Induced pluripotent stem cells in cartilage tissue engineering: a literature review

Abstract Osteoarthritis (OA) is a long-term, persistent joint disorder characterized by bone and cartilage degradation, resulting in tightness, pain, and restricted movement. Current attempts in cartilage regeneration are cell-based therapies using stem cells. Multipotent stem cells, such as mesenchymal stem cells (MSCs), and pluripotent stem cells, such as embryonic stem cells (ESCs), have been used to regenerate cartilage. However, since the discovery of human-induced pluripotent stem cells (hiPSCs) in 2007, it was seen as a potential source for regenerative chondrogenic therapy as it overcomes the ethical issues surrounding the use of ESCs and the immunological and differentiation limitations of MSCs. This literature review focuses on chondrogenic differentiation and 3D bioprinting technologies using hiPSCS, suggesting them as a viable source for successful tissue engineering. Methods: A literature search was conducted using scientific search engines, PubMed, MEDLINE, and Google Scholar databases with the terms ‘Cartilage tissue engineering’ and ‘stem cells’ to retrieve published literature on chondrogenic differentiation and tissue engineering using MSCs, ESCs, and hiPSCs. Results: hiPSCs may provide an effective and autologous treatment for focal chondral lesions, though further research is needed to explore the potential of such technologies. Conclusions: This review has provided a comprehensive overview of these technologies and the potential applications for hiPSCs in regenerative medicine.


Introduction
Cartilage is an avascular, aneural, and alymphatic white connective tissue covering the end of long bones.Once damaged, it lacks intrinsic healing ability due to its avascular nature [1,2].Articular cartilage (AC) provides wear resistance and frictionless, lubricated, and compressible surfaces for joint movement [3,4].AC comprises chondrocytes and an extracellular matrix (ECM), which includes water, proteoglycans, and collagen [5] Osteoarthritis (OA) is a degenerative disease that results from AC injury, leading to severe joint deformity, stiffness, and limited mobility [6,7].However, OA is now widely accepted as a whole-joint disease in which several tissues, including synovium, menisci, ligaments, fat pads, and tendons, play key roles [8].According to World Health Organization (WHO), by 2023, OA is expected to be the fourth leading cause of disability [9].Finding ways to regenerate AC and restore normal joint function is essential to treat and improve the quality of life of those suffering from OA.
Cartilage destruction occurs as chondrocytes produce proteolytic enzymes that degrade collagen and proteoglycans.Factors such as age, genetics, metabolic syndrome, trauma, injury, and stress play a role in the progression of OA [10].In the United States, OA has been reported to affect approximately 10-15% of people over 60 [11].A severe case of OA may require knee replacement in addition to pharmacological and non-pharmacological treatments [12].Cartilage matrix homeostasis is essential as chondrocytes respond to cytokines, stimuli, and growth factors [13].An imbalance in homeostasis leads to the degradation of ECM, resulting in OA [14].Modifications brought about by OA directly influence the biomechanical responses of various joint tissues, particularly the AC.Where they are critical in bearing biomechanical strain due to their load-bearing surfaces that offer minimal friction and wear resistance and exhibit gliding characteristics.The physiological changes induced by OA also affect chondrocytes and chondrons, which alter their biomechanical nature.Interestingly, in comparison with healthy chondrocytes, OA chondrocytes have a lower elastic modulus and viscosity [15,16].
Depending on the type and size of the affected area, various treatments are available for OA.Pharmaceutical treatments aim only to reduce pain and not to restore cartilage damage [17,18].In addition to pharmaceutical pain management, surgical therapies such as arthroscopy, arthroplasty, microfracture, mosaicplasty, and autologous chondrocyte implantation (ACI) can restore damaged tissue [2,19].However, surgical therapies are invasive and carry risks, such as infection, bleeding, and nerve damage, and also the recovery from surgery is lengthy with extensive physical therapy [20].ACI is the only proven therapy that can temporarily restore normal function in people with cartilage degeneration and delay the need for joint replacement surgery.Unfortunately, this technique cannot effectively treat the widespread cartilage damage that occurs in OA [21].New approaches, such as cell-based therapies using mesenchymal stromal cells (MSCs) isolated from adipose tissue, bone marrow, synovium fluid, or umbilical cord, have shown promising results in cartilage regeneration in OA [22].OA-MSCs have been successfully used to produce 3D cartilage tissue since 2007 [23].Due to their chondrogenic potential, preclinical and clinical studies have investigated several treatments for OA that involve the introduction of MSCs into damaged cartilage regions [21,24].However, the low efficiency of MSCs in differentiation, researchers have investigated the use of embryonic stem cell-derived mesenchymal stem cells (ES-MSCs) as therapy for various disorders [25,26].Multiple injections of ES-MSCs have been shown to improve OA symptoms in rat models, and ES-MSCs could be an effective, potentially endless cellular source for OA treatment [22].
Since the introduction of induced pluripotent stem cells (iPSCs) by the Yamanaka group in 2006 has revolutionized regenerative medicine and created incredible opportunities, as these cells exhibit properties similar to ESCs overcoming the ethical issues surrounding the use of embryonic material [2,27,28].In the future, iPSCs could treat cartilage defects in clinical settings [29].Recent studies have demonstrated a variety of protocols for chondrogenic differentiation, such as high-density mass, micro mass, monolayer culture, embryoid body formation, and directed differentiation methods [2].To help treat OA, new technologies such as hiPSCs and 3D bioprinting are being developed and explored [28].This review discusses the various methods employed in chondrogenic differentiation and the recent trends of 3D bioprinting used to regenerate cartilage which could be a possible approach for treating OA.

Methodology
The present review article focused on attaining knowledge on chondrogenic differentiation using hiPSCs and its recent trends in 3D printing technology.A literature search was conducted by two separate reviewers using scientific search engines like PubMed, MEDLINE, and Google Scholar databases with the terms 'cartilage tissue engineering,' 'osteoarthritis treatment,' 'hiPSCs,' and '3D bioprinting' to retrieve published literature on chondrogenic differentiation and their different protocols.The inclusion criteria for the current literature review were studies published in English, studies on chondrogenic differentiation, and tissue engineering using hiPSCs, with in vitro and in vivo models This review discusses the current cell-based therapies for cartilage tissue engineering, chondrogenic differentiation protocols, 3D bioprinting also offers promising treatment options for OA.This article highlights the potential of iPSCs in regenerative medicine, emphasizing the need for continued research and innovation to improve outcomes for patients with OA.

Chondrogenic differentiation of hiPSCs
An exciting new approach to cartilage tissue engineering involves chondrogenic differentiation of hiPSCs.As a result of cartilage tissue formation and maintenance, chrondrocytes play a vital role.Repairing cartilage defects using chondrocytes is challenging, as they have limited self-renewal and tissue regeneration capacity; here hiPSCs take advantage of these challenges [30].In this context, hiPSCs are valuable alternatives, as they can differentiate into chondrocytes and produce cartilage [13,31].Different methods can be used to induce cartilage differentiation in hiPSCs, including the use of growth factors such as transforming growth factor-β (TGF-β), bone morphogenetic protein (BMP), and fibroblast growth factor (FGF) [32].Several studies have reported successful chondrogenic differentiation of hiPSCs in vitro and in vivo, using different approaches.Growth factors activate specific signaling pathways that promote the expression of chondrogenic markers, including SOX9 (SRY (sex determining region Y-box 9), COL2A1 (Collagen type 2), and ACAN (AGGRECAN), these markers can be validated under in vitro conditions in induced hiPSC cell lines [33].Differentiating hiPSCs into functional cartilage tissue can also be improved using mechanical stimulation and 3D culture systems [7].For example, a study by Ko et al. demonstrated that hiPSC-derived chondrocytes could produce cartilage-like tissue in a mouse model of OA [34].Similarly, another study by Toh et al. showed that hiPSC-derived chondrocytes could repair cartilage defects in a rat model of OA [35].These findings suggest that the chondrogenic differentiation of hiPSCs holds great promise for cartilage tissue engineering and regenerative medicine.
Differentiation protocols must consider several important factors, such as growth factor concentration, cell density in culture, type of cell population, and biomaterials that mimic the in vivo microenvironment [36].Some of the protocols used to differentiate iPSCs into chondrocyte-like cells include the following: (1) Embryoid bodies (EBs) formation (2) Coculture (3) Direct differentiation [7] To evaluate successful chondrogenic differentiation, certain markers should be considered.Table 1 lists the chondrogenic differentiation markers used to assess chondrogenic differentiation.

Embryoid bodies (EB) formation
One of the most established stem cell differentiation protocols involves the formation of EBs.In addition, hiPSCs can also be differentiated into EBs with respect to OA differentiation.As hiPSCs aggregate spontaneously, the resulting structures resemble early embryos in three dimensions [37].EBs are aggregates of 3D cells that reflect embryonic differentiation into the three germ layers (endoderm, mesoderm, and ectoderm) [38] (Table 2).EBs are obtained by culturing iPSCs in ultra-low attachment flasks to prevent surface attachment and promote cell aggregation [39].Without exogenous growth factors, EBs can differentiate into chondrocytes, thus providing an added advantage compared with other chondrogenic differentiation methods [38].Chondrogenic differentiation of hiPSCs into cartilage involves two critical steps: induction of mesenchymal-like outgrowth cells and accumulation of chondrogenic pellets [34,40].According to Rim et al. hiPSCs can be induced in EBs that can differentiate into chondrocytes that express chondrogenic markers, such as SOX9 and COL2A1 [41].Costa et al. used TGF-3 and BMP-6 to induce chondrogenesis in hiPSC-derived EB and showed that the resulting chondrocytes produced cartilage tissue in vitro [42].Kim et al. successfully used EB formation as the first protocol for chondrogenesis [43].In the EB protocol, EBs are cultured in a standard chondrogenic medium supplemented with specific growth factors that induce chondrogenic differentiation, such as TGF-β1, TGF-β3, BMP-2, BMP-4, BMP-6, and BMP-7 [36].Auguyniak et al. used hiPSCs to generate EBs cultured in a chondrogenic medium containing TGF-β1, BMP-2, and insulin-like growth factor-1 (IGF-1) and observed a promising outcome [44] Rim et al. used TGF-β1, BMP-2, and dexamethasone to induce chondrogenic differentiation of hiPSC-derived Ebs [41].Compared with primary human chondrocytes, chondrocytes derived from this method had similar gene expression profiles (Table 2).
Lee et al. reported that a cocktail of defined growth factors allowed more efficient differentiation of hiPSCs into chondrocytes in fibronectin-coated plates within 14 days using EB [45].Despite this advantage, this protocol requires many growth factors at high concentrations (up to 100 ng/ml), making it expensive and limiting its potential clinical application [45].Despite its advantages, such as its ability to mimic early embryonic development and promote cellular interactions, the use of EBs for chondrogenic differentiation has some limitations, which mainly include heterogeneity, little control over the differentiation process, time-consuming, and risk for teratoma formation [45][46][47]    Abbreviations: CDM, chondrogenic differentiation medium; NS, not significant; NT, not tested.

Coculturing protocol
Another method is coculturing hiPSCs with chondrocytes.This procedure creates a microenvironment that promotes chondrogenic differentiation by growing hiPSCs alongside chondrocytes (Table 2).Furthermore, coculture systems mimic the in vivo stimuli environment of cartilage tissue, making them more physiologically relevant for chondrogenic differentiation.Wei et al. used a two-step protocol where EB formation was cocultured with primary human chondrocytes in a chondrogenic medium with alginate for 28 days [3].The differentiated cells exhibited chondrocyte-like morphology and expressed AGGRECAN and COL-II.Biochemical assays showed that differentiated cells produced components of the extracellular matrix typical of hyaline cartilage, including glycosaminoglycan and collagen.A study by Qu et al. used the coculture method supplemented with dexamethasone, L-ascorbic acid 2-phosphate, proline, pyruvate, and TGF-β3 [49].Cells were cultured for 21 days and observed up-regulation of chondrogenic markers, including collagen type II and AGGRECAN, and down-regulation of markers associated with undifferentiated cells, such as OCT4 and SOX2.The alginate matrix was a suitable scaffold for the chondrogenic differentiation of iPSC.The results suggest that this approach could be further developed to produce functional cartilage tissue for clinical applications [3].Table 2 highlights the articles that used coculturing protocol in the chondrogenic differentiation.

Directed differentiation
Directed differentiation of human-induced pluripotent stem cells (hiPSCs) into chondrocytes is a promising approach to cartilage tissue engineering as it bypasses the stage of MSCs [50].Oldershaw et al. initially described a method to direct human embryonic stem cells (hESCs) to differentiate into chondrocytes, which form and maintain cartilage tissue: a step-by-step protocol-induced mesoderm formation, chondrogenic differentiation, and chondrocyte maturation [51].As a result, chondrocytes produced cartilage-like tissue in vitro and expressed chondrogenic markers (SOX9, ACAN, and COL2A1).This approach offers a more controlled and efficient method for producing chondrocytes for cartilage tissue engineering and regenerative medicine than other methods of chondrogenic differentiation.Based on this technology, protocols for iPSCs were developed to induce chondrogenesis by chemical definition.Table 2 provides an overview of the research using directed differentiation methods.Later, Toh et al. used TGF-β3 and BMP-6 to induce chondrogenesis in hiPSCs and showed that the resulting chondrocytes could repair cartilage defects in a rat model of osteoarthritis [35].Another study by Nakagawa et al. used a combination of TGF-β1 and BMP-2 to induce chondrogenic differentiation of hiPSCs and demonstrated the successful formation of hyaline cartilage in vitro [52].Direct differentiation of hiPSCs into chondrocytes, without contamination-prone coculture systems, is possible.Nejadnik et al. reported a method to differentiate iPSCs into chondrocytes in approximately 42 days using a chondrogenic differentiation medium and TGF-β3 [53].Similarly, Matsumoto et al. reported a successful protocol with a chondrogenic differentiation medium and TGF-β3 and BMP-2 [54].In another study by Saito et al. suggested that they induced chondrogenic differentiation of hiPSC and formed hyaline cartilage tissue when implanted subcutaneously into immunodeficient mice [33].However, the study highlighted the risk of tumorigenesis due to the formation of teratomas in the implanted tissue.The directed differentiation method is chemically defined, making it more reproducible than other differentiation methods.Also, coculture with ESCs provides a more natural and physiological environment, producing more homogeneous chondrogenic cells [55].Several reports also suggested the high levels of gene expression of COL2A1, SOX-9, and ACAN in 14-15 days of differentiation [54,56,57].

3D cartilage tissue engineering using hiPSC
Regenerative medicine uses tissue engineering to develop functional tissues or organs that can replace or repair damaged tissues or organs.This field involves the creation of 3D structures that mimic the structure and function of native tissues and organs using cells, biomaterials, and biochemical factors.It was reported that hiPSCs are an effective approach for repairing damaged cartilage and restoring joint functions [2].Compared with other cell sources, hiPSCs can differentiate into chondrocytes, the main cells responsible for cartilage formation and maintenance [58].
In 3D bioprinting, the two main approaches are scaffold-based and scaffold-free are used to create tissue constructs [59].Using hiPSCs, a scaffold-based system can engineer 3D cartilage tissue.As a result of the scaffold, hiPSCs can grow and organize into functional cartilage tissues [60].A scaffold provides mechanical support, cell guidance, and a platform for delivering biochemical factors that promote tissue growth [61].In contrast, scaffold-free tissue engineering does not require the use of scaffolds, and instead, they rely on cell self-assembly and organization.This approach uses cell sheet engineering and spheroid and organoid cultures to create 3D structures without scaffolds [61].
Scaffold-based tissue engineering presents multiple benefits compared with scaffold-free methods.One key advantage is the ability to manage the framework and mechanical characteristics of the scaffold, which can be customized to align with the features of the original tissue.This enables the creation of tissue-engineered constructs that accurately imitate the structure and functionality of native tissues [62].In addition, they facilitate the delivery of biochemical elements to cells, which encourages tissue development and differentiation.This is especially valuable for tissues that require complex microenvironments, such as bone or cartilage, which require the delivery of growth factors and other signaling molecules to foster tissue generation [63].However, scaffold-free tissue engineering can produce 3D structures that closely resemble native tissues without requiring a scaffold.This enables the creation of tissue-engineered constructs that closely mimic the architecture and functionality of natural tissue.
Additionally, scaffold-free tissue engineering eliminates the possibility of immune rejection because it does not incorporate foreign materials that may be identified by the host's immune system [64].Scaffold-based and scaffold-free methods present unique advantages and disadvantages, with the chosen technique relying on the specific tissue or organ being engineered and its intended clinical use [65].Further investigation is required to enhance both methodologies and to devise novel tissue engineering strategies to address the limitations of the field.
Bioprinting is an interdisciplinary field that combines the strengths and advantages of 3D printing and regenerative medicine to bridge the gap between biology and engineering.Using traditional additive manufacturing techniques, bioprinting technology creates microscale tissues by precisely deploying bioinks containing cells [66].Bioprinting has garnered interest in hiPSCs because of their capacity to generate intricate 3D formations that resemble the architecture and functionality of natural tissues or organs [67].3D bioprinting is a burgeoning technology that presents a promising method for tissue engineering using hiPSCs, where hydrogels, ECM proteins, and synthetic polymers are used [68].Employing hiPSCs in 3D bioprinting provides numerous benefits compared with other cell sources, such as their ability to transform into various cell types and their potential for limitless growth and accessibility [69].Multiple studies have documented the successful bioprinting of hiPSCs for tissue engineering.Extrusion-based, Inkjet, and laser-assisted techniques are used in 3D bioprinting [70,71].
Nguyen and colleagues employed a 3D bioprinting technique to create iPSCs with a composite bio-ink made of nanofibrillated cellulose and alginate (NFC/ALG) combined with irradiated human chondrocytes.After 5 weeks, they observed the formation of hyaline-like cartilaginous tissue, accompanied by an increased presence of chondrocytes within the bioprinted structures.These findings imply that the optimal bioink for 3D bioprinting of iPSCs, and their direct conversion to chondrocytes, may offer a novel regenerative treatment for damaged cartilage [72].A bioink was created by mixing COL type I and agarose (AG) with sALG in 2018, which embedded chondrocytes to 3D print cartilage tissue in vitro, which enabled higher mechanical strength [73].Hontani et al. developed a unique approach to chondrogenic differentiation, which involves using ultra-purified alginate gel as a 3D scaffold and gradually transforming iPSCs into chondrocytes through the MSC-like cell phase.As iPSC-MSCs were cultivated, SOX9, Col2A1, and aggrecan expression increased significantly [74].In another study, Bioink based on nanofibrillated cellulose (NFC) composite was used for 3D bioprinting methods to fabricate cartilage tissue structures from iPSCs with irradiated human chondrocytes (iCHons) [75].The hyaline-like cartilage tissue maintained pluripotency for five weeks, as confirmed by the expression of collagen type II and the tumor-causing gene OCT4 [54].Nakagawa et al. used a combination of TGF-β1 and BMP-2 to initiate the chondrogenic differentiation of hiPSCs and successfully formed hyaline cartilage in vitro [52].
Furthermore, the study discovered that incorporating a poly(lactic-co-glycolic acid) (PLGA) scaffold enhanced the chondrogenic differentiation of hiPSCs and the development of functional cartilage tissue.Figure 1 illustrates the combined approach to OA repair using cell-based therapies and 3D bioprinting technology.iPSCs are derived from adult somatic cells to avoid the ethical issues of using ESCs.Furthermore, they can be derived from tissue sources, such as fibroblasts, and do not require invasive methods such as bone marrow or adipose tissue biopsies [75].The NFC/ALG composite ink formulation has been brought to market under the brand name CELLINK ® .It has undergone in vitro and in vivo testing with human chondrocytes and iPSC cells originating from chondrocytes [76].
Lee et al. conducted a study using a 3D bioprinting technique to create a customized cardiac tissue structure by incorporating hiPSC-derived cardiomyocytes and a hydrogel predominantly composed of collagen.Using a rat model of myocardial infarction, the engineered cardiac tissue was shown to demonstrate characteristics similar to native cardiac tissue and to improve cardiac function [77].Similarly, Zhang et al. employed a 3D bioprinting approach to producing a cartilage tissue structure using hiPSCs and a hydrogel mainly composed of silk fibroin.The study concluded that the fabricated cartilage tissue exhibited properties similar to those of natural cartilage tissue and could correct cartilage defects in a rabbit model of OA [78].Additionally, the study demonstrated the feasibility of using magnetic nanoparticles to enhance the targeting and retention of hiPSCs in damaged cartilage tissue [78].Table 3 lists some articles in which different 3D printing technologies have been successfully used to achieve optimal results in chondrogenesis.In summary, 3D bioprinting with hiPSCs holds great promise as a strategy for treating OA and regenerating a damaged cartilage tissue.However, further research is necessary to optimize 3D bioprinting and develop new techniques to fabricate complex tissues or organs utilizing hiPSCs.
The results of this review suggest that hiPSCs can regenerate cartilage successfully.However, further research is required to address the potential risk of tumorigenesis associated with hiPSCs.Hence, other studies are needed to address potential risks associated with hiPSCs and ensure their safety.Moreover, in vivo studies could assess the Abbreviations: ALG, alginate; CS, chitosan; CTE, cartilage tissue engineering; GAG, glycosaminoglycan; Gel, gelatine; GF, growth factor; HA, hyaluronic acid; HAP, hydroxyapatite; NFC, nanofibrillated cellulose.
long-term safety and efficacy of hiPSC-derived cartilage and the molecular and genetic changes occurring during iPSC generation and differentiation.

Conclusion
The ability of stem cells to differentiate into chondrocytes, the cells responsible for forming, and maintaining cartilage, is one of the major advantages of hiPSCs for cartilage tissue engineering.Further, hiPSCs combined with bioprinting technology could lead to a revolution in cartilage tissue engineering.Using bioprinting, complex 3D structures mimicking the native tissue can be designed with precise placement of cells and biomaterials.From hiPSCs, cartilage tissue can be created with greater accuracy, reproducibility, and efficiency using bioprinting than traditional methods.In addition, custom scaffolds made according to the geometry of the defect site might improve cartilage regeneration and repair outcomes.HiPSCs combined with bioprinting could overcome many limitations of the current cartilage tissue engineering strategies and provide an alternative to treat cartilage defects and osteoarthritis.However, other innovative bioinks, rich in chondrogenic cells and growth factors, are still needed to optimize cartilage tissue engineering strategy and promote cartilage repair.However, this technology offers a new frontier for bio-fabrication, demonstrating the potential for revolutionizing 3D bioprinting.

Figure 1 .
Figure 1.A schematic representation of cell-based therapies and 3D bioprinting practice in osteoarthritis

COL2A1, P<0.05 COL1A1, NT ACAN, P<0.05
© 2024 The Author(s).This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY).