Lucienne A. Vonk

Is Magnetic Resonance Imaging Reliable in Predicting Clinical Outcome After Articular Cartilage Repair of the Knee? A Systematic Review and Meta-analysis

Background: While MRI can provide a detailed morphological evaluation after articular cartilage repair, its additional value in determining clinical outcome has yet to be determined. Purpose: To evaluate the correlation between MRI and clinical outcome after cartilage repair and to identify parameters that are most important in determining clinical outcome. Study Design: Systematic review and meta-analysis. Methods: A systematic search was performed in Embase, MEDLINE, and the Cochrane Collaboration. Articles were screened for relevance and appraised for quality. Guidelines in the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) Statement were used. Chi-square tests were performed to find variables that could determine correlation between clinical and radiological parameters. Results: A total of 32 articles (total number of patients, 1019) were included. A majority (81%) were case series or cohort studies that used similar standardized MRI techniques. The mean Coleman score was 63 (range, 42-96). For the majority of MRI parameters, limited or no correlation was found. Nine studies (28%) found a correlation between clinical outcome and the composite magnetic resonance observation of cartilage repair tissue (MOCART) or Henderson score and 7 (22%) with defect fill. In 5 studies, a weak to moderate correlation was found between clinical outcome and the T2 index (mean Pearson coefficient r = .53). Conclusion: Strong evidence to determine whether morphological MRI is reliable in predicting clinical outcome after cartilage repair is lacking. Future research aiming specifically at clinical sensitivity of advanced morphological and biochemical MRI techniques after articular cartilage repair could be of great importance to the field.

Single-Stage Cell-Based Cartilage Regeneration Using a Combination of Chondrons and Mesenchymal Stromal Cells Comparison With Microfracture

Background: Autologous chondrocyte implantation (ACI) is traditionally a 2-step procedure used to repair focal articular cartilage lesions. With use of a combination of chondrons (chondrocytes in their own territorial matrix) and mesenchymal stromal cells (MSCs), ACI could be innovated and performed in a single step, as sufficient cells would be available to fill the defect within a 1-step surgical procedure. Chondrons have been shown to have higher regenerative capacities than chondrocytes without such a pericellular matrix. Purpose: To evaluate cartilage formation by a combination of chondrons and MSCs in vitro and in both small and large animal models. Study Design: Controlled laboratory study. Methods: Chondrons and MSCs were cultured at different ratios in vitro containing 0%, 5%, 10%, 20%, 50%, or 100% chondrons (n = 3); embedded in injectable fibrin glue (Beriplast); and implanted subcutaneously in nude mice (n = 10; ratios of 0%, 5%, 10%, and 20% chondrons). Also, in a 1-step procedure, a combination of chondrons and MSCs was implanted in a freshly created focal articular cartilage lesion (10% chondrons) in goats (n = 8) and compared with microfracture. The effect of both treatments, after 6-month follow-up, was evaluated using biochemical glycosaminoglycan (GAG) and GAG/DNA analysis and scored using validated scoring systems for macroscopic and microscopic defect repairs. Results: The addition of MSCs to chondron cultures enhanced cartilage-specific matrix production as reflected by a higher GAG production (P < .03), both in absolute levels and normalized to DNA content, compared with chondrocyte and 100% chondron cultures. Similar results were observed after 4 weeks of subcutaneous implantation in nude mice. Treatment of freshly created cartilage defects in goats using a combination of chondrons and MSCs in Beriplast resulted in better microscopic, macroscopic, and biochemical cartilage regeneration (P ≤ .02) compared with microfracture treatment. Conclusion: The combination of chondrons and MSCs increased cartilage matrix formation, and this combination of cells was safely applied in a goat model for focal cartilage lesions, outperforming microfracture. Clinical Relevance: This study describes the bench-to-preclinical development of a new cell-based regenerative treatment for focal articular cartilage defects that outperforms microfracture in goats. In addition, it is a single-step procedure, thereby making the expensive cell expansion and reimplantation of dedifferentiated cells, as in ACI, redundant.

Overexpression of hsa-miR-148a promotes cartilage production and inhibits cartilage degradation by osteoarthritic chondrocytes

OBJECTIVE Hsa-miR-148a expression is decreased in Osteoarthritis (OA) cartilage, but its functional role in cartilage has never been studied. Therefore, our aim was to investigate the effects of overexpressing hsa-miR-148a on cartilage metabolism of OA chondrocytes. DESIGN OA chondrocytes were transfected with a miRNA precursor for hsa-miR-148a or a miRNA precursor negative control. After 3, 7, 14 and 21 days, real-time PCR was performed to examine gene expression levels of aggrecan (ACAN), type I, II, and X collagen (COL1A1, COL2A1, COl10A1), matrix metallopeptidase 13 (MMP13), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and the serpin peptidase inhibitor, clade H (heat shock protein 47), member 1 (SERPINH1). After 3 weeks, DNA content and proteoglycan and collagen content and release were determined. Type II collagen was analyzed at the protein level by Western blot. RESULTS Overexpression of hsa-miR-148a had no effect on ACAN, COL1A1 and SERPINH1 gene expression, but increased COL2A1 and decreased COL10A1, MMP13 and ADAMTS5 gene expression. Luciferase reporter assay confirmed direct interaction of miR-148a and COL10A1, MMP13 and ADAMTS5. The matrix deposited by the miR-148a overexpressing cells contained more proteoglycans and collagen, in particular type II collagen. Proteoglycan and collagen release into the culture medium was inhibited, but total collagen production was increased. CONCLUSION Overexpression of hsa-miR-148a inhibits hypertrophic differentiation and increases the production and deposition of type II collagen by OA chondrocytes, which is accompanied by an increased retention of proteoglycans. Hsa-miR-148a might be a potential disease-modifying compound in OA, as it promotes hyaline cartilage production.

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

Concise Review: Unraveling Stem Cell Cocultures in Regenerative Medicine: Which Cell Interactions Steer Cartilage Regeneration and How?

Cartilage damage and osteoarthritis (OA) impose an important burden on society, leaving both young, active patients and older patients disabled and affecting quality of life. In particular, cartilage injury not only imparts acute loss of function but also predisposes to OA. The increase in knowledge of the consequences of these diseases and the exponential growth in research of regenerative medicine have given rise to different treatment types. Of these, cell‐based treatments are increasingly applied because they have the potential to regenerate cartilage, treat symptoms, and ultimately prevent or delay OA. Although these approaches give promising results, they require a costly in vitro cell culture procedure. The answer may lie in single‐stage procedures that, by using cell combinations, render in vitro expansion redundant. In the last two decades, cocultures of cartilage cells and a variety of (mesenchymal) stem cells have shown promising results as different studies report cartilage regeneration in vitro and in vivo. However, there is considerable debate regarding the mechanisms and cellular interactions that lead to chondrogenesis in these models. This review, which included 52 papers, provides a systematic overview of the data presented in the literature and tries to elucidate the mechanisms that lead to chondrogenesis in stem cell cocultures with cartilage cells. It could serve as a basis for research groups and clinicians aiming at designing and implementing combined cellular technologies for single‐stage cartilage repair and treatment or prevention of OA.

Caprine articular, meniscus and intervertebral disc cartilage: an integral analysis of collagen network and chondrocytes

Cartilage is a tissue with only limited reparative capacities. A small part of its volume is composed of cells, the remaining part being the hydrated extracellular matrix (ECM) with collagens and proteoglycans as its main constituents. The functioning of cartilage depends heavily on its ECM. Although it is known that the various (fibro)cartilaginous tissues (articular cartilage, annulus fibrosus, nucleus pulposus, and meniscus) differ from one each other with respect to their molecular make-up, remarkable little quantitative information is available with respect to its biochemical constituents, such as collagen content, or the various posttranslational modifications of collagen. Furthermore, we have noticed that tissue-engineering strategies to replace cartilaginous tissues pay in general little attention to the biochemical differences of the tissues or the phenotypical differences of the (fibro)chondrocytes under consideration. The goal of this paper is therefore to provide quantitative biochemical data from these tissues as a reference for further studies. We have chosen the goat as the source of these tissues, as this animal is widely accepted as an animal model in orthopaedic studies, e.g. in the field of cartilage degeneration and tissue engineering. Furthermore, we provide data on mRNA levels (from genes encoding proteins/enzymes involved in the synthesis and degradation of the ECM) from (fibro)chondrocytes that are freshly isolated from these tissues and from the same (fibro)chondrocytes that are cultured for 18 days in alginate beads. Expression levels of genes involved in the cross-linking of collagen were different between cells isolated from various cartilaginous tissues. This opens the possibility to include more markers than the commonly used chondrogenic markers type II collagen and aggrecan for cartilage tissue-engineering applications.

Preservation of the chondrocyte's pericellular matrix improves cell-induced cartilage formation

The extracellular matrix surrounding chondrocytes within a chondron is likely to affect the metabolic activity of these cells. In this study we investigated this by analyzing protein synthesis by intact chondrons obtained from different types of cartilage and compared this with chondrocytes. Chondrons and chondrocytes from goats from different cartilage sources (articular cartilage, nucleus pulposus, and annulus fibrosus) were cultured for 0, 7, 18, and 25 days in alginate beads. Real‐time polymerase chain reaction analyses indicated that the gene expression of Col2a1 was consistently higher by the chondrons compared with the chondrocytes and the Col1a1 gene expression was consistently lower. Western blotting revealed that Type II collagen extracted from the chondrons was cross‐linked. No Type I collagen could be extracted. The amount of proteoglycans was higher for the chondrons from articular cartilage and nucleus pulposus compared with the chondrocytes, but no differences were found between chondrons and chondrocytes from annulus fibrosus. The expression of both Mmp2 and Mmp9 was higher by the chondrocytes from articular cartilage and nucleus pulposus compared with the chondrons, whereas no differences were found with the annulus fibrosus cells. Gene expression of Mmp13 increased strongly by the chondrocytes (>50‐fold), but not by the chondrons. Taken together, our data suggest that preserving the pericellular matrix has a positive effect on cell‐induced cartilage production. J. Cell. Biochem. 110: 260–271, 2010.

Autologous, allogeneic, induced pluripotent stem cell or a combination stem cell therapy? Where are we headed in cartilage repair and why: a concise review

The evolution of articular cartilage repair procedures has resulted in a variety of cell-based therapies that use both autologous and allogeneic mesenchymal stromal cells (MSCs). As these cells are increasingly available and show promising results both in vitro and in vivo, cell-based strategies, which aim to improve ease of use and cost-effectiveness, are progressively explored. The use of MSCs in cartilage repair makes it possible to develop single-stage cell-based therapies. However, true single-stage procedures rely on one intervention, which will limit cell sources to fraction concentrates containing autologous MSCs or culture-expanded allogeneic MSCs. So far, it seems both autologous and allogeneic cells can safely be applied, but clinical studies are still ongoing and little information on clinical outcome is available. Further development of cell-based therapies may lead to clinical-grade, standardized, off-the-shelf products with easy handling for orthopedic surgeons. Although as of yet no preclinical or clinical studies are ongoing which explore the use of induced pluripotent stem cells for cartilage repair, a good manufacturing practice-grade induced pluripotent stem cell line might become the basis for such a product in the future, providing that cell fate can be controlled. The use of stem cells in clinical trials brings along new ethical issues, such as proper controls and selecting primary outcome measures. More clinical trials are needed to estimate detailed risk-benefit ratios and trials must be carefully designed to minimize risks and burdens for patients while choosing outcome measures that allow for adequate comparison with results from similar trials. In this review, we discuss the different aspects of new stem cell-based treatments, including safety and ethical issues, as well as provide an overview of current clinical trials exploring these approaches and future perspectives.

Collagen-induced expression of collagenase-3 by primary chondrocytes is mediated by integrin α1 and discoidin domain receptor 2: a protein kinase C-dependent pathway

OBJECTIVES To investigate whether maintaining the chondrocytes native pericellular matrix prevents collagen-induced up-regulation of collagenase-3 (MMP-13) and whether integrin α1 (ITGα1) and/or discoidin domain receptor 2 (DDR2) modulate MMP-13 expression and which signalling pathway plays a role in collagen-stimulated MMP-13 expression. METHODS Goat articular chondrocytes and chondrons were cultured on collagen coatings. Small interfering RNA (siRNA) oligonucleotides targeted against ITGα1 and DDR2 were transfected into primary chondrocytes. Chemical inhibitors for mitogen-activated protein kinase kinase (MEK1) (PD98059), focal adhesion kinase (FAK) (FAK inhibitor 14), mitogen-activated protein kinase 8 (JNK) (SP600125) and protein kinase C (PKC) (PKC412), and a calcium chelator (BAPTA-AM) were used in cell cultures. Real-time PCR was performed to examine gene expression levels of MMP-13, ITGα1 and DDR2 and collagenolytic activity was determined by measuring the amount of hydroxyproline released in the culture medium. RESULTS Maintaining the chondrocytes native pericellular matrix prevented MMP-13 up-regulation and collagenolytic activity when the cells were cultured on a collagen coating. Silencing of ITGα1 and DDR2 reduced MMP-13 gene expression and collagenolytic activity by primary chondrocytes cultured on collagen. Incubation with the PKC inhibitor strongly reduced MMP-13 gene expression levels. Gene expression levels of MMP-13 were also decreased by chondrocytes incubated with the MEK, FAK or JNK inhibitor. CONCLUSION Maintaining the native pericellular matrix of chondrocytes prevents collagen-induced up-regulation of MMP-13. Both ITGα1 and DDR2 modulate MMP-13 expression after direct contact between chondrocytes and collagen. PKC, FAK, MEK and JNK are involved in collagen-stimulated expression of MMP-13.

Treatment and Prevention of (Early) Osteoarthritis Using Articular Cartilage Repair-Fact or Fiction? A Systematic Review.

Early osteoarthritis (OA) is increasingly being recognized in patients who wish to remain active while not accepting the limitations of conservative treatment or joint replacement. The aim of this systematic review was to evaluate the existing evidence for treatment of patients with early OA using articular cartilage repair techniques. A systematic search was performed in EMBASE, MEDLINE, and the Cochrane collaboration. Articles were screened for relevance and appraised for quality. Nine articles of generally low methodological quality (mean Coleman score 58) including a total of 502 patients (mean age range = 36-57 years) could be included. In the reports, both radiological and clinical criteria for early OA were applied. Of all patients included in this review, 75% were treated with autologous chondrocyte implantation. Good short-term clinical outcome up to 9 years was shown. Failure rates varied from 8% to 27.3%. The conversion to total knee arthroplasty rate was 2.5% to 6.5%. Although a (randomized controlled) trial in this patient category with long-term follow-up is needed, the literature suggests autologous chondrocyte implantation could provide good short- to mid-term clinical outcome and delay the need for total knee arthroplasty. The use of standardized criteria for early OA and implementation of (randomized) trials with long-term follow-up may allow for further expansion of the research field in articular cartilage repair to the challenging population with (early) OA.