Background

Focal cartilage lesions represent a major challenge in orthopedic and knee surgery because they are highly prevalent, clinically debilitating, exhibit limited intrinsic repair capacity, and tend to progress to osteoarthritis.

The first use of cell-based therapy for cartilage repair dates to 1987 and was described in 1994 by Brittberg et al. [1], introducing autologous chondrocyte implantation (ACI). In this procedure, autologous chondrocytes harvested from the patient’s articular cartilage via arthroscopic biopsy are expanded in vitro and subsequently reimplanted into the chondral defect to promote the formation of hyaline-like repair tissue. The cultured cells are injected under a periosteal flap sutured over the defect during a second surgery. Later generations of ACI replaced the periosteum with biomaterial scaffolds, such as decellularized collagen membranes or hyaluronic acid matrices, pre-seeded with autologous cells [2]. These advances have allowed cartilage regeneration beyond the limited capacity of native tissue repair. ACI is widely recognized as an effective treatment option for focal chondral lesions, showing favorable long-term outcomes.

Progenitor cells have been identified in various adult tissues [3], including articular cartilage. Chondroprogenitors are less differentiated than mature chondrocytes and are thought to participate in cartilage regeneration, representing a promising cell source for articular cartilage repair [4]. These cells display a strong chondrogenic phenotype, low hypertrophic potential, high proliferative capacity, and maintenance of chondrogenic potential during extended in vitro expansion [5–8]. Chondroprogenitors have emerged as a promising alternative for cell-based therapy in cartilage repair. In 2004, chondroprogenitor cells were first isolated using a fibronectin adhesion assay [9]. Subsequently, alternative isolation methods were described, including migration-based selection [10] and sorting by surface markers [11–13].

The fibronectin adhesion assay selects cartilage cells that attach rapidly to fibronectin-coated substrates; it was among the first practical methods used to prospectively enrich cartilage progenitor-like cells and remains widely applied in cartilage progenitor research. This approach is relatively simple, can yield clonogenic progenitor clones, and selects based on a behaviorally relevant property (adhesion). The limitations are the heterogeneity of the cells obtained, the differing performance by cartilage source, and protocol variability [14,15].

Surface marker sorting aims to prospectively isolate chondroprogenitors by antigen expression, enabling more defined populations when robust markers are available. It presents high specificity when validated markers exist, the ability to combine markers for refined subpopulations, and high-purity populations for downstream assays [16]. As limitations, it requires cytometry infrastructure, can be time-consuming, and surface antigen expression can be contextually variable or lost during expansion [17].

The explant-based isolation of chondroprogenitor cells offers several biological and methodological advantages over enzymatic or adhesion-based protocols. In explant cultures, cells remain initially embedded within their native extracellular matrix (ECM), preserving key cell–matrix interactions that support a chondrogenic phenotype and reduce dedifferentiation typically observed during monolayer expansion [16]. Moreover, explant systems naturally select for migratory chondroprogenitors—cells capable of leaving the tissue and migrating into the surrounding environment—which may reflect an intrinsic regenerative response to cartilage injury. These cells exhibit enhanced proliferative capacity, low hypertrophic potential, and stable expression of chondrogenic genes such as SOX9, ACAN, and COL2A1 even after prolonged culture [16,18]. Furthermore, when tested in ex vivo models, chondroprogenitors isolated from explants retained high viability and regenerative capacity within platelet-rich plasma scaffolds, forming a cartilage-like matrix rich in glycosaminoglycans and type II collagen [19]. Together, these findings highlight that explant-based approaches not only preserve the native microenvironment but also enrich for a physiologically relevant progenitor population with promising potential for cartilage repair strategies. Obtaining chondroprogenitors by explant is an important way to obtain cells for both scientific research and use in ACI. Therefore, we present a protocol for obtaining chondroprogenitors by explants.