Subcutaneous adipose tissue of arms, thighs, and abdomen are the most clinically relevant sources of ASCs (Bacakova et al., 2018; Louwen et al., 2018). The proliferation and differentiation of ASCs isolated from different physiological depots can vary (Fraser et al., 2007). For example, ASCs isolated from the medial thigh, trochanteric, or superficial abdominal regions and upper thigh show higher apoptosis than ASCs isolated from superficial abdominal regions (Schipper et al., 2008). It has been also reported that the isolation procedures can impact the ASC’s plasticity, functionality, and quality (Argentati et al., 2018; Palumbo et al., 2018). The main function of white adipose tissue is the storage of excess energy in the form of triglycerides and is found in visceral and subcutaneous adipose tissues (Himms-Hagen et al., 2000; Barbatelli et al., 2010). In terms of yield, the highest number of ASCs is obtained from arm adipose tissue while the highest plasticity is found in the white inguinal adipose tissue. In adults, brown adipose tissue is less abundant than white adipose tissues; however, brown adipose tissues can be found in neonates in the area of the neck, mediastinum, and interscapular tissues. In contrast to white adipose tissues, the primary function of beige/brown adipose tissues is thermogenesis or non-shivering heat generation. Consequently, ASCs derived from beige/brown adipose tissue display distinct characteristics compared with ASCs derived from white adipose tissue, including the ability to undergo skeletal myogenic differentiation (Raposio and Bertozzi, 2017).

In tissue engineering and regenerative medicine, ASCs have significant utility because of their capability to differentiate along multiple lineages. The ASCs can efficiently differentiate into mesenchymal lineages such as bone, fat, and cartilage when exposed in vitro to appropriate inductive culture conditions. This is consistent with their ability to regenerate bone, fat, and cartilage in vivo. While there have been reports of ASC differentiation along non-mesodermal lineages (neurons, cardiomyocytes, and hepatocytes), the efficiency of non-mesodermal lineage ASC differentiation in vivo remains less well documented (Bunnell, 2021).

Compared with bone marrow-derived mesenchymal stem cells (BMSC), ASCs are considered inferior in the case of osteogenic differentiation in vitro (Brennan et al., 2017). However, it is well-established that ASCs undergo osteogenic differentiation both in vitro (Girolamo et al., 2007; Kuterbekov et al., 2018) and in vivo (Supronowicz et al., 2011; Schubert et al., 2013; Lo et al., 2017). Moreover, the differentiation of ASCs into bone and cartilage has also exhibited promising results in clinical applications (Sándor, 2012; Vériter et al., 2015; Farré-Guasch et al., 2018). It has been observed that the OC differentiation of ASCs can be enhanced by exogenous biological factors. The addition of growth factors such as transforming growth factor β1 (TGF-β1), TGF-β3 (Fan et al., 2010), and bone morphogenic protein 4 (BMP4) (Lópiz-Morales et al., 2010) enhance the OC differentiation of ASCs. It has also been shown in pre-clinical studies that the activation of fibroblast growth factor (FGF2) signaling enhances MSC-mediated cartilage repair (Grafe et al., 2018). Furthermore, additional studies have identified a correlation between cell culture oxygen levels and the differentiation and proliferation of ASCs. Generally, native chondrocytes and cartilage are cultured in low oxygen conditions ranging from 2–7% saturation, comparable with oxygen levels experienced by cartilage in vivo (Zhou et al., 2004). Consistent with these observations, ASCs display enhanced chondrogenesis when cultured in vitro under 5% oxygen (Merceron et al., 2010; Weijers et al., 2011).

An optimal scaffold for OC regeneration should have the ability to support osteogenesis, chondrogenesis, and angiogenesis, and have space for nutrient and cell infiltration (Roddy et al., 2018). Scaffolds based on polymers, ceramics, and decellularized ECM have shown promising results so far for the regeneration of OC defects (Ghassemi et al., 2018; Roddy et al., 2018). The shape, size, total porosity, and pore interconnectivity significantly influence the therapeutic effect of a scaffold (Yamane et al., 2007). The pore structure is a critical feature among these properties since it is the literal “gatekeeper” of cell migration, growth, and nutrient flow (Zeltinger et al., 2001). Pore sizes that are too small will restrict cell migration, nutrient diffusion and waste removal and adversely affect cell viability, whereas pore sizes that are too large can diminish cell attachment and biomechanical stiffness of the scaffold (Karageorgiou and Kaplan, 2005). Presently, the two types of scaffolds used in bone and cartilage tissue engineering are synthetic and natural scaffolds.

Chitosan has the ability to support cartilaginous ECM deposition and chondrocyte attachment which makes it a favorable scaffold for OC regeneration. The in vitro and in vivo attachment and proliferation of ASCs, BMSC, and chondrocyte have been reported by several studies (Griffon et al., 2006; Elder et al., 2013; Faikrua et al., 2013; Sheehy et al., 2015; Huang et al., 2019; Scalzone et al., 2019). ASCs seeded on a composite hydrogel of silanized-hydroxypropyl methylcellulose (Si-HPMC) mixed with silanized chitosan were found to remain viable both in vitro and after subcutaneous implantation in nude mice. Moreover, this ASC-seeded hydrogel exhibited significant regeneration in a canine model of OC defect (Boyer et al., 2020).

In another study, the investigators analyzed ASC-seeded chitosan/gelatin hydrogel and cancellous bone composite scaffold for the regeneration of bone and cartilage. The ASCs were induced to chondrocytes and osteoblasts before implantation in the hydrogel and their subsequent viability, proliferation, and ECM deposition were analyzed. The cells displayed significant proliferation in the composite scaffold and deposited cell-specific ECM which was confirmed by staining and scanning electron microscopy (SEM) (Song et al., 2016). These findings are significant since they can host cells and allow remodeling of its microenvironment, a highly desirable trait for a scaffold.

Articular cartilage relies on its biomechanical and biochemical interplay with the subchondral bone to maintain tissue health (Lories and Luyten, 2011). Therefore, for the regeneration of OC defects, a scaffold that supports the regeneration of cartilage and bone simultaneously is ideal, which is a feature often found missing in general scaffolds. To mitigate this challenge, a poly(l-glutamic acid/chitosan and hydroxyapatite-graft-poly (l-glutamic acid)) scaffold was prepared for the regeneration of both hyaline-like cartilage and underlying subchondral bone. The scaffold was found to support the chondrogenic differentiation of ASCs and spheroid formation. In addition, a chondrogenic ASC spheroid-seeded scaffold successfully regenerated hyaline cartilage along with the underlying subchondral bone in a rabbit-based model of OC defects (Zhang et al., 2016).

Alginate-based scaffolds with growth factors are widely used for bone (Kolambkar et al., 2011) and cartilage repair (Mierisch et al., 2002). Stem cells are facilitated to differentiate into bone and cartilage cells by using BMP4. It also facilitates the cells to deposit collagen type I and collagen type II and the in vivo regeneration of bone and cartilage (Kuroda et al., 2006; Zhang et al., 2008). Calcium alginate (CaAlg) hydrogels fabricated with BMP4-transduced ASCs were found to reconstruct the subchondral bone along with the formation of smooth and flat cartilage surfaces. Because of the upregulation of cytoplasmic BMP4, the secretion of collagen I, collagen II, and alkaline phosphatase was also increased. The deposition of these materials enhanced the differentiation of bone and cartilage cells (Chen et al., 2019).

Type 1 collagen is the most abundant component of the ECM (Yoneno et al., 2005) and a useful material for tissue engineering (Dong and Lv, 2016). A study has reported that the use of scaffolds along with growth factors such as BMP2 enhances the proliferation, attachment, and differentiation of ASCs (Lin et al., 2013). ASCs seeded on a collagen type 1 scaffold have displayed enhanced differentiation to OC lineage compared to two-dimensional (2D) cultures. The differentiation further increased when an ASC-collagen composite was cultured with media supplemented with platelet-rich plasma (PRP) and insulin. The expression of beta-1/beta-3 integrin was increased while it was found that the differentiation was independent of the mammalian target of rapamycin (mTOR) signaling and insulin-like growth factor 1 receptor (IGF-1R) (Scioli et al., 2016).

Cartilage-based scaffolds have advantages over other scaffold materials since they contain the native cartilaginous ECM materials crucial for proliferation, attachment, and providing cues for the differentiation of cells. ASCs seeded on cartilage ECM-derived particles (CEDPs) differentiate into chondrocytes without the addition of any exogenous growth factor and with higher efficiency than the 2D-cultured ASCs. ASC-laden CEDPs displayed robust regeneration of rabbit hyaline cartilage, which was limited to fibrous tissue repair only when CEDP was used in the absence of ASCs (Yin et al., 2018).

A biodegradable porous sponge cartilage (BPSC) scaffold has been developed for the regeneration of hyaline-like cartilage. The BPSC scaffold was supplemented with ASCs or its secretome. The BPSC scaffolds fabricated with ASCs were found to regenerate the OC defect more efficiently than the scaffold fabricated with secretome alone (Widhiyanto et al., 2020). Kim et al. (2021) prepared PCL nanofibrils filled with decellularized cartilage ECM which were used along with ASCs. The ASCs seeded on the nanofibrils displayed chondrogenic differentiation without using any exogenous factors and cytokines, which was confirmed by the upregulation of cartilage marker genes. The ASC–nanofibril composite formed a clay-like structure that compactly filled the OC defect in rats and regenerated the cartilage and underlying bone.

Like articular cartilage, auricular cartilage comprises a GAG-collagen type II matrix with limited cell distribution. In contrast to articular cartilage, it has an elastic fiber network which surrounds the cells enabling the uniform distribution of cells. When enzymes are applied to remove the cells, it forms a hollow channel network which enables uniform distribution of cells on repopulation of the decellularized matrix. Bovine auricular cartilage scaffolds were repopulated with bovine and human chondrocytes in monoculture or co-culture with ASCs, which were found to increase cartilage regeneration with a high cell repopulation efficiency (Nürnberger et al., 2019).

The oligo (PEG) fumarate (OPF) scaffold has been well characterized in previous studies (Dadsetan et al., 2012). The OPF scaffold has previously shown positive regenerative results in porcine OC defect models, where the scaffold was seeded with BMSCs before implantation (Lim et al., 2013). The OPF scaffold when seeded with autologous and human-derived ASCs exhibited a good quality of regeneration in a pig-based OC defect model compared to an unseeded scaffold. It was also found that type II collagen was expressed at higher levels in the ASCs seeded on an OPF scaffold, while the formation of mature subchondral bone was also observed, characterized by type I collagen expression (De Girolamo et al., 2015).

Because of its biocompatibility, flexibility, and biodegradability, polycaprolactone (PCL) is one of the most commonly used polyesters in medical applications. PCL-based scaffolds display slow degradation and maintenance of long-term structural integrity during in vitro culture. In addition, MSCs derived from the umbilical cord, bone marrow, and adipose tissue differentiate and synthesize bone and cartilaginous matrix when seeded on a PCL scaffold (Kim et al., 2010; Xue et al., 2017). Im and Lee (2010) investigated the comparison of a PCL scaffold seeded with ASCs and immobilized growth factors TGF-β2 and BMP-7 for the regeneration of OC defects in rabbits. Interestingly, the use of growth factors significantly improved the macroscopic scores of the OC defect but failed to improve the histological scores. It was also noted that the ASC-seeded scaffolds had an uncertain effect on the cartilage repair outcome, which was not found to be significantly better than the scaffold alone. The comparable performance of the scaffold alone could have been due to the infiltration of bone marrow stem cells into the scaffold from the surrounding tissue (Im and Lee, 2010).

Since it is essential to recapitulate the complex fiber arrangement and pore size for optimal TE, 3D bioprinting has emerged as a useful tool to attain these requirements. A recent study has reported the development of 3D-printed scaffolds that are capable of hosting ASCs and regenerating site-specific OC defects. To drive site-specific ASC osteogenesis and chondrogenesis, a scaffold was fabricated using 3D-bioplotting of biodegradable PCL with TCP, or decellularized bovine cartilage ECM (dECM). The PCL-TCP scaffolds were found to be osteo-inductive whereas the PCL-dECM scaffolds favored chondrogenic differentiation of ASCs. Furthermore, a triphasic full-thickness OC scaffold was developed containing layers of PCL, PCL-TCP, and PCL-dECM to mimic the OC unit. ASCs were seeded on the triphasic scaffold and the histochemical analysis of the scaffold after 28 days of culture revealed that the scaffold was positive for calcium and GAGs in the PCL-TCP and PCL-dECM segments, respectively (Mellor et al., 2020). The in vivo evaluation of the same triphasic scaffold in the pig OC defect model revealed that lesions filled with scaffolds along with ASCs showed improved therapeutic effects compared to open lesions. However, although the scaffold facilitated subchondral bone regeneration, cartilage regeneration was found to be limited (Nordberg et al., 2021).

The calcified layer of cartilage present above the subchondral plate acts as a barrier between the bone and cartilage (Findlay and Kuliwaba, 2016). Some studies have suggested that the communication between the calcified cartilage and subchondral bone occurs through numerous vascular canals (Clark and Huber, 1990). The holes present in the subchondral plate open into the bone marrow space that connects the OC unit (Duncan et al., 1987). Moreover, upon culturing bovine osteochondral explants, it was found that the chondrocytes died more rapidly after 7 days in the absence of subchondral bone, whereas when cultured in the presence of subchondral bone, the chondrocytes remained viable. This phenomenon was potentially a result of the survival factor(s) provided by the bone as observed between the bone and cartilage under physiological conditions (Amin et al., 2009).

Recapitulating the interface between the bone and cartilage is one of the most significant challenges in OC TE. Earlier, some biphasic grafts were developed to support the growth of both bone and cartilage as separate tissues (Chu et al., 1995; Gao et al., 2001; Malda et al., 2005; Getgood et al., 2012). However, they do not optimally mimic the native OC interphase, since they partially support bone and cartilage regeneration in separated scaffold layers (Schek et al., 2004; Keeney and Pandit, 2009). Based on the structure of OC tissue, there is a need for matrices that support tissue regeneration and present an interactive bone–cartilage interface (Athanasiou et al., 2009). To cope with this problem, gradient scaffolds are being developed that support osteogenesis and chondrogenesis, while providing a native tissue-like transition between cartilage and bone layers. These scaffolds have been evaluated both in vitro and in vivo (Sherwood et al., 2002; Martin et al., 2007; Wang et al., 2009; Mohan et al., 2011; Declercq et al., 2014; Yousefi et al., 2015). A gradient scaffold comprising poly(D, L-lactic-co-glycolic acid), containing TGFβ1 and BMP2, with or without hydroxyapatite, showed a significant extent of OC regeneration in rabbits (Mohan et al., 2011). Several other gradient scaffolds in combination with various cell types have shown promising results for the treatment of OC defects (Du et al., 2017; Gao et al., 2018).

To better understand the intra-articular tissue cross-talk and the etiology of OC defects, Li et al. (2022) have developed an MSC-derived miniature joint system containing OC tissue, fibrous tissue, and adipose tissue. Such systems will not only enhance the understanding of the OC unit but can potentially be adapted to engineer patient-specific OC units for transplantation in the future.

ASCs are a good candidate for OC defect regeneration because of their easy availability, rich source, and biocompatibility with various types of scaffolds. However, some limitations must be addressed for the successful clinical translation of ASC-scaffold composites. The use of autologous ASCs is considered safe for therapeutic applications as they are not expected to elicit an immune response. However, obtaining a sufficient amount of tissue to isolate a clinically efficacious number of cells is a major challenge (Zhang et al., 2015). To overcome this issue, ASCs can be culture-expanded to increase the cell number (Kharbanda et al., 2014). Another potential solution is the use of allogeneic stem cells. However, the availability of adipose tissue is dependent on surgical procedures, which limits the availability of tissue from healthy donors. Another limitation in using allogeneic ASCs is the variability at the cellular and molecular levels as a consequence of donor sex, age, BMI, and tissue depot (Shu et al., 2012; Bodle et al., 2014; Ock et al., 2016; Abbo et al., 2017).

To produce clinical-grade ASCs, good manufacturing practice (GMP)-based manufacturing facilities need to be established where ASCs obtained from healthy donors are isolated and culture-expanded under aseptic conditions. The ASCs isolated from multiple donors can then be pooled to minimize donor-based variability in the final batch of cells to be used for therapeutic purposes (Kuçi et al., 2016) (Ankrum et al., 2014; Patrikoski et al., 2014). A major impediment in the large-scale culture of clinical-grade cells is the availability of a non-xenogeneic source of growth factors. Animal-derived serum is presently the gold standard for experimental cell culture; however, for clinical translation, human serum and platelet lysate are being analyzed.

The optimal concentration of ASCs to be seeded on scaffolds for OC regeneration is another area that needs increased focus and harmonization. A wide range of ASC doses has been tested for OC regeneration thus far, details of which are shown in Table 1. For the clinical translation of ASC-scaffold composites, it is imperative that future studies are conducted to determine the dose-specific effect of these composites on OC regeneration.

Immunomodulatory and anti-inflammatory capacities are the most clinically relevant properties of ASCs. Several studies have confirmed that ASCs carry out these activities via paracrine signaling via the upregulation of anti-inflammatory cytokines while suppressing pro-inflammatory molecules (Melief et al., 2013; Carrade Holt et al., 2014; Bowles et al., 2017). ASCs have been found to suppress the proliferation and migration of activated inflammatory cells in case of arthritis, thus preventing bone and cartilage degradation (Ter Huurne et al., 2012; Manferdini et al., 2017; Ueyama et al., 2020). Although the role of ASC paracrine activity in tissue generation via modulation of immune and inflammatory response is well established, the present literature relating to the use of ASC-scaffold composites for OC regeneration has placed very little emphasis on the elucidation of these mechanisms. Further studies are mandatory to establish the understanding of molecular mechanisms involved in the regeneration of OC defects by these composites. Moreover, it will be interesting to evaluate if ASCs display enhanced or diminished paracrine activity when implanted alone or in combination with scaffolds.