Cartilage Tissue Engineering: An Update on Multi-Component Approach

Cartilage injury and osteoarthritis are big clinical challenges as self-healing potential of cartilaginous tissue is very limited. The need for a multi-disciplinary approach in order to establish new strategies for cartilage healing has been addressed by many scientists from the fields of orthopaedic surgery or biomedical engineering in the last two decades. With a focus on the very preclinical research in this field, this review covers the multitude of approaches, ranging from cellbased to scaffold-based strategies and also including growth factors, precondition approach, mechanical stimulation-that have been combined to assess their potential to develop effective concepts for the treatment of cartilage injury or osteoarthritis. Citation: Zong Z, Wu X, Su Z, Wang Z, Zhao Z, et al. Cartilage Tissue Engineering: An Update on Multi-Component Approach. J Orthopedics Rheumatol. 2019; 6(1): 9. J Orthopedics Rheumatol 6(1): 9 (2019) Page 02 ISSN: 2334-2846 scenarios [14]. Amount of all, operations like arthroscopic lavage, debridement, microfracture, Autologous Chondrocyte Implantation (ACI), and Osteochondral Autograft Transplantation (OAT) are most widely used nowadays encountering to the cartilage lesions [15]. These reparative methods are tended to stimulate the formation of new fibrocartilage tissue by facilitating access to the vascular system and bringing new progenitor cells capable of chondrogenesis (e.g., microfracture procedure and drilling). Reconstructive methods fill up the defects with autologous, homologous, or other tissue (e.g., autologous chondrocyte implantation and osteochondral autologous transplantation) [16,17]. Such methods may associated with good outcomes after surgery, but according to a systematic review of level I and II studies on OAT procedures and microfracture surgery showing that, patients with small lesions who returned to higher-demand activities had an higher progressive failure rate and only 52% of athletes returned to sports after received microfracture surgery, 37% of them retained their same level of sports 10-year after operation [18,19]. Besides, another systematic review reported by Filardo et al. revealed that 33.7% failure rate at a mean was recorded follow-up of 8.5 years after ACI surgeries (5-12 years post-surgery) in 193 patients [20]. The therapeutic strategies for OA are distinct from acute cartilage injuries. Chronic pain relief could be achieved with lifestyle modification and medication such as Non-Steroidal AntiInflammatory Drugs (NSAIDs) or glucocorticoid. NSAIDs are the most widely prescribed pharmacological medications and were recommended in the guidelines in the treatment of OA but longterm administration are associated with serious side effects including bleeding and perforated gastric ulcers [21-23]. Long-term use of glucocorticoid may cause several side effects such as immunodeficiency, osteoporosis, peptic ulcer disease or gastrointestinal bleeding [24,25]. Viscosupplementation with hyaluronic acid through intra-articular injection helps to reduce OA caused pain through its lubricating action, but recent clinical studies showed that the use of hyaluronic acid did not improve clinical outcomes compared to the placebo group significantly [26,27]. However, these current treatments are not promising solution to prevent articular cartilage from further progressive destruction, thus OA patients may need joint replacement to regain reasonable joint movement at the expense of potential complications. Although the shelf life of prosthetics for joint replacement is significantly improved, this surgery remains less suitable for young OA patients [28,29]. Thus, there is a burning need for alternative approaches to manage cartilage lesions, which would prevent the early onset of OA and to reduce the need for total joint replacement. Biological Solutions for Cartilage Repair Autologous Chondrocyte Implantation (ACI) is a convincing and effective method for the treatment of cartilage lesions [30,31]. The usefulness of allogeneic chondrocytes as alternative source was constrained because of the reported immunogenicity [32]. Furthermore, in vitro expansion of chondrocytes can lead to rapid dedifferentiation and a fibroblastic phenotype [30], resulting in an inferior tissue-engineered cartilage. Mesenchymal Stem Cells (MSCs) are a promising and readily available cell source showing chondrogenic differentiation potential and forming cartilage-like tissues in vitro induced by specific growth factors without compromising its low immunogenicity [33-37]. MSCs can be derived from various types of tissues, including bone marrow [38,39], adipose tissue [40], tendon [41,42], synovial membrane [43], dental pulp [44], umbilical cord blood [45], placenta [46,47], etc. Autologous MSCs are currently the major cell source because of ethical and immunological concerns. However, a major drawback of their clinical use is the aging-related decline in MSCs proliferation and chondrogenic differentiation potential from aged patients (donors) and in vitro cell culturing as several studies had reported that MSC isolated from older donors exhibited a slower proliferation rate throughout the entire in vitro expansion compared with the younger donors. And the shorter average length of telomere, loss of telomere length after cell passage and lower levels of telomerase activity may contribute to such phenomenon. Besides, the expression of p16INK4A is also strongly associated with cell senescence [4851]. Furthermore, instable MSCs phenotypes such as formation of mineralized deposits within cartilage. Current available strategies for enhancing plasticity of MSCs included genetic modification [5254], hypoxia stimulation [55,56], etc. However, safety and ethical concerns are existed for genetic modification approach, which is left far behind clinical use, and hypoxia could only promote cell proliferation at this stage. Hence, it is mandatory to find out a simple and feasible manipulation for promoting plasticity of MSCs including proliferation, chondrogenesis and viability. Dedifferentiation Reprogrammed MSCs for Tissue Regeneration Cellular dedifferentiation is cellular regression from a more differentiated stage back to a less differentiated stage from within its own lineage that confers pluripotency, giving rise to reminiscent of stem cells [57,58]. Based on this definition, cellular dedifferentiation is not only initiating from a completely differentiated stage, but also initiating from partially differentiated stage. Similarly, cellular dedifferentiation could result in partially or fully pluripotent cells, depends on the different time points. This process is more commonly studied in plants and more primitive creatures. Several non-mammalian vertebrate species, such as zebra fish and urodele amphibians [59-65], possess a remarkable capacity to regenerate heart tissue or limb, respectively. Apart from natural conditions, researchers found that inducible dedifferentiation is an appropriate strategy to promote regeneration in mammalian tissues that lack of this ability. Studies have reported the occurrence of cell dedifferentiation during tissue regeneration both in vitro and in vivo [66-70]. Recent studies have demonstrated that dedifferentiation reprogramming is a reliable method to improve properties of stem cells and promote lineage differentiation commitment [71-73]. Previous data revealed that a population of MSCs with enhanced viability in vitro and improved therapeutic efficacy in a cerebral ischemia model could be attained via neuronal differentiation and dedifferentiation reprogramming [72]. Recently we reported that, compared with untreated MSCs, MSCs which manipulated with osteogenic differentiation medium exhibited a better osteogenic differentiation potential, improved cell migratory capacity and upregulated expression of genes Nanog, Oct4 and Sox2 [74]. And we Citation: Zong Z, Wu X, Su Z, Wang Z, Zhao Z, et al. Cartilage Tissue Engineering: An Update on Multi-Component Approach. J Orthopedics Rheumatol. 2019; 6(1): 9. J Orthopedics Rheumatol 6(1): 9 (2019) Page 03 ISSN: 2334-2846 also proved that such improvements were inducted by decreased methylation and accrual of activating histone marks of promoters on Nanog and Oct4.Besides, after preconditioned with chondrogenic differentiation medium and complete medium, the Manipulated MSCs (M-MSCs) also showed an improved cell clonogenicity, proliferation, survivability and chondrogenic property. And the results of epigenetic analysis revealed the central role of Nanog in maintaining the multipotency of the manipulated MSCs [75]. Furthermore, we also revealed that neocartilage formation of M-MSC-laden constructs implanted in the nude mice was significantly promoted after dynamic compressive applied in the bioreactor and the constructs laden with M-MSCs were also significantly promoted the cartilage healing process of osteochondral defect of a rat model [76]. Growth Factors for Chondrogenic Differentiation In the hyaline cartilage, growth factors regulate homeostasis and integrity, as well as development [77]. Growth factors also play an important role in the process of chondrogenic differentiation of MSCs. Table 1 summarizes some representable endogenous bioactive cytokines, including Transforming Growth Factor β (TGF-β) superfamily with respect to cartilage tissue engineering are TGF-β1, TGF-β3, Bone Morphogenetic Protein 2(BMP-2), BMP-4, BMP-6, BMP-7, BMP-9 and Growth Differentiation factor-5 (GDF5) [78-81], which are reported to stimulate MSCs proliferation and differentiation. Among of these, TGF-β1 and TGF-β3 are the most frequently used cytokines in experimental studies to promote chondrogenic differentiation and synthesis of corresponding Extracellular Matrix (ECM) production [79,81-83] (Table 1). Biomaterials for Cartilage Repair Various materials in the form of sponges, hydrogels, electrospun fibers, and microparticles have been 


Epidemiology of Cartilage Injury and Osteoarthritis
Cartilage injury (also called chondral injury) is known as the lesion within cartilage layer, while osteochondral injury is the fullthickness lesion extending to the subchondral bone. Cartilage injury or osteochondral injury is common in sport injuries [1], road traffic accidents [2], and other trauma. An epidemiological study on 31,516 knee arthroscopies in USA reported that 63% of patients had chondral lesions (averaging 2.7 lesions per knee) and 20% had fullthickness lesions, with 5% of these occurring in patients less than 40 years of age [3], 65% of them were accompanied with meniscal or ligament lesions, mostly anterior crucial ligament (ACL) tear [3,4]. In subgroup analysis, 75% of young patients below 40 years old had solitary chondral lesions and; the remaining 25% had multiple lesions. Another similar study conducted in Poland examining a total of 25,124 knee arthroscopies, reported that chondral lesions were found in 60% of these patients. Medial meniscus tear (37%) and ACL injury (36%) were the most frequent associated factors [5].
Cartilage is categorized into three types including hyaline cartilage, elastic cartilage, and fibro cartilage according to its composition. Articular cartilage is a tough but flexible hyaline cartilage that covers the ends of bones at a joint, which functions as a cushion allowing smooth joint movement. As articular cartilage injuries can occur focally, which is localized and contained, or globally, which can finally lead to joint Osteoarthritis (OA) -the most common chronic joint disease. OA is a chronic degenerative disease mainly happened in elderly with destruction of articular cartilage and subchondral bone sclerosis, which is distinct from acute cartilage injury. Data from 2010 to 2012 showed that one in five, or 52.5 million, USA adults had arthritis; one in nine, or 22.7 million, had arthritis-attributable activity limitations [6]. Recently, it was reported that more than fifty million of the population over 60 years old in mainland China were affected by joint pain that may be attributed to osteoarthritis [7]. A local survey in Hong Kong on men aged 50 years and above revealed that 17% and 7% had persistent knee pain and OA, respectively. The prevalence in women was higher, being 24% and 13%, respectively [8].

Healing Process of Cartilage Injury
Cartilage is an avascular tissue with minimal supply of nutrients and progenitor cells from circulation, and composed of limited number of chondrocytes with low mitotic potential, making cartilage a poor self-regenerating tissue in response to injury [9]. In cartilage, nutrients and wastes exchange are achieved through synovial fluid perfusion, which also allow the delivery of various factors participating in healing [9]. Scarce resident stem cells in cartilage are identified recently, which require considerable manipulation efforts to generate cartilage in vitro [10]. Chondroclasts have only been described for calcified or hypertrophic matrices, which are proposed to play a role in cartilage remodeling. Tiny defects are healed by migration of chondrocytes, while large defects are healed by formation of biomechanically incompetent fibrocartilage [11]. Hence, cartilage lesions seldom heal spontaneously and thus constitute one of the main causes of joint disease and disability [12,13]. Given that persistent cartilage defects gradually lead to degeneration of the articular cartilage and osteoarthritis [11], the restoration of cartilage integrity through the promotion of cartilage regeneration has been a research question over the decades.

Traditional Treatments for Cartilage Injury or Osteoarthritis
Primary treatments options including protecting from further injury, ice cooling, and analgesic may help to settle the initial pain and swelling after acute cartilage injury. Further surgical treatments are subjected to the severity of cartilage lesion. Several surgical techniques are readily available to treat cartilage injuries of the knee upon different ISSN: 2334ISSN: -2846 scenarios [14]. Amount of all, operations like arthroscopic lavage, debridement, microfracture, Autologous Chondrocyte Implantation (ACI), and Osteochondral Autograft Transplantation (OAT) are most widely used nowadays encountering to the cartilage lesions [15]. These reparative methods are tended to stimulate the formation of new fibrocartilage tissue by facilitating access to the vascular system and bringing new progenitor cells capable of chondrogenesis (e.g., microfracture procedure and drilling). Reconstructive methods fill up the defects with autologous, homologous, or other tissue (e.g., autologous chondrocyte implantation and osteochondral autologous transplantation) [16,17]. Such methods may associated with good outcomes after surgery, but according to a systematic review of level I and II studies on OAT procedures and microfracture surgery showing that, patients with small lesions who returned to higher-demand activities had an higher progressive failure rate and only 52% of athletes returned to sports after received microfracture surgery, 37% of them retained their same level of sports 10-year after operation [18,19]. Besides, another systematic review reported by Filardo et al. revealed that 33.7% failure rate at a mean was recorded follow-up of 8.5 years after ACI surgeries (5-12 years post-surgery) in 193 patients [20].
The therapeutic strategies for OA are distinct from acute cartilage injuries. Chronic pain relief could be achieved with lifestyle modification and medication such as Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) or glucocorticoid. NSAIDs are the most widely prescribed pharmacological medications and were recommended in the guidelines in the treatment of OA but longterm administration are associated with serious side effects including bleeding and perforated gastric ulcers [21-23]. Long-term use of glucocorticoid may cause several side effects such as immunodeficiency, osteoporosis, peptic ulcer disease or gastrointestinal bleeding [24,25]. Viscosupplementation with hyaluronic acid through intra-articular injection helps to reduce OA caused pain through its lubricating action, but recent clinical studies showed that the use of hyaluronic acid did not improve clinical outcomes compared to the placebo group significantly [26,27].
However, these current treatments are not promising solution to prevent articular cartilage from further progressive destruction, thus OA patients may need joint replacement to regain reasonable joint movement at the expense of potential complications. Although the shelf life of prosthetics for joint replacement is significantly improved, this surgery remains less suitable for young OA patients [28,29]. Thus, there is a burning need for alternative approaches to manage cartilage lesions, which would prevent the early onset of OA and to reduce the need for total joint replacement. Autologous MSCs are currently the major cell source because of ethical and immunological concerns. However, a major drawback of their clinical use is the aging-related decline in MSCs proliferation and chondrogenic differentiation potential from aged patients (donors) and in vitro cell culturing as several studies had reported that MSC isolated from older donors exhibited a slower proliferation rate throughout the entire in vitro expansion compared with the younger donors. And the shorter average length of telomere, loss of telomere length after cell passage and lower levels of telomerase activity may contribute to such phenomenon. Besides, the expression of p16INK4A is also strongly associated with cell senescence [48][49][50][51]. Furthermore, instable MSCs phenotypes such as formation of mineralized deposits within cartilage. Current available strategies for enhancing plasticity of MSCs included genetic modification [52-54], hypoxia stimulation [55,56], etc. However, safety and ethical concerns are existed for genetic modification approach, which is left far behind clinical use, and hypoxia could only promote cell proliferation at this stage. Hence, it is mandatory to find out a simple and feasible manipulation for promoting plasticity of MSCs including proliferation, chondrogenesis and viability.

Dedifferentiation Reprogrammed MSCs for Tissue Regeneration
Cellular dedifferentiation is cellular regression from a more differentiated stage back to a less differentiated stage from within its own lineage that confers pluripotency, giving rise to reminiscent of stem cells [57,58]. Based on this definition, cellular dedifferentiation is not only initiating from a completely differentiated stage, but also initiating from partially differentiated stage. Similarly, cellular dedifferentiation could result in partially or fully pluripotent cells, depends on the different time points. This process is more commonly studied in plants and more primitive creatures. Several non-mammalian vertebrate species, such as zebra fish and urodele amphibians [59][60][61][62][63][64][65], possess a remarkable capacity to regenerate heart tissue or limb, respectively. Apart from natural conditions, researchers found that inducible dedifferentiation is an appropriate strategy to promote regeneration in mammalian tissues that lack of this ability. Studies have reported the occurrence of cell dedifferentiation during tissue regeneration both in vitro and in vivo [66][67][68][69][70].
Recent studies have demonstrated that dedifferentiation reprogramming is a reliable method to improve properties of stem cells and promote lineage differentiation commitment [71][72][73]. Previous data revealed that a population of MSCs with enhanced viability in vitro and improved therapeutic efficacy in a cerebral ischemia model could be attained via neuronal differentiation and dedifferentiation reprogramming [72]. Recently we reported that, compared with untreated MSCs, MSCs which manipulated with osteogenic differentiation medium exhibited a better osteogenic differentiation potential, improved cell migratory capacity and upregulated expression of genes Nanog, Oct4 and Sox2 [74]. And we ISSN: 2334ISSN: -2846 also proved that such improvements were inducted by decreased methylation and accrual of activating histone marks of promoters on Nanog and Oct4.Besides, after preconditioned with chondrogenic differentiation medium and complete medium, the Manipulated MSCs (M-MSCs) also showed an improved cell clonogenicity, proliferation, survivability and chondrogenic property. And the results of epigenetic analysis revealed the central role of Nanog in maintaining the multipotency of the manipulated MSCs [75]. Furthermore, we also revealed that neocartilage formation of M-MSC-laden constructs implanted in the nude mice was significantly promoted after dynamic compressive applied in the bioreactor and the constructs laden with M-MSCs were also significantly promoted the cartilage healing process of osteochondral defect of a rat model [76].

Biomaterials for Cartilage Repair
Various materials in the form of sponges, hydrogels, electrospun fibers, and microparticles have been fabricated as scaffolds to support chondrogenic differentiation [89]. Natural biomaterials, derived from either polymer (agarose, alginate, chitosan, and hyaluronan) or protein (collagen, gelatin, fibrin, and silk) are biocompatible but have poor mechanical strength and relatively high degradation rate in most cases without proper modification [90,91]. Synthetic biodegradable polymers offer some important advantages such as controllable degradation rate, high reproducibility, high mechanical strength, and easy manipulation into specific shapes. However, the cell recognition signals are usually missing in such scaffolds [92]. When stem cells are applied to cartilage defects, direct administrations of stem cells into cartilage defects often lead to limited cartilage regeneration due to significant cell loss and death as a result of the harsh mechanical loading and catabolic factors in the diseased joints [93]. The lack of a functional carrier material to provide physical retention and biochemical cues to the delivered cells in the cartilage defects results in poor retention, significant death and unsatisfactory differentiation of the cells [94]. Therefore, there exists a huge demand for effective carrier biomaterials that afford not only physical support but also biochemical signals to the delivered cells in order to promote the cartilage repair. As articular cartilage is totally covered by the articular capsule, it will be much helpful to deliver the cells through a minimal invasive way, such as intra-articular injection.
Among all of these materials, natural polymer like Hyaluronic Acid (HA) has been intensively investigated. HA can be modified to photo-crosslink into 3D hydrogels that confers chondrogenesis properties of MSCs [95]. The superior mechanical stiffness and network porosity and permeability have positive impact on the differentiation of encapsulated MSCs [96][97][98][99], distribution of newly synthesized cartilage matrix, and nutrition transportation [100,101]. Previous data showed enhanced chondrogenic differentiation and inhibited hypertrophy could be achieved by modulating cross linking density of HA macromer [102,103]. Besides, after modified Quantum Dots (QDs) with β-Cyclodextrin (β-CD) and RGD peptide, the manipulated nanocarrier gained the ability of carrying hydrophobic small molecules such as kartogenin in the hydrophobic pockets to induce chondrogenic differentiation of human mesenchymal stem cells [104]. Moreover, after conjugated sulfate groups to HA, these modified sulfated HA exhibit a higher protein affinity and significantly slower degradation by hyaluronidase with no negative effect on the viability of human Mesenchymal Stem Cells (hMSCs) compared to the wild type HA hydrogel, which results the avert of cartilage abrasion and hypertrophy in the osteoarthritis joints of a rat model of OA [105].
Compared with HA, after proper modification, gelatin hydrogel also exhibited an excellent capacity of self-healing and improved

TGF-β1
Increases proliferation and cartilaginous ECM production, downregulates collagen type I gene expression [79] TGF-β3 Increases cartilaginous ECM production [82] BMP-2 Turns on the chondrogenic pathway in the appropriate chondrogenic precursor cell pool and Repairs cartilage-bone interface tissue defects [79,80] GDF-5 Increases cartilaginous ECM production [78] IGF-1 Increases proliferation and cartilaginous ECM production, additive effect on chondrogenesis with TGF-β1 and BMP-7 [84] BMP-4 Accelerates the progression of cartilage differentiation to maturation [78] BMP-6 upregulates chondrogenic genes and downregulates genes associated with chondrocyte hypertrophy and endochondral ossification [80] BMP-7 Inhibits cell proliferation, induces chondrogenic differentiation, additive effect on chondrogenesis with TGF-β1 and IGF-1 [85,86] BMP-9 Maintains the expression of chondrocyte-specific ECM molecules in the presence of OA-related physiological levels of IL-1β [80] FGF-2 Increases proliferation, increases proteoglycan production [87] FGF-18 Inhibits cell proliferation, induces chondrogenic differentiation [88]  physical and biological properties. Recently, cyclodextrin-based host-guest interact with gelatin are of great interest because of its effectiveness and specificity of host-gust molecular recognition under physiological condition which can be facilitated to form supramolecular hydrogels. In our recent study, we revealed that thought crosslinked acrylated β-cyclodextrins (Ac-β-CDs) with the aromatic residues of gelatin by in situ formed multivalent host-guest nanoclusters under UV-initiated oligomerization, the as-prepared hydrogel shown a significantly enhanced mechanical strength thanks to its reversible nature of the host-guest interactions. Those interactions enables the hosts and guests moieties to re-form the hostguest cross-links thus preventing the early rupture of the polymer. Besides, the Host-Guest Macromer (HGM) hydrogels also exhibited improved compressive properties with much faster stress relaxation rate. Such enhanced compressibility and fast stress relaxation property facilitate the HGM hydrogels to fit into irregular geometries without compromising the hydrogel integrity [106]. Moreover, the Host-Guest Macromer (HGM) hydrogels were also able to sustain release of encapsulated therapeutic growth factors and deliver therapeutic cells. In animal study, we also demonstrated that such novel HGM hydrogel could significantly promoted the cartilage regeneration in a rat model [106]. In our subsequent study, we also demonstrated that the injectable stem cell-laden HGM hydrogels could remarkably boost the regeneration of both cartilage and subchondral bone in an osteochondral defect model after encapsulated human Bone Marrowderived Mesenchymal Stem Cells (hBMSCs) with small molecule (Kartogenin) and proteinaceous chondrogenic agents (TGF-β1). Data also showed that the injection process only has a minor negative impact on cell viability and chondrogenic differentiation capacity of the cells encapsulated in the hydrogels which indicated that such biomaterial and cell delivery method could greatly facilitate stem cell therapies [107].

Mechanical Stimulation and Chondrogenic Differentiation
Mechanical stimulation with bioreactors on cell-seeded constructs is a well-established cue for improving the mechanical properties of tissue-engineered cartilage [108,109]. Direct confined or unconfined compression and hydrostatic pressure are the two most investigated loading regimes in cartilage tissue engineering studies. Direct dynamic compression applied to chondrocyte-seeded constructs generally increased ECM production and proliferation of chondrocytes, and improved compressive properties of the engineered tissue [110][111][112][113][114][115][116][117]. Mechanical forces generated intrinsically within the cell in response to its extracellular environment, and extrinsic mechanical signals imposed upon the cell by the extracellular environment, play a critical role in determining the fate of MSCs [118][119][120]. Mechanical signals have also been reported to induce chondrogenesis of bone marrowderived MSCs and inhibit subsequent hypertrophy as effectively as TGF-β1 stimulation [121][122][123][124][125]. Compressive loading is the most frequently used protocol for promoting chondrogenesis of MSCs. A combination of TGF-β1 and compressive loading presents a synergistic effect on chondrogenic differentiation [126]. Apart from compressive loading, fluid flow has also been shown to upregulate Sox9 gene expression in murine C3H10T1/2 MSCs plated onto glass slides [127]; tensile strain regulated chondrogenic differentiation and GAG synthesis by MSCs embedded in collagen-GAG [128].

Conclusion
With aging and rising of obesity, cartilage injury and osteoarthritis has become major healthcare problems worldwide. The biological approaches showed a great therapeutic potential in the treatment of cartilage injury or OA. However, open questions and challenges are existed and remained to be settled, as most of the studies are still at early stage and evidences such as long-term and large-scale study are still needed. Besides, the problem of stability of the growth factors, survival rate of the cells encapsulated in the biomaterial and large-scale fabrication are still challenging the process of final commercialization. Taken all these together, till now, even bioactive scaffold cannot completely meet every request in the clinical application; we still believe that biological functionalization solutions are the future direction for the treatment of cartilage injury and osteoarthritis.