytes and chondrons were cocultured with MSCs at different cell ratios (100/0, 50/50, 70/30, 0/100) in two-dimensional (2D) and three-dimensional (3D) culture systems. High proliferation of cells with chondrocyte morphology, enhanced GAG production and expression of cartilage genes (aggrecan, type II collagen, and SOX-9) were observed in chondrocytes/MSCs cocultures. In contrast, fibroblastoid cells, down-regulation of cartilage gene expression and reduction of GAG production were observed in chondrons/MSCs cocultures. Chondrocytes within cartilage lacunae and surrounded by extracellular matrix were observed in chondrocytes/MSC pellets. MSCs promote the proliferation of functional chondrocytes in 2D and 3D culture systems. Transplantation of chondrogenic construct based on MSCs and chondrocytes may constitute a potential treatment for inducing cartilage repair.

Combination of chondrocytes and chondrons improves extracellular matrix production to promote the repairs of defective knee cartilage in rabbits.

BACKGROUND: Chondrons are composed of chondrocytes and the surrounding pericellular matrix (PCM) and function to enhance chondrocyte-mediated cartilage tissue engineering. This study aimed at investigating the potential effect of combined chondrocytes with chondrons on the production of proteoglycan and collagen-II (Col-2) and the repair of defective knee cartilage in rabbits. METHODS: Chondrocytes and chondrons were isolated from the knee cartilage of rabbits, and cultured alone or co-cultured for varying periods in vitro. Their morphology was characterized by histology. The levels of aggrecan (AGG), Col-2 and glycosaminoglycan (GAG) expression were quantified by qRT-PCR, Alcian blue-based precipitation and ELISA. The effect of combined chondrocytes with chondrons in alginate spheres on the repair of defective knee cartilage was examined in rabbits. RESULTS: The isolated chondrocytes and chondrons displayed unique morphology and began to proliferate on day 3 and 6 post culture, respectively, accompanied by completely degenerated PCM on day 6 post culture. Evidently, chondrocytes had stronger proliferation capacity than chondrons. Longitudinal analyses indicated that culture of chondrons, but not chondrocytes, increased AGG mRNA transcripts and GAG levels with time and Col-2 mRNA transcripts only on day 3 post culture. Compared with chondrocytes or chondrons alone, co-culture of chondrocytes and chondrons significantly up-regulated AGG and Col-2 expression and GAG production, particularly at a ratio of 1:1. Implantation with chondrocytes and chondrons at 1:1 significantly promoted the repair of defective knee cartilage in rabbits, accompanied by reduced the Wakiteni scores with time. CONCLUSION: Combined chondrons with chondrocytes promoted the production of extracellular matrix and the repair of defective knee cartilage in rabbits. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: This study explores that the combination of chondrons and chondrocytes may be new therapeutic strategy for cartilage tissue engineering and repair of defective cartilage.

Protocol for the Isolation of Intact Chondrons from Healthy and Osteoarthritic Human Articular Cartilage.

Chondrons are the main functional microanatomical units in cartilage, consisting of chondrocytes and the directly surrounding pericellular matrix (PCM). They have attracted attention as a more physiological and biomimetic in vitro model for evaluating chondrocyte function and metabolism as compared to single chondrocytes. Chondrons may be more suitable for in vitro studies than primary chondrocytes that have been isolated without PCM since their in situ and in vivo states remain intact: chondrocytes within their PCM do not undergo the rapid dedifferentiation that proliferating single chondrocytes undergo in culture. Therefore, chondrons may be a better model for studying chondrocyte biology and responses to pro-inflammatory and anti-inflammatory cytokines, growth factors and novel therapeutics. In this chapter, we present a concise and unified protocol for enzymatic isolation of intact chondrons from human articular cartilage and determination of their viability.

Scientific Developments and Clinical Applications Utilizing Chondrons and Chondrocytes with Matrix for Cartilage Repair.

Injuries to articular cartilage of the knee are increasingly common. The operative management of these focal chondral lesions continues to be problematic for the treating orthopedic surgeon secondary to the limited regenerative capacity of articular cartilage. The pericellular matrix (PCM) is a specialized, thin layer of the extracellular matrix that immediately surrounds chondrocytes forming a unit together called the chondron. The advancements in our knowledge base with regard to the PCM/chondrons as well as interterritorial matrix has permeated and led to advancements in product development in conjunction with minced cartilage, marrow stimulation, osteochondral allograft, and autologous chondrocyte implantation (ACI). This review intends to summarize recent progress in chondrocytes with matrix research, with an emphasis on the role the PCM/extracellular matrix (ECM) plays for favorable chondrogenic gene expression, as a barrier/filtration unit, and in osteoarthritis. The bulk of the review describes cutting-edge and evolving clinical developments and discuss these developments in light of underlying basic science applications. Clinical applications of chondrocytes with matrix science include Reveille Cartilage Processor, Cartiform, and ACI with Spherox (which was recently recommended for the treatment of grade III or IV articular cartilage defects over 2 cm2 by the National Institute of Health and Care Excellence [NICE] in the United Kingdom). The current article presents a comprehensive overview of both the basic science and clinical results of these next-generation cartilage repair techniques by focusing specifically on the scientific evolution in each category as it pertains with underlying chondrocytes with matrix theory.

Efficacy of one-stage cartilage repair using allogeneic mesenchymal stromal cells and autologous chondron transplantation (IMPACT) compared to nonsurgical treatment for focal articular cartilage lesions of the knee: study protocol for a crossover randomized controlled trial.

BACKGROUND: Articular cartilage defects in the knee have poor intrinsic healing capacity and may lead to functional disability and osteoarthritis (OA). "Instant MSC Product accompanying Autologous Chondron Transplantation" (IMPACT) combines rapidly isolated recycled autologous chondrons with allogeneic MSCs in a one-stage surgery. IMPACT was successfully executed in a first-in-man investigator-driven phase I/II clinical trial in 35 patients. The purpose of this study is to compare the efficacy of IMPACT to nonsurgical treatment for the treatment of large (2-8 cm2) articular cartilage defects in the knee. METHODS: Sixty patients will be randomized to receive nonsurgical care or IMPACT. After 9 months of nonsurgical care, patients in the control group are allowed to receive IMPACT surgery. The Knee Injury and Osteoarthritis Outcome Score (KOOS), pain (numeric rating scale, NRS), and EuroQol five dimensions five levels (EQ5D-5 L) will be used to compare outcomes at baseline and 3, 6, 9, 12, and 18 months after inclusion. Cartilage formation will be assessed at baseline, and 6 and 18 months after inclusion using MRI. An independent rheumatologist will monitor the onset of a potential inflammatory response. (Severe) adverse events will be recorded. Lastly, the difference between IMPACT and nonsurgical care in terms of societal costs will be assessed by monitoring healthcare resource use and productivity losses during the study period. A health economic model will be developed to estimate the incremental cost-effectiveness ratio of IMPACT vs. nonsurgical treatment in terms of costs per quality adjusted life year over a 5-year time horizon. DISCUSSION: This study is designed to evaluate the efficacy of IMPACT compared to nonsurgical care. Additionally, safety of IMPACT will be assessed in 30 to 60 patients. Lastly, this study will evaluate the cost-effectiveness of IMPACT compared to nonsurgical care. TRIAL REGISTRATION: NL67161.000.18 [Registry ID: CCMO] 2018#003470#27 [EU-CTR; registered on 26 March 2019] NCT04236739 [ ClinicalTrials.gov ] [registered after start of inclusion; 22 January 2020].

Allogeneic MSCs and Recycled Autologous Chondrons Mixed in a One-Stage Cartilage Cell Transplantion: A First-in-Man Trial in 35 Patients.

MSCs are known as multipotent mesenchymal stem cells that have been found capable of differentiating into various lineages including cartilage. However, recent studies suggest MSCs are pericytes that stimulate tissue repair through trophic signaling. Aimed at articular cartilage repair in a one-stage cell transplantation, this study provides first clinical evidence that MSCs stimulate autologous cartilage repair in the knee without engrafting in the host tissue. A phase I (first-in-man) clinical trial studied the one-stage application of allogeneic MSCs mixed with 10% or 20% recycled defect derived autologous chondrons for the treatment of cartilage defects in 35 patients. No treatment-related serious adverse events were found and statistically significant improvement in clinical outcome shown. Magnetic resonance imaging and second-look arthroscopies showed consistent newly formed cartilage tissue. A biopsy taken from the center of the repair tissue was found to have hyaline-like features with a high concentration of proteoglycans and type II collagen. DNA short tandem repeat analysis delivered unique proof that the regenerated tissue contained patient-DNA only. These findings support the hypothesis that allogeneic MSCs stimulate a regenerative host response. This first-in-man trial supports a paradigm shift in which MSCs are applied as augmentations or "signaling cells" rather than differentiating stem cells and opens doors for other applications. Stem Cells 2017;35:1984-1993.

Allogeneic Mesenchymal Stem Cells Stimulate Cartilage Regeneration and Are Safe for Single-Stage Cartilage Repair in Humans upon Mixture with Recycled Autologous Chondrons.

Traditionally, mesenchymal stem cells (MSCs) isolated from adult bone marrow were described as being capable of differentiating to various lineages including cartilage. Despite increasing interest in these MSCs, concerns regarding their safety, in vivo behavior and clinical effectiveness have restrained their clinical application. We hypothesized that MSCs have trophic effects that stimulate recycled chondrons (chondrocytes with their native pericellular matrix) to regenerate cartilage. Searching for a proof of principle, this phase I (first-in-man) clinical trial applied allogeneic MSCs mixed with either 10% or 20% recycled autologous cartilage-derived cells (chondrons) for treatment of cartilage defects in the knee in symptomatic cartilage defect patients. This unique first in man series demonstrated no treatment-related adverse events up to one year postoperatively. At 12 months, all patients showed statistically significant improvement in clinical outcome compared to baseline. Magnetic resonance imaging and second-look arthroscopies showed completely filled defects with regenerative cartilage tissue. Histological analysis on biopsies of the grafts indicated hyaline-like regeneration with a high concentration of proteoglycans and type II collagen. Short tandem repeat analysis showed the regenerative tissue only contained patient-own DNA. These findings support the novel insight that the use of allogeneic MSCs is safe and opens opportunities for other applications. Stem cell-induced paracrine mechanisms may play an important role in the chondrogenesis and successful tissue regeneration found. Stem Cells 2017;35:256-264.

Indirect coculture of stem cells with fetal chondrons using PCL electrospun nanofiber scaffolds.

In vitro coculture system provides a powerful tool for tissue engineering. In this study, we evaluated the gene expressions of human adipose-derived stem cells (ASCs) on polycaprolactone (PCL) scaffold in coculture model with fetal chondrons. Electrospun PCL scaffolds (900 nm fiber diameter) were created and human infrapatellar fat pad-adipose-derived stem cells (IPFP-ASCs) were seeded on these scaffolds. Scanning electron microscopy (SEM) showed attachment of human IPFP-ASCs to scaffold. IPFP-ASCs on scaffolds were cocultured with fetal chondrons in transwell. Gene expressions were investigated using real-time polymerase chain reaction (real-time PCR). In comparison with control group, the expression level of collagen type 2 and aggrecan were significantly decreased but Indian Hedgehog(IHH) significantly increased (P < 0.05).These findings may interpreted that IPFP-ASCs seeded on PCL scaffold, in cocultures with fetal chondrons are tending toward osteogenesis rather than chondrogenesis.

Arthroscopic airbrush assisted cell implantation for cartilage repair in the knee: a controlled laboratory and human cadaveric study.

OBJECTIVE: The objective of this study was to investigate the feasibility of arthroscopic airbrush assisted cartilage repair. METHODS: An airbrush device (Baxter) was used to spray both human expanded osteoarthritic chondrocytes and choncrocytes with their pericellular matrix (chondrons) at 1 × 10(6) cells/ml fibrin glue (Tissucol, Baxter) in vitro. Depth-dependent cell viability was assessed for both methods with confocal microscopy. Constructs were cultured for 21 days to assess matrix production. A controlled human cadaveric study (n = 8) was performed to test the feasibility of the procedure in which defects were filled with either arthroscopic airbrushing or needle extrusion. All knees were subjected to 60 min of continuous passive motion and scored on outline attachment and defect filling. RESULTS: Spraying both chondrocytes and chondrons in fibrin glue resulted in a homogenous cell distribution throughout the scaffold. No difference in viability or matrix production between application methods was found nor between chondrons and chondrocytes. The cadaveric study revealed that airbrushing was highly feasible, and that defect filling through needle extrusion was more difficult to perform based on fibrin glue adhesion and gravity-induced seepage. Defect outline and coverage scores were consistently higher for extrusion, albeit not statistically significant. CONCLUSION: Both chondrons and chondrocytes can be evenly distributed in a sprayed fibrin glue scaffold without affecting viability while supporting matrix production. The airbrush technology is feasible, easier to perform than needle extrusion and allows for reproducible arthroscopic filling of cartilage defects.

Enhanced cell-induced articular cartilage regeneration by chondrons; the influence of joint damage and harvest site.

OBJECTIVE: Interactions between chondrocytes and their native pericellular matrix provide optimal circumstances for regeneration of cartilage. However, cartilage diseases such as osteoarthritis change the pericellular matrix, causing doubt to them as a cell source for autologous cell therapy. METHODS: Chondrons and chondrocytes were isolated from stifle joints of goats in which cartilage damage was surgically induced in the right knee. After 4 weeks of regeneration culture, DNA content and proteoglycan and collagen content and release were determined. RESULTS: The cartilage regenerated by chondrons isolated from the damaged joint contained less proteoglycans and collagen compared to chondrons from the same harvest site in the nonoperated knee (P < 0.01). Besides, chondrons still reflected whether they were isolated from a damaged joint, even if they where isolated from the opposing or adjacent condyle. Although chondrocytes did not reflect this diseased status of the joint, chondrons always outperformed chondrocytes, even when isolated from the damaged joints (P < 0.0001). Besides increased cartilage production, the chondrons showed less collagenase activity compared to the chondrocytes. CONCLUSION: Chondrons still outperform chondrocytes when they were isolated from a damaged joint and they might be a superior cell source for articular cartilage repair and cell-induced cartilage formation.