Achieving Nasal Septal Cartilage In Situ Regeneration: Focus on Cartilage Progenitor Cells

The nasal septal cartilage plays an important role in preventing the collapse of the nasal bones and maintaining the appearance of the nose. In the context of inherent difficulties regarding septal cartilage repair and the shortage of cartilage graft resources for regeneration, tissue engineering, especially the in situ strategy based on scaffolds, has become a new prospect and become one of the most promising approaches. Given that it is difficult for chondrocytes to achieve directional migration and secrete matrix components to participate in tissue repair after cartilage injury, cartilage progenitor cells (CPCs), with great migratory ability and stem cell characteristics, have caught the attention of researchers and brought hope for nasal septal cartilage in situ regeneration. In this review, we first summarized the distribution, characteristics, isolation, and culture methods of nasal septal CPCs. Subsequently, we described the roles of migratory CPCs in cartilage regeneration. Finally, we reviewed the existing studies on CPCs-based cartilage tissue engineering and summarized the strategies for promoting the migration and chondrogenesis of CPCs so as to provide ideas for achieving nasal septal cartilage in situ regeneration.


Introduction
The nasal septal cartilage plays an important role in preventing the collapse of the nasal bones and maintaining the appearance of the nose.Clinically, trauma, surgery, tumor, infection, and congenital factors may cause nasal septal deformity and defect, which affects the respiratory function of the patient's nose and results in morphological deformity, impacting the patient's daily life [1].Therefore, severe cases often require surgical intervention and the implantation of new cartilage.Nasal septal cartilage offers easy accessibility compared to rib cartilage and articular cartilage and shows better tissue stability and higher chondrogenic capacity [2,3].Therefore, nasal septal cartilage has become one of the important donor sites for autologous cartilage transplantation and is widely used, not only in plastic surgery, but also in joint surgery [3][4][5].
In the context of the inherent difficulties for septal cartilage self-repair and the shortage of cartilage graft resources for transplant surgery, tissue engineering strategies have revealed a new prospect and become one of the most promising approaches.In the study by Bermueller et al. [6], an orthotopic rat model for nasal septal repair was developed in which the marine collagen chondrocyte-seeded/unseeded scaffolds were evaluated.In similar rabbit models, elastin-gelatin-hyaluronic acid and decellularized porcine nasal septal cartilage scaffolds were also evaluated, both using auricular cartilage as seed cells [7,8].However, traditional tissue engineering methods based on seed cells and scaffolds often required multiple steps, including tissue acquisition, seed cell isolation and culture, scaffold implantation, in vitro culture, and construct implantation.This causes patients to undergo at least two surgical procedures, with significant time spent in between, and to suffer from donor-site injury, accompanying pain, and risk of donor-site complications.In situ regeneration is one of the most sought-after tissue engineering methods, requiring only the implantation of a scaffold and the recruitment of autologous in situ cells to achieve tissue regeneration.The absence of additional seed cells ensures biosafety and reduces the number of pre-implantation procedures, which facilitates clinical translation.The successful recruitment of autologous in situ cells is the key to in situ regeneration.However, it is difficult for chondrocytes harbored in the pyknotic cartilage matrix to achieve directed migration and secrete matrix components to achieve satisfying tissue repair after cartilage injury.
Cartilage progenitor cells (CPCs) were first reported to be found in bovine joints [9], and subsequently, nasal CPCs (NCPCs) were isolated and identified in human nasal septal cartilage in 2011 [10].NCPCs were proven to exhibit the basic characteristics of stem cells, such as self-renewal, multidirectional differentiation potential, and clonal formation; more importantly, they also showed an even a stronger migratory capacity than bone marrowderived mesenchymal stem cells (BMSCs) [11].Therefore, these cells bring new hope for nasal septal cartilage in situ regeneration [10,11].However, the natural recruitment of NCPCs is insufficient to achieve the complete repair of cartilage defects [12].Therefore, researchers hope to design scaffolds with high CPCs recruitment capabilities, or to screen and utilize biochemical additives such as chemokines, to effectively improve cartilage in situ regeneration.
This review is aimed at summarizing the existing basic information on NCPCs and existing CPC-related tissue engineering designs to provide support and references for the in situ regeneration of nasal septal cartilage (Figure 1).Specifically, we first summarized the distribution, isolation, and culture methods of NCPCs.Subsequently, we described the roles of migratory CPCs in cartilage regeneration.Finally, we reviewed the existing studies on CPCs-based cartilage tissue engineering and summarized the strategies for promoting the migration and chondrogenesis of CPCs to provide ideas for achieving nasal septal cartilage in situ regeneration.
Biomolecules 2023, 13, x FOR PEER REVIEW 2 undergo at least two surgical procedures, with significant time spent in between, an suffer from donor-site injury, accompanying pain, and risk of donor-site complicatio In situ regeneration is one of the most sought-after tissue engineering methods quiring only the implantation of a scaffold and the recruitment of autologous in situ to achieve tissue regeneration.The absence of additional seed cells ensures biosafety reduces the number of pre-implantation procedures, which facilitates clinical transla The successful recruitment of autologous in situ cells is the key to in situ regenera However, it is difficult for chondrocytes harbored in the pyknotic cartilage matri achieve directed migration and secrete matrix components to achieve satisfying tissu pair after cartilage injury.
Cartilage progenitor cells (CPCs) were first reported to be found in bovine joints and subsequently, nasal CPCs (NCPCs) were isolated and identified in human nasal tal cartilage in 2011 [10].NCPCs were proven to exhibit the basic characteristics of s cells, such as self-renewal, multidirectional differentiation potential, and clonal forma more importantly, they also showed an even a stronger migratory capacity than bone m row-derived mesenchymal stem cells (BMSCs) [11].Therefore, these cells bring new h for nasal septal cartilage in situ regeneration [10,11].However, the natural recruitme NCPCs is insufficient to achieve the complete repair of cartilage defects [12].There researchers hope to design scaffolds with high CPCs recruitment capabilities, or to sc and utilize biochemical additives such as chemokines, to effectively improve cartilag situ regeneration.
This review is aimed at summarizing the existing basic information on NCPCs existing CPC-related tissue engineering designs to provide support and references fo in situ regeneration of nasal septal cartilage (Figure 1).Specifically, we first summar the distribution, isolation, and culture methods of NCPCs.Subsequently, we descr the roles of migratory CPCs in cartilage regeneration.Finally, we reviewed the exis studies on CPCs-based cartilage tissue engineering and summarized the strategies promoting the migration and chondrogenesis of CPCs to provide ideas for achieving n septal cartilage in situ regeneration.

Histological Distribution and Characteristics of NCPCs
Nasal cartilage mainly includes the central septal cartilage, two upper lateral cartilages, and two lower lateral cartilages.These cartilaginous structures are made up of hyaline cartilage, consisting of an extracellular matrix (ECM) and chondrocytes, and which was formerly considered to be the only existing cell component [13].With the perichondrium on its surface and a sandwich-like structure, as opposed to a structure transiting into the subchondral bone, the nasal cartilage is obviously different from the articular cartilage (Figure 2).When approaching the deeper zone, the cells gradually become larger, more rounded, and less frequent [13].The perichondrium is rich in type I collagen, thus distinguishing it from the cartilage tissue, which contains type II only [14].The perichondrium, however, is not a membrane that can be easily separated from cartilage tissue.In previous histological studies, the demarcation between the perichondrium and the cartilage surface was not easily distinguished, but exhibited a more transitional evolution [14,15], and the demarcation between the cartilage and the perichondrium and between the perichondrium and the lamina propria was more regular in the posterior versus the anterior septum [16].Therefore, it is difficult to distinguish the flat cells in the superficial layer of cartilage from those in the deep layer of the perichondrium.But it is confirmed that a subpopulation of CPCs exists among these flat cells, playing an important role in cartilage regeneration.Compared to the differentiated chondrocytes, in addition to the morphological differences, CPCs exhibit the basic characteristics of stem cells (such as self-renewal, multidirectional differentiation potential, and clonal formation) and the stem cell markers on their surfaces, along with a stronger migratory capacity [11].
engineering, especially the in-situ strategy based on scaffolds, has become one of the most p ing approaches.CPCs, with great migratory ability and stem cell characteristics, have caug attention of researchers and brought hope for nasal septal cartilage in situ regeneration.Stra for promoting the migration and chondrogenesis of CPCs are constantly being improved to more CPCs to migrate to tissue engineering scaffolds and to subsequently differentiate in or achieve satisfactory cartilage in situ regeneration.Created with BioRender.com(accessed on 9 2023).

Histological Distribution and Characteristics of NCPCs
Nasal cartilage mainly includes the central septal cartilage, two upper lateral lages, and two lower lateral cartilages.These cartilaginous structures are made up aline cartilage, consisting of an extracellular matrix (ECM) and chondrocytes, and w was formerly considered to be the only existing cell component [13].With the peri drium on its surface and a sandwich-like structure, as opposed to a structure tran into the subchondral bone, the nasal cartilage is obviously different from the articula tilage (Figure 2).When approaching the deeper zone, the cells gradually become l more rounded, and less frequent [13].The perichondrium is rich in type I collagen distinguishing it from the cartilage tissue, which contains type II only [14].The peri drium, however, is not a membrane that can be easily separated from cartilage tiss previous histological studies, the demarcation between the perichondrium and the lage surface was not easily distinguished, but exhibited a more transitional evo [14,15], and the demarcation between the cartilage and the perichondrium and be the perichondrium and the lamina propria was more regular in the posterior versu anterior septum [16].Therefore, it is difficult to distinguish the flat cells in the supe layer of cartilage from those in the deep layer of the perichondrium.But it is confi that a subpopulation of CPCs exists among these flat cells, playing an important r cartilage regeneration.Compared to the differentiated chondrocytes, in addition morphological differences, CPCs exhibit the basic characteristics of stem cells (such a renewal, multidirectional differentiation potential, and clonal formation) and the ste markers on their surfaces, along with a stronger migratory capacity [11].Early in 1995, in the study of Pirsig et al. [17], an implant of demineralized b bone matrix enfolded in a pedicled flap of autogenic ear perichondrium was transfo into the autogenic cartilage of a boy's nose.Tcacencu et al. reported that in the c Early in 1995, in the study of Pirsig et al. [17], an implant of demineralized bovine bone matrix enfolded in a pedicled flap of autogenic ear perichondrium was transformed into the autogenic cartilage of a boy's nose.Tcacencu et al. reported that in the cricoid cartilage defect rabbit models, new cartilage originated from the mesenchymal progenitor cells of a host perichondrium adhered to cricoid cartilage by treatment with recombinant human bone morphogenetic protein-2 (BMP-2) [18].In the study of Kaiser et al. [12], a 1.0 cm × 2.5 cm central basal segment of the rabbit's septal cartilage was removed, and both sides of the septal mucosa were then reapproximated, resulting in the substantial regrowth of cartilage in the defect covered by the perichondrium.
Similar flat cells are also distributed on the surface of articular cartilage and other cartilage tissues.In early studies, researchers only speculated that there were progenitor cell subsets on the surface of the cartilage [19].In 2004, Dowthwaite et al. isolated articular CPCs (ACPCs) from the surface zone of bovine articular cartilage and identified them adequately for the first time [9].Later in 2011, Shafiee et al. first isolated and identified NCPCs in human nasal septal cartilage [10].Currently, CPCs have been isolated from different species and cartilage tissue of different body organs.However, research on NCPCs is still limited, and only a few articles, which are summarized in the following table (Table 1), isolate human NCPCs and discuss their features and functions.Like CPCs from other sites, NCPCs possessed properties of adult stem cells, such as self-renewal capacity, multilineage differentiation potential, and MSC-related surface markers (Figure 3) [25].NCPCs possessed the ability to differentiate into adult chondrocytes without additional growth factors, which did not decrease with 10 passages [10,11,15,[20][21][22][23][24].Additionally, NCPCs were shown to differentiate into osteoblasts in the osteogenic-inducing medium [10,11,15,20,21,23,24].However, the adipogenesis capacity of CPCs was controversial.In some studies, CPCs failed to differentiate into adipocytes and accumulate cytoplasmic lipid droplets [10,15,20], while possessing adipogenesis differentiation potential in others [11,23,24].In addition, in the study of Shafiee et al. [10], CPCs possessed the capacity to differentiate into neural-like cell types, with positive expression of the mature neural markers b-tubulin III (b-TUB) and microtubule-associated protein-2 (MAP-2), nestin, and neural-specific enolase (NSE).
were negative for CD14, C19, CD31, CD34, and CD45 (Table 1).In the study by Sha al. [10,21], the higher expression of CD133 was distinctly different in NCPCs com with the other cell types, including adipose-derived stem cells (ADSCs), BMSCs, an PCs.However, in other studies, NCPCs were negative for CD133 [11,20].Moreover, study by do Amaral et al. [15], NCPCs were negative for CD146, which was a d marker of CPCs in most studies.

The Characteristic-Based Isolation of CPCs
Different methods could be used to isolate CPCs, including fibronectin adhesi say, method of migration, cell sorting, enzymatic digestion, and colony screening (F 4).Before the isolation, the cartilage tissue was usually washed with phosphate saline (PBS) and cut into small 1 mm 2 pieces.The most commonly used fibronectin sion assay protocol is based on the strong adhesion to fibronectin of the CPCs, w bronectin receptors, integrin-α5 and integrin-β1, on their surface [9].The cells d from cartilage tissue by enzymatic hydrolysis were seeded on the fibronectin-coated for 20 min.Afterward, the nonadherent cells were removed, and the CPCs were harv This is an attractive method, as it is time-saving and stable, while pure chondrocyt be simultaneously harvested.Moreover, with a strong migration ability, CPCs cou grate out of the cartilage tissue mass, with or without stimulation.Therefore, takin vantage of this property, CPCs could be isolated from the small pieces of cartilage within 7-14 days of in vitro culture, with or without being triggered by cartilage [11,26].Alternatively, CPCs could also be sorted from the total cell population v sorting methods like immunomagnetic separation or fluorescence-activated cell s (FACS), based on the specific expression of CPC surface markers, mainly CD105, C and CD166, individually or in combination [27][28][29][30][31].In addition, collagenase was us isolation, combined with colony screening.Some articles used collagenase type I alo isolation, and the obtained cells were usually derived from the perichondrium, whic tains collagen type I; thus, it was worth investigating whether or not they were

The Characteristic-Based Isolation of CPCs
Different methods could be used to isolate CPCs, including fibronectin adhesion assay, method of migration, cell sorting, enzymatic digestion, and colony screening (Figure 4).Before the isolation, the cartilage tissue was usually washed with phosphate buffer saline (PBS) and cut into small 1 mm 2 pieces.The most commonly used fibronectin adhesion assay protocol is based on the strong adhesion to fibronectin of the CPCs, with fibronectin receptors, integrin-α5 and integrin-β1, on their surface [9].The cells derived from cartilage tissue by enzymatic hydrolysis were seeded on the fibronectin-coated plate for 20 min.Afterward, the nonadherent cells were removed, and the CPCs were harvested.This is an attractive method, as it is time-saving and stable, while pure chondrocytes can be simultaneously harvested.Moreover, with a strong migration ability, CPCs could migrate out of the cartilage tissue mass, with or without stimulation.Therefore, taking advantage of this property, CPCs could be isolated from the small pieces of cartilage tissue within 7-14 days of in vitro culture, with or without being triggered by cartilage injury [11,26].Alternatively, CPCs could also be sorted from the total cell population via cell sorting methods like immunomagnetic separation or fluorescence-activated cell sorting (FACS), based on the specific expression of CPC surface markers, mainly CD105, CD146, and CD166, individually or in combination [27][28][29][30][31].In addition, collagenase was used for isolation, combined with colony screening.Some articles used collagenase type I alone for isolation, and the obtained cells were usually derived from the perichondrium, which contains collagen type I; thus, it was worth investigating whether or not they were CPCs [6,15,22].In the other studies, the cells digested by the enzyme were later cultured at a low density.While chondrocytes in culture rapidly lost their phenotype, CPCs expressed as highefficiency colony-forming cells, with multi-differentiation potential [32]; thus, the forming colony could be harvested as CPCs [33].
However, CPCs isolated in different ways may possess different characteristics.In the study of Elizabeth et al. [34], CPCs isolated through migration (MCPCs) displayed lower levels of hypertrophy markers (RUNX2 and COL1A1), higher levels of chondrogenic markers (Aggrecan and COL2A1/COL1A1 ratio), and higher levels of GAG/DNA, with stronger staining.These results suggested that MCPCs showed a greater potential for cartilage regeneration, especially in situ tissue engineering, due to their basic migration features.[6,15,22].In the other studies, the cells digested by the enzyme were later cultured at a low density.While chondrocytes in culture rapidly lost their phenotype, CPCs expressed as high-efficiency colony-forming cells, with multi-differentiation potential [32]; thus, the forming colony could be harvested as CPCs [33].However, CPCs isolated in different ways may possess different characteristics.In the study of Elizabeth et al. [34], CPCs isolated through migration (MCPCs) displayed lower levels of hypertrophy markers (RUNX2 and COL1A1), higher levels of chondrogenic markers (Aggrecan and COL2A1/COL1A1 ratio), and higher levels of GAG/DNA, with stronger staining.These results suggested that MCPCs showed a greater potential for cartilage regeneration, especially in situ tissue engineering, due to their basic migration features.

The Migrating CPCs in Cartilage
CPCs have been shown to play an important role in the process of chondrogenesis and regeneration.When we talk about the physiological and pathological functions of CPCs, their migration is often the focus of our attention.

Migrating CPCs in the Injury Cartilage and Inflammatory Environment
After injury in the bovine osteochondral explant, CPCs migrated to the injury site in 7-14 days, via the increase in high mobility group box chromosomal protein 1 (HMGB-1) and the receptor for advanced glycation end products (RAGE)-mediated chemotaxis, and promoted the repair of chondral damage [26].In the study of Wagner et al. [35], HMBG-1 release by chondrocytes was shown to have a migratory effect on CPCs, mediated via RAGE and toll-like receptor 4 (TLR-4).Using the collagenase-caused enzymatic injury properly, short-term enzymatic treatment could help to loosen cartilage tissue and accelerate CPCs migration into injured cartilage, differentiating into chondrocyte-like cells [36].In subsequent studies, many other factors released after cartilage injury could also induce the migration of CPCs to the injury site, including stromal cell-derived factor 1 (SDF-1)

The Migrating CPCs in Cartilage
CPCs have been shown to play an important role in the process of chondrogenesis and regeneration.When we talk about the physiological and pathological functions of CPCs, their migration is often the focus of our attention.

Migrating CPCs in the Injury Cartilage and Inflammatory Environment
After injury in the bovine osteochondral explant, CPCs migrated to the injury site in 7-14 days, via the increase in high mobility group box chromosomal protein 1 (HMGB-1) and the receptor for advanced glycation end products (RAGE)-mediated chemotaxis, and promoted the repair of chondral damage [26].In the study of Wagner et al. [35], HMBG-1 release by chondrocytes was shown to have a migratory effect on CPCs, mediated via RAGE and toll-like receptor 4 (TLR-4).Using the collagenase-caused enzymatic injury properly, short-term enzymatic treatment could help to loosen cartilage tissue and accelerate CPCs migration into injured cartilage, differentiating into chondrocyte-like cells [36].In subsequent studies, many other factors released after cartilage injury could also induce the migration of CPCs to the injury site, including stromal cell-derived factor 1 (SDF-1) [37], insulin-like growth factor 1 (IGF-1), and platelet-derived growth factor (PDGF) [38], suggesting that the migration was likely to be regulated by multiple injury-/inflammatory-related signaling pathways.
Danger/damage-associated molecular patterns (DAMPs) not only triggered the chemotaxis, but also elicited the immunomodulatory response of CPCs, showing a significant increase in pro-and anti-inflammatory gene expression [39].Moreover, compared to chondrocytes, CPCs were more sensitive in their response to endogenous DAMPs which were released from dead cells, represented by the systemic inflammation mediator HMGB1 (a nuclear DAMP) and mitochondrial DAMPs (MTDs), with a higher upregulation of MMP-13, IL-6, and IL-8 in the latter group [40].Moreover, CPCs also played a macrophage-like role in injured cartilage.Compared to chondrocytes, CPCs showed significantly higher rates of fluorophore-labeled fibronectin fragment engulfment, greater lysosome activity and mass, over-expressed phagocytosis-related genes and proteins, and increased cathepsin B-dependent clearance of cell debris [41].

Migrating CPCs in Osteoarthritis
With the increased frequency and MSC surface markers in osteoarthritic cartilage, CPCs are recognized to be involved in the pathogenesis of osteoarthritis (OA) [27].CPCs were reported to be mainly distributed on the surface of articular cartilage.When cartilage was damaged, CPCs tended to migrate to the injury site, as they did in the case of OA.However, the migration of CPCs was significantly inhibited by OA-associated cytokines, such as Interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α), contributing to the low regenerative potential of cartilage in vivo [38].Additionally, Xia et al. revealed that in the instance of downregulated MiR-31-5p and miR-424-5p, the reduced percentage and proliferation ability and the increased osteogenic potential of CPCs contributed to the homeostasis imbalance in OA cartilage [28].Although the migration was inhibited, the distribution and activation of CPCs were also observed in the middle-deep layers of OA articular cartilage, suggesting that the maldistribution of CPCs was closely related to the progression of OA [42,43].
In the OA articular cartilage, the phenotype of CSPCs correlating with the degree of ECM degeneration is likely to be different at different stages of OA.Tong et al. disclosed that CPCs were activated and exhibited a transient proliferative response in early OA as an initial attempt for self-repair, gradually quieting as the OA process was mediated by IL-1β via the NF-κB pathway [44].In the study by Mantripragada et al. [45], although OA grade 3-4 cartilage showed greater cell concentration than did grade 1-2 cartilage, it harbored fewer CPCs.Moreover, compared to grade 1-2 CPCs, grade 3-4 CPCs expressed a higher CD271 and exhibited enhanced osteo-adipogenic activities, decreased chondrogenic capacity, and stronger cell migration in response to OA synovial fluids [46].In addition to migration, the aging of CPC is also closely related to the occurrence and development of OA.In the study of Jacob et al., OA-CPCs exhibited elevated reactive oxygen species (ROS) levels, along with a relatively high percentage of senescent cells compared to non-OA CPCs, mediated via the release of the pro-inflammatory cytokines, IL-6 and IL-8 [47].Interestingly, Zhao et al. revealed the relationship between obesity and OA: high doses of leptin decreased the ability of the CPCs to migrate, inhibited their chondrogenic potential, increased their osteogenic potential, and induced cell cycle arrest and senescence by activating the p53/p21 pathway and inhibiting the Sirt1 pathway [48].
The migration of CPCs is undoubtedly an objective pathological manifestation.When we design a treatment plan, whether to promote the migration of CPCs to achieve better cartilage regeneration or to inhibit migration to restore disturbed tissue architecture is a vital question whose answer depends, at least in part, on the stage of OA.At present, the roles of CSPCs in the onset and progression of OA have not been clarified.More research and evidence are urgently needed to determine the related mechanisms and devise CPC-targeted therapies.

The Migratory CPCs and Cartilage Regeneration
The stronger migration ability is shared by CPCs obtained via different methods and is an essential characteristic that distinguishes CPCs from MSCs.Migratory CPCs (MCPCs), which are defined by the isolation method (method of migration) with outstanding migration capacity, are of particular interest for research.To determine what role they play in cartilage regeneration and to achieve better cartilage repair, many researchers have targeted MCPCs for study (Table 2).MCPCs were first isolated and identified from human OA-articular cartilage by Keoling et al. [49].Most of the subsequent studies were conducted in the context of OA and attempted to clarify the relationship between the progression of OA and the role of MCPCs.In the process of exploring the mechanisms, researchers hoped to find therapeutic targets for OA and reverse the stage of OA.In addition, OA-MCPCs were compared with MCPCs from rheumatoid arthritis cartilage (RA-CPCs) [54].These cells shared basic characteristics of MCPCs, but still exhibited the following main differences: RA-MCPCs expressed IL-17 receptors and resulted in the upregulation of IL-6 and matrix metalloproteinase 3 (MMP-3) and exhibited a reduced chondrogenic potential, indicating that IL-17 blockade might be a new therapeutic option for RA [54].
Other researchers constructed cartilage injury models (blunt impact, enzyme) and isolated MCPCs that migrated after stimulation.In the study by Seol et al. [26], blunt impact injury caused local chondrocyte death and induced the homing of MCPCs via HMGB-1 and RAGE-mediated chemotaxis.There are also studies using the previously mentioned method of migration to obtain MCPCs and exploring the basic phenotype and functions of MCPCs in non-disease states.Moreover, some studies have focused on comparing CPCs with cartilage-derived cells (a mixture of CPCs and chondrocytes) and chondrocytes, which are derived from MCPCs-depleted cartilage (non-MCPCs) or isolated for not adhering to the fibronectin-coated plate.This comparison is interesting because, in past studies, cartilage-derived cells were often referred to as chondrocytes.Compared with non-MCPCs, MCPCs expressed significantly more baseline mRNAs of MMP-13, CXCL12, and IL-6 and were more sensitive than non-MCPCs in response to proinflammatory damage-associated molecular patterns (DAMPs), especially mitochondrial DAMPs (MTDs) [40].Unfortunately, the study on nasal septal MCPCs is limited.Using the method of migration in the nasal field for the first time, Elsaeaaer et al. showed the characterizations of the nasal septal MCPCs and clarified their progenitor cell features and potential for nasal cartilage in situ regeneration [11].

CPCs-Based Cartilage Tissue Engineering Strategies
Based on CPCs, many researchers have constructed tissue-engineered cartilage using a variety of scaffold materials, conducting in vivo experiments (Table 2).With the help of growth factors or functional scaffolds, the researchers realized bionics in structure and function through their ingenious design.
However, there are few studies regarding tissue-engineered cartilage based on NCPCs, and most of the studies only focus on the characteristics or functions of NCPCs.At the same time, because of the difficulty of constructing a nasal septal defect model, there are currently no in vivo experiments devising tissue-engineered nasal septal cartilage based on CPCs.Shafiee et al. seeded NCPCs on aligned electrospun nanofibrous Poly (L-lactide) (PLLA)/Polycaprolactone (PCL) hybrid scaffolds, and the proliferation and chondrogenic differentiation of NCPCs were significantly enhanced compared to those obtained from samples seeded on random scaffolds [20].In the later research, Shafiee et al. compared the four popular candidates for cartilage tissue engineering, including NCPCs, ACPCs, BMSCs, and ADSCs [21].Seeded on the previous nanofibrous scaffolds, their self-renewal potential, immunophenotype, chromosomal stability, chondrogenic potential, and proliferation rate were compared, and the superiority of NCPCs was confirmed [21].

Strategies to Promote CPCs Migration and Chondrogenesis
In order to achieve in situ regeneration, the migration of cells around the defective tissue is the most important link.CPCs have a strong ability for basic migration, and could respond to inflammatory factors and migrate to the injured site, which was fully discussed in the previous section.However, despite possessing resident progenitors committed to chondrogenesis, cartilage does not show strong intrinsic self-repair ability, indicating that migration and regeneration of the natural physiological/pathological levels is insufficient to repair cartilage defects [42].Therefore, it is necessary for us to use scaffolds or factors (biological, chemical or physical) to induce more CPCs to migrate to tissue engineering scaffolds and then differentiate in order to promote the proliferation of chondrocytes in the target site and achieve cartilage in situ regeneration (Figure 5).

Strategies to Promote CPCs Migration and Chondrogenesis
In order to achieve in situ regeneration, the migration of cells around the defe tissue is the most important link.CPCs have a strong ability for basic migration, and c respond to inflammatory factors and migrate to the injured site, which was fully discu in the previous section.However, despite possessing resident progenitors committ chondrogenesis, cartilage does not show strong intrinsic self-repair ability, indicating migration and regeneration of the natural physiological/pathological levels is insuffi to repair cartilage defects [42].Therefore, it is necessary for us to use scaffolds or fa (biological, chemical or physical) to induce more CPCs to migrate to tissue engine scaffolds and then differentiate in order to promote the proliferation of chondrocyt the target site and achieve cartilage in situ regeneration (Figure 5).

Promoting the Migration of CPCs
Stromal cell-derived factor 1 (SDF-1) is a key chemokine regulating stem cell m tion and homing to sites of tissue damage through binding to the cell surface rece CXCR4.In the study by Jayasuriya et al. [89], treating CPCs with CXCR4 blo AMD3100 disrupted cell homing to injured sites and prevented fibrocartilage rege tion in meniscus tissue.In this way, SDF-1 has become a promising molecule for in Figure 5. Strategies for promoting the migration and chondrogenesis of CPCs: After the tissueengineered cartilage scaffold implantation into the cartilage defect, resident CPCs in the surrounding tissue are expected to undergo migration, proliferation, and differentiation to achieve the in situ regeneration of the cartilage.Some factors have been found to promote the migration of CPCs to the scaffold, and some other factors could promote the proliferation and chondrogenic differentiation of CPCs, while among these, some could promote both migration and chondrogenesis.Created with BioRender.com(accessed on 15 December 2022).

Promoting the Migration of CPCs
Stromal cell-derived factor 1 (SDF-1) is a key chemokine regulating stem cell migration and homing to sites of tissue damage through binding to the cell surface receptor CXCR4.In the study by Jayasuriya et al. [89], treating CPCs with CXCR4 blocker AMD3100 disrupted cell homing to injured sites and prevented fibrocartilage regeneration in meniscus tissue.In this way, SDF-1 has become a promising molecule for in situ cartilage regeneration by recruiting endogenous CPCs.Using a recombinant human SDF-1α-loaded fibrin/HA scaffold, Yu et al. achieved the functional repair of full-thickness articular cartilage via the homing of CPCs in bovine femoral condyle osteochondral explants [68].Moreover, hydroxypropyl cellulose (HPC) scaffolds pretreated with SDF-1 stimulated CPCs migration and meniscal fibrocartilage repair in human explant tissue [87].Fibronectin (FN) is a dimeric glycoprotein that exists in the plasma and ECM of various tissues.As the ligand of integrin α5β1, FN provides the molecule binding sites and participates in important processes including cellular migration, adhesion, and differentiation.With high expression levels of the integrin α5β1 (CD54 and CD29) [11,26,38,54], CPCs are promising for responding positively to FN and achieving ideal recruitment to the target site.In the study by Kalkreuth et al. [90], human plasma-derived FN stimulated the migration and proliferation of human subchondral progenitor cells.Additionally, Tao et al. demonstrated that FN could enhance cartilage repair by activating CPCs through the integrin α5β1 receptor in the knee of earlystage OA mouse models [75].High mobility group box 1 protein (HMGB-1) is a protein primarily located in the nucleus, and it can be translocated to the cytoplasm and secreted during cell activation and cell death.In the study by Seol et al. [26], MCPCs were more active than CCs in chemotaxis assays and responded to HMGB-1.In the exploration of OA mechanism, Wagner et al. found that HMGB1 could be released by OA-CCs and exhibited migratory effects on CPCs via its receptors toll-like receptor 4 (TLR4) and the receptor for advanced glycation end products (RAGE) [35].Moreover, in the study by He et al. [91], link protein N-terminal peptide (LPP) could stimulate the proliferation, site-directional migration, and chondrogenic differentiation of CPCs.
Natural combinations of growth factors, including platelet lysate (PL), platelet-rich plasma (PRP), and extracellular vesicles (EVs), could also promote CPCs migration, and at the same time, they have a prominent effect of promoting the chondrogenesis of CPCs.The platelet membrane is always ruptured in the PRP suspension to obtain the PL [62].As products based on platelets, both possess a high content of platelet-derived growth factors and bioactive proteins, making them promising clinical therapeutic agents to restore damaged cartilage [92].In the study by Carluccio et al. [62], CPCs recruited by PL were able to migrate in response to inflammatory stimuli, showed paracrine activity in attracting other cells to the injured sites, and displayed high chondrogenic potential and resistance to hypertrophy.In the PRP-based scaffold, the proliferation and chondrogenesis in CPCs were stimulated, and were stronger in CPCs than in BMSCs and chondrocytes [60].EVs are membrane-bound nanovesicles, ranging in size from 30 to 500 nm, and containing bioactive molecules that migrate throughout the local microenvironment, exerting their effects on target cells and inducing endogenous repair mechanisms [93].It has been proved that progenitor cells could activate resident cells to mediate tissue regeneration through paracrine mechanisms, and CPCs-derived EVs might play a comparable role in CPCs-mediated cartilage regeneration [94].Moreover, ADSCs/BMSCs-derived EVs could promote the migration, proliferation, and chondrogenic differentiation of CPCs for cartilage tissue engineering as well [95][96][97].Therefore, EVs from multiple sources could be used as effective additives.
In addition, some mechanical stimulation can also promote the migration of CPCs.Low-intensity pulsed ultrasound (LIPUS) promoted the migration of CPCs to the injured site via the focal adhesion kinase pathway [51].Under the stimulation of intermittent hydrostatic pressure (IHP), the migration, proliferation and chondrogenic differentiation of CPCs were significantly promoted [74].
Similarly, the promotion of migration of chondrocytes, which had not yet been identified to be CPCs, was explored in some studies [98][99][100].However, these cells migrated in response to the factors and showed characteristics similar to CPCs (especially MCPCs), appearing to play an important role in cartilage regeneration.Therefore, these studies may still be relevant when exploring the migration of CPCs.

Promoting the Chondrogenesis of CPCs
Compared to the promotion of chondrogenic progenitor cell migration, the promotion of chondrogenesis seems to be easier, as the previous systems of research on the induction of chondrogenic differentiation of stem cells and the promotion of chondrocyte secretion of cartilage matrix are more fully developed.In the study by Padmaja et al. [101], bone morphogenic protein 9 (BMP-9) could enhance the chondrogenic potential of CPCs in expansion, without affecting their low hypertrophic tendency, and the presence of the parathyroid hormone during expansion was shown to mitigate hypertrophy.In the study of Shen et al. [102], basic fibroblast growth factor (bFGF) could promote the proliferation and chondrogenesis of CPCs and inhibit osteogenesis, while a culture system containing 2 ng/mL bFGF was optimal.Kartogenin (KGN), a popular small molecule in cartilage regeneration, could repair full-thickness cartilage defects [103], and the effect of cartilage regeneration was partially stimulated by the IL-6/Stat3-dependent proliferation of CPCs [104].Moreover, some of the factors promoting the chondrogenesis of CPCs have been described in the previous introduction of the promotion of migration.Moreover, some modifications of the scaffold material can also improve cartilage regeneration, and the related studies are summarized in Table 3.
In addition to factors that have been shown to promote CPCs chondrogenesis, other factors that could promote the chondrogenic differentiation of BMSCs and the proliferation of chondrocytes may also be considered as potentially effective additions [94,96,97,99,105,106].However, while research ideas are provided, further research is needed to prove their efficacy and effectiveness.The implant composed of ACPCs-laden hydrogel reinforced with an MEW scaffold retained its personalized shape, improved its compressive properties, and supported neocartilage formation.The addition of bioglass improved the cell adherence, cartilage matrix formation, and biomechanical performance.

Summary and Future Outlook
Increasing evidence has shown that CPCs are promising seed cells for cartilage tissue engineering and have superior migration ability and in situ distribution compared to other seed cells, which have been fully discussed in the previous section.Therefore, the use of CPCs is expected to achieve in situ regeneration, which has great potential for clinical application and brings hope for the treatment of cartilage injury-related diseases.Currently, studies on articular cartilage progenitor cells are more comprehensive, while studies on nasal CPCs are yet to be further developed.The easier availability and larger surface layer in which CPCs are mainly distributed both illustrate the advantages of using NCPCs for in situ regeneration of nasal cartilage compared to those using articular cartilage.Moreover, with perichondrium on the surface, it is not clear whether perichondrium-derived progenitor cells or superficial cartilage-derived progenitor cells are the main force in nasal cartilage regeneration.Moreover, given that CPCs from the deep zone of articular cartilage possess greater chondrogenic and osteogenic potential [33], the differences of CPCs from different zones of nasal septal cartilage (superficial versus medial; posterior versus anterior) and different nasal cartilage (lobular versus septum) are worth exploring.
At the same time, nasal septal cartilage is often used as a source of cartilage autograft due to its easy accessibility and superior mechanical properties.Therefore, if regeneration of nasal septal cartilage can be achieved, it can not only solve the problem of the nasal septal defect, but also serve as a regenerable cartilage donor area, indirectly solving the problem of insufficient cartilage sources faced when diseases in other parts of the body require treatment with cartilage grafts, such as contracted nose deformity, cleft lip nasal deformity, microtia, osteoarthritis, etc.
However, studies on NCPCs and even CPCs are still insufficient in terms of the overall perspective.The study of different functional subpopulations of CPCs is still at a preliminary stage, and the mechanisms of the migration and participation of CPCs in tissue repair and the interaction and transformation of CPCs with mature chondrocytes are still unclear.More systematic and in-depth studies need to be completed to further elucidate the mechanisms involved, with subsequent improvement of the cartilage scaffold (modified polymer/hydrogel/biological scaffold) to increase its ability to recruit CPCs.The unresolved status of these issues not only affects the judgment of CPCs-based strategy security, but also limits the development of new strategies.Moreover, as one of the most important aspects of achieving in situ cartilage regeneration, the efficacy of CPCs recruitment requires further improvement.New strategies for improving the cartilage scaffold (modified polymer/hydrogel/biological scaffold), as well as new additives, are constantly emerging.However, none of these has yet showed absolute superiority.In addition, whether these strategies have other side effects is also an important issue to be determined.Additional in vivo and in situ animal experiments need to be completed, which would lay a foundation for clinical application and ultimately provide help for patients with nasal cartilage defects in the future.

Figure 1 .Figure 1 .
Figure 1.Achieving nasal septal cartilage in situ regeneration with a focus on CPCs: Facing diffi ties for septal cartilage repair and the shortage of cartilage graft resources for regeneration, tFigure 1. Achieving nasal septal cartilage in situ regeneration with a focus on CPCs: Facing difficulties for septal cartilage repair and the shortage of cartilage graft resources for regeneration, tissue engineering, especially the in-situ strategy based on scaffolds, has become one of the most promising approaches.CPCs, with great migratory ability and stem cell characteristics, have caught the attention of researchers and brought hope for nasal septal cartilage in situ regeneration.Strategies for promoting the migration and chondrogenesis of CPCs are constantly being improved to induce more CPCs to migrate to tissue engineering scaffolds and to subsequently differentiate in order to achieve satisfactory cartilage in situ regeneration.Created with BioRender.com(accessed on 9 April 2023).

Figure 2 .
Figure 2. (a) The four-layer structure over the subchondral bone of the articular cartilage: the ficial zone, the transition zone, the deep zone, and the calcified zone.(b) The three-layer str of the nasal cartilage and the perichondrium covering it.Created with BioRender.com(acces 15 December 2022).

Figure 2 .
Figure 2. (a) The four-layer structure over the subchondral bone of the articular cartilage: the superficial zone, the transition zone, the deep zone, and the calcified zone.(b) The three-layer structure of the nasal cartilage and the perichondrium covering it.Created with BioRender.com(accessed on 15 December 2022).

Figure 3 .
Figure 3.The surface markers on NCPCs and the multilineage differentiation of CPCs.Created with BioRender.com(accessed on 27 January 2023).

Figure 5 .
Figure 5. Strategies for promoting the migration and chondrogenesis of CPCs: After the tissu gineered cartilage scaffold implantation into the cartilage defect, resident CPCs in the surroun tissue are expected to undergo migration, proliferation, and differentiation to achieve the i regeneration of the cartilage.Some factors have been found to promote the migration of CPCs scaffold, and some other factors could promote the proliferation and chondrogenic different of CPCs, while among these, some could promote both migration and chondrogenesis.Created BioRender.com(accessed on 15 December 2022).

Table 1 .
Studies on human NCPCs and their isolation/culture methods and characteristics.