MicroRNAs in Chondrogenesis,Articular Cartilage,and Osteoarthritis: Implications for Tissue Engineering

Abstract

Coordinated actions of various regulators, including morphogens are required for chondrogenesis and maintenance of articular cartilage function. Bone morphogenetic proteins, and related signaling molecules and transcription factors form a complex regulatory network. MicroRNAs (miRNAs) are noncoding small RNAs that negatively regulate the expression of downstream targets by repressing the translation or inducing the cleavage of messenger RNAs (mRNAs). Increasing evidence indicates that miRNAs are an integral part of the regulatory network in chondrocyte differentiation and cartilage function. The aim of this article is to review the progress in miRNA expression and target genes in cartilage differentiation, homeostasis, and in the pathobiology of osteoarthritis. The recent progress in miRNAs in cartilage has implications for tissue engineering.

Introduction

Adult articular cartilage has a limited capacity for regeneration and repair due to avascular and aneural nature and perhaps, the paucity of mesenchymal stem cells (MSCs). Thus, damage of articular cartilage due to degenerative joint disease or traumatic injury can lead to loss of function and may ultimately lead to symptomatic joint degeneration culminating in osteoarthritis (OA). Recent tissue engineering strategies have combined cell-based therapy (chondrocytes and stem cells) with scaffolds and various growth and differentiation signals to obtain functional tissue-engineered cartilage.¹ Accumulating advances in gene regulation during chondrogenesis and in articular cartilage function have aided in obtaining functionally improved tissue-engineered cartilage. However, several challenges remain. There is an unmet need to develop methods to induce complete regeneration and repair of articular cartilage. Increasing evidence indicates that miRNAs are an integral part of the regulatory network in chondrocyte differentiation and homeostasis. A better understanding of the mechanisms that promote and maintain the differentiated phenotype has implications for cartilage regeneration and restoration of cartilage function.

MiRNAs are a class of small (20–25 nucleotides) single-stranded noncoding RNA molecules that normally bind to the 3′-untranslated region (3′-UTR) of mRNAs and lead to translational repression or mRNA degradation.² The biogenesis of a miRNA begins in the nucleus as a miRNA gene is transcribed by RNA polymerase II or III (Fig. 1). The primary transcript (pri-miRNA) consists of a stem-loop structure that contains the sequence for the mature miRNA. The pri-miRNA is subjected to cleavage by Drosha, a nuclear RNase III enzyme, to form a short stem-loop of 70–100 nucleotides called precursor miRNA (pre-miRNA) in the nucleus.³ Pre-miRNA is exported to the cytoplasm by exportin 5,⁴ and further processed by Dicer, a second RNase III enzyme that catalyzes the loss of the terminal loop and generates a double-stranded miRNA duplex of approximately 22 nucleotides in length. This duplex binds to proteins of the Argonaute family, which are parts of the RNA-induced silencing complex (RISC). One strand (passenger miRNA) of the miRNA duplex is degraded, while the other strand (mature miRNA) then directs RISC to the target mRNA. Mature miRNA binds to the 3′-UTR of the target mRNA, which is partially or fully complementary to the seed sequence of the mature miRNA (nucleotides in position 2–9), although in rare cases the miRNA–mRNA interaction takes place at the 5′-UTR and protein coding regions.⁵ RISC cleaves the target mRNA when the miRNA–mRNA interaction is a perfect pairing. When the interaction is partially complementary, RISC does not cleave the mRNA, but either represses the target mRNA translation or leads to decapping and/or deadenylation of the mRNA. Mechanisms of the miRNA biogenesis pathway are beyond the scope of this review and details can be found in excellent reviews.^6,7 Computational estimates indicate that there are over 800 miRNAs in the human genome, comprising ∼5% of human genes, and that they may target one-third of mammalian mRNAs.^2,8 The importance of miRNAs is becoming increasingly clear in almost all biological processes, including developmental onset, cell proliferation, cell differentiation, morphogenesis, metabolism, and apoptosis.⁹

FIG. 1.

Schematics of miRNA biogenesis and the mechanisms of target gene regulation. See text for the detailed description. Color images available online at www.liebertpub.com/teb

Since the importance of miRNAs in chondrogenesis and skeletal development was first shown in Dicer-null mice in 2008,¹⁰ a number of studies have discovered various miRNAs in the cartilage. There have been various studies on the expression profiling of miRNAs in various conditions, such as in mouse development, in-vitro chondrogenesis, MSCs and chondrocytes, and normal and osteoarthritic cartilages. An increasing number of studies have reported specific miRNAs that were shown to play a role in regulating chondrocyte differentiation and gene expression. Here, we focus on the findings and progress in miRNA research in chondrogenesis, articular cartilage functions and disease pathogenesis and discuss the potential implications of miRNAs for tissue engineering. Disease pathogenesis will be focused on OA as recent reviews have summarized miRNAs in rheumatoid arthritis (RA).^11,12 Individual miRNAs will be discussed in terms of their differential expression, target genes, and physiological relevance. Articular cartilage is the main focus of this review as no publications discuss meniscal cartilage and temporomandibular cartilage.

miRNAs in Skeletal Development

Chondrogenesis is the earliest phase of skeletal development, involving mesenchymal cell condensation, chondrocyte differentiation, and maturation. These processes lead to the formation of cartilage and bone during endochondral ossification.^13,14 During endochondral ossification, successive steps of chondrogenesis occur where cells undergo a program of proliferation, maturation, hypertrophy, and ultimately, apoptosis and replacement by bone. These processes are precisely regulated not only by various cytokines, growth factors, transcription factors, and extracellular matrix (ECM) components, but also by miRNAs, although the exact roles of individual miRNA are not clear.

Global reduction of miRNAs in chondrocytes by using chondrocyte-specific deletion of Dicer, an essential component for miRNA biogenesis, causes severe skeletal growth defects and premature death of mice.¹⁰ Growth defects in the Dicer-null growth plate are caused by reduction of proliferating chondrocytes and accelerating differentiation into postmitotic hypertrophic chondrocytes.¹⁰ This finding suggests that miRNAs play a critical role in maintaining proliferating chondrocytes and inhibiting premature differentiation into hypertrophic chondrocytes in the growth plate, eventually affecting skeletal development.

The developmental-stage-specific and tissue-specific expression is critical for appropriate miRNA function. A combinatorial approach of in-situ hybridization and microarray has shown that a number of miRNAs are associated with the head skeleton and cartilage of developing zebrafish embryos in a highly tissue-specific manner.¹⁵ Ten miRNAs are highly expressed in the pharyngeal arches (miR-23a, miR-27a, miR-27b, miR-140, miR-140*, miR-145, miR-146, miR-199a, miR-199a*, and miR-214), indicating that these miRNAs may be associated with cartilaginous tissues.¹⁵ An analysis of the miRNA expression in other species, including developing medaka and chicken embryos, showed that miR-140 and miR-199a are specifically expressed in developing cartilage, while other miRNAs are expressed in multiple tissues.¹⁶ Additionally, miRNA expression profiling studies have identified subsets of miRNAs expressed tissue-specific¹⁰ or developmental-stage-specific.¹⁷ MiRNA expression profiling between growth plates (chondrocytes) and calvariae (osteoblasts) of neonatal mice identified several miRNAs that are preferentially expressed in chondrocytes compared to osteoblasts, such as miR-196a, miR-196b, miR-433, and miR-202, whereas other miRNAs showed similar expression levels.¹⁰ Recent a more comprehensive analysis of miRNA by using Solexa sequencing identified groups of miRNAs that are upregulated (miR-26a, miR-29a/b/c, miR-140, miR-210, miR-150, and miR-181a) and downregulated (miR-1) during articular cartilage development in rats.¹⁷ These expression profiling studies provide a possible pool of miRNAs that may contribute to articular cartilage development and chondrocyte differentiation. However, many of the individual targets of miRNA in chondrocytes have not yet been identified and further systematic investigation is needed.

Expression of miRNAs During In-Vitro Chondrogenesis

In-vitro differentiation of stem cells into chondrogenic cells is an important technique not only to study the mechanisms of in-vivo chondrogenesis, but also to approach creation of tissue-engineered cartilage for replacement of damaged cartilage. A few miRNA microarray studies have identified the specific miRNAs during in-vitro chondrogenic differentiation of MSCs from human and mouse. Chondrogenic differentiation of mouse MSCs results in upregulation of miR-199a and miR-124a and downregulation of miR-96.¹⁸ Another group reported that miR-140* and miR-30a are upregulated, while two miRNA clusters, miR-143/-145 and miR-132/-212 are downregulated during chondrogenic differentiation of mouse MSCs.¹⁹ Further work on miR-145 has shown that miR-145 negatively regulates chondrogenic differentiation by directly targeting the Sox9 transcription factor.²⁰ In human MSCs, chondrogenic differentiation resulted in upregulation of miR-130b, miR-152, miR-28, miR-26b, and miR-193b.²¹ Groups of miRNAs up- or downregulated during in-vitro chondrogenesis were different among the reports, and different reports showed an even opposite expression pattern for the same species of miRNAs. This inconsistency might be due to the heterogeneous nature of the MSCs and various methods used for MSC culture and chondrogenic induction. MiR-140 is the prominent one that demonstrates the increasing expression pattern during in-vitro chondrogenesis in multiple reports and its target genes have been identified in multiple laboratories.^22–26 MiR-199a* expression was shown to be regulated in BMP-2-induced chondrogenesis and its target gene was identified.²⁷

miRNAs in Articular Cartilage

Articular cartilage provides a durable friction-free tissue for locomotion of limbs in joints. The function of articular cartilage is achieved by cartilage-specific ECM. Type II collagen and aggrecans are two major components of the ECM and matrix-degrading enzymes, such as matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases with thrombospondin motifs (ADAMTSs) are associated with ECM degradation. A fine balance of these catabolic and anabolic processes is critical for cartilage homeostasis. Recently, the role of miRNAs in the maintenance of cartilage homeostasis is increasingly recognized as regulators for ECM components and matrix-degrading enzymes.

Recent studies have identified several miRNAs that may play a role in maintaining cartilage homeostasis. MiR-140 and miR-675 were independently identified to be specifically and abundantly expressed in cartilage, indicating their roles in maintaining cartilage phenotypes.^25,28 MiR-140 knockout (−/−) mice showed grossly normal skeletal development, but developed age-related OA-like change, indicating its role in maintaining cartilage tissue homeostasis.²³ No knockout-mouse study has been done for miR-675, but expression of miR-675 was associated with the upregulation of ECM molecules, such as type II collagen.²⁸

Mechanical signals are critical for chondrocyte differentiation and cartilage tissue homeostasis. Moderate physiological mechanical loading maintains the integrity of articular cartilage, whereas mechanical overloading and complete unloading induce degradative changes in cartilage.²⁹ Dunn et al. identified several miRNAs as potential regulators of the articular cartilage mechanotransduction pathway by showing that a set of miRNAs, including miR-221 and miR-222, are differentially expressed in the weight-bearing anterior medial condyle as compared with the nonweight-bearing posterior medial condyle.³⁰ More recently, miR-365 was identified as a mechanosensitive miRNA in chondrocytes and its possible role in chondrocyte differentiation was further demonstrated.³¹

The articular chondrocyte phenotype progressively disappears during in-vitro monolayer culture, through the process termed dedifferentiation, a critical challenge in cartilage tissue engineering. Chondrocytes isolated from articular cartilage rapidly dedifferentiate into fibroblast-like cells during monolayer expansion. During dedifferentiation, chondrocytes change from original spherical morphology to a flat spindle shaped fibroblast-like phenotype,³² and attendant changes in gene expression occur in a variety of genes, including genes encoding ECM proteins, transcription factors, and growth factors.^33,34 Two recent studies analyzed miRNAs during dedifferentiation of chondrocytes from healthy human cartilages.^35,36 Downregulation (miR-451, miR-140-3p, and miR-210) and upregulation (miR-17, miR-31, miR-145, miR-132, miR-138, miR-221, and miR-222) were observed in the dedifferentiated cells at day 28 in monolayer culture compared with uncultured primary chondrocytes.³⁶ Another group identified miRNAs that are downregulated (miR-491-3p, miR-140-5p, miR-222, miR-26a, and let-7d) and upregulated (miR-548e, miR-146a, miR-18a, and miR-1248) in the dedifferentiated cells at their fifth passage compared with uncultured primary chondrocytes.³⁵

miRNAs in OA

The chondrocyte phenotype and cartilage homeostasis are altered in OA. It is generally accepted that the balance between synthesis and degradation of ECM is perturbed in OA, resulting in cartilage degeneration.³⁷ In addition to changes in metabolism, increased apoptosis of cells is observed in OA cartilage.^38–40 A number of studies have shown that altered levels of mRNAs and proteins are associated with the phenotypic changes in OA chondrocytes. Recent miRNA studies have suggested possible correlations between miRNAs and pathological conditions in cartilage, adding more complexity in OA pathology.

Expression profiling studies have identified altered expression of specific miRNAs in OA cartilage compared with normal controls.^41,42 Further studies have shown a molecular link between altered miRNA and phenotypic changes in OA. Iliopoulos et al. identified miRNAs that are upregulated (miR-483, miR-22, miR-377, miR-103, miR-16, miR-223, miR-30b, miR-23b, and miR-509) and miRNAs that are downregulated (miR-29a, miR-140, miR-25, miR-337, miR-210, miR-26a, and miR-373) in OA cartilage using microarrays and following the real-time polymerase chain reaction analysis.⁴¹ One of the downregulated miRNAs in OA, miR-140, has been linked to altered homeostasis in OA cartilage by another group.²² Another miRNA expression profiling reported by Jones et al. showed miRNAs upregulated (miR-9, miR-25, miR-34a, miR-34b, miR-98, miR-137, miR-182, miR-185, miR-200a, miR-211, miR-299, and miR-342) and miRNAs downregulated (miR-107, miR-130b, miR-146, miR-148, and miR-149) in OA cartilage.⁴² Overexpression of miR-9, miR-98, and miR-146 reduced interleukin (IL)-1β-mediated production of tumor necrosis factor (TNF), indicating their roles in inflammatory diseases.⁴² One of the upregulated miRNAs, miR-34a, has been related to chondrocyte apoptosis by another group.⁴³

Proteolytic degradation of cartilage is a hallmark of OA, and the OA chondrocytes actively produce cartilage degrading enzymes, such as MMP-13 and ADAMTS-5 in OA joints,^44,45 of which, expression is further stimulated by proinflammatory cytokines, such as IL-1β. Recent miRNA studies also have correlated the expression of specific miRNA with inflammation and catabolic changes in OA. MiR-140 inhibited IL-1β-induced ADAMTS-5 expression and IL-1β suppressed miR-140 expression in chondrocytes.²² Increased level of miR-22 has been implicated with the increased MMP-13 level indirectly via downregulation of BMP-7 and peroxisome proliferator-activated receptor (PPARA) in OA cartilage.⁴¹ MiR-27a was shown to indirectly downregulate MMP-13,²⁴ whereas miR-27b appears to directly target MMP-13.⁴⁶

Individual miRNAs

In this section, individual miRNAs in cartilage are discussed and summarized in Table 1. The word direct target is used for mRNAs that contain sequences matching to a seed sequence of miRNA in their 3′-UTR and miRNA/mRNA expression levels are strongly reverse-correlated in cells. Experimental validation for the direct target gene of a miRNA was generally done in two ways, the 3′-UTR luciferase reporter assay and miRNA-gain-of-function and loss-of-function experiments.

Table 1.

Summary of MicroRNAs in Cartilage

miRNAs	Targets	Cellular process	References
miR-1	N.I.	chondrocyte differentiation (−)	Sumiyoshi et al., 2010⁴⁷
miR-18a	CCN2	chondrocyte differentiation (−)	Ohgawara et al., 2009⁴⁸
miR-22	PPARA	OA pathogenesis (+)	Iliopoulos et al., 2008⁴¹
	BMP7	OA pathogenesis (+)	Iliopoulos et al., 2008⁴¹
miR-27b	MMP-13	OA pathogenesis (−)	Akhtar et al., 2010⁴⁶
miR-34a	N.I.	OA pathogenesis (+)	Abouheif et al., 2010⁴³
miR-140	HDAC4	chondrocyte differentiation	Tuddenham et al., 2006²⁵
	CXCL12	chondrocyte differentiation	Nicolas et al., 2008⁵⁵
	IGFBP-5	OA pathogenesis	Tardif et al., 2009²⁴
	ADAMTS-	OA pathogenesis (−)	Miyaki et al., 2010²³
	SMAD3	chondrocyte differentiation (−)	Pais et al., 2010⁵⁶
	BMP2	endochondral bone formation	Nicolas et al., 2011⁵⁷
	SP1	chondrocyte proliferation (+)	Yang et al., 2011²⁶
	DNPEP	endochondral bone formation (−)	Nakamura et al., 2011⁵²
miR-145	SOX9	chondrocyte differentiation (−)	Yang et al., 2011²⁰
	SOX9	cartilage homeostasis (−)	Martinez-Sanchez et al., 2012⁶⁰
miR-146	N.I.	OA pathogenesis	Jones et al., 2009⁴²
miR-199^*	SMAD1	chondrocyte differentiation (−)	Lin et al., 2009²⁷
miR-221	MDM2	chondrocyte differentiation (−)	Kim et al., 2011⁶⁵
miR-365	HDAC4	chondrocyte differentiation (+)	Guan et al., 2011³¹
miR-675	N.I.	cartilage homeostasis (+)	Dudek et al., 2010²⁸

Target genes, cellular processes, and references are summarized for miRNAs whose roles have been implicated in chondrogenesis and/or cartilage function.

N.I., not identified; PPARA, peroxisome proliferator-activated receptor; MMP, matrix metalloproteinases; HDAC4, histone deacetylase 4; CXCL12, chemokine ligand 12; IGFBP-5, Insulin-like growth factor binding protein 5; ADAMTS, a disintegrin and metalloproteinases with thrombospondin motifs; DNPEP, aspartyl aminopeptidase; OA, osteoarthritis.

(+), stimulates; (−), inhibits the cellular processes.

MiR-1

MiR-1 was suggested as the most repressed miRNA upon hypertrophic differentiation by showing its most dramatic decrease in the chondrocytes from upper sternum (hypertrophic) compared to lower sternum (proliferating) cells of the sterna cartilage of a chicken embryo.⁴⁷ The direct target of miR-1 in chondrocytes is not known, but aggrecan expression was repressed indirectly in the miR-1-gain-of-function experiment.

MiR-18a

MiR-18a was connected to chondrocyte differentiation by directly targeting the CCN family protein 2/connective tissue growth factor (CCN2/CTGF).⁴⁸ CCN2 is involved in chondrocyte differentiation and angiogenesis during skeletal development.⁴⁹ Among several miRNAs, which are predicted to target CCN2, miR-18a was most strongly downregulated in human chondrocytic HCS-2/8 cells compared to HeLa cells. CCN2 was validated as a miR-18a target by using the 3′-UTR reporter assay and the miR-18a-gain-of-function experiment.

MiR-22

MiR-22 was identified as one of the upregulated miRNAs in human OA cartilage.⁴¹ The same authors further showed that miR-22 directly targets PPARAα and BMP7. MiR-22 and its targets are implicated with inflammatory and catabolic changes in OA chondrocytes as overexpression of miR-22, and knockout of either PPARA or BMP7 in normal chondrocytes resulted in increased IL-1β and MMP-13.

MiR-27

MiR-27b was identified as one of the downregulated miRNAs in IL-1β-stimulated human OA chondrocytes.⁴⁶ Expression of miR-27b was significantly lower in OA cartilage compared with normal control. Additionally, MMP-13 was identified as a direct target of miR-27b by using the 3′-UTR reporter assay and gain- or loss-of-function experiments.

MiR-34a

MiR-34a has been implicated in antiproliferative potential as miR-34a is induced by p53 tumor suppressor, subsequently leading the gene expression change toward cell apoptosis or cell cycle arrest.^50,51 MiR-34a was identified as one of the upregulated miRNAs in human OA cartilage,⁴² and its expression was increased by IL-1β in rat primary chondrocytes.⁴³ A direct target of miR-34a is not known in chondrocytes, but its expression is implicated with inflammation and catabolic changes as silencing of miR-34a reduced IL-1β-induced apoptosis and significantly prevented IL-1β-induced downregulation of type II collagen (Col2A1) as well as IL-1β-induced upregulation of inducible nitric oxide synthase.

MiR-140

MiR-140 is expressed in cartilaginous tissues during embryogenesis^15,25 and also in adult articular cartilage.^22,41 Its expression is increased during in-vitro chondrogenesis,^22,26 and decreased in OA cartilage.^22,24,41 During mouse development, miR-140 expression was detected specifically in the cartilaginous tissues, including limbs, ribs, skull, and sternum throughout development.²⁵ In adult cartilage, miR-140 exhibited the largest expression difference between human articular chondrocytes and human MSCs.²² In addition, miR-140 expression increased during in-vitro chondrogenesis of MSCs from human²² and mouse,²⁶ although another group reported that miR-140 expression remained constant during in-vitro chondrogenesis.¹⁸ Importantly, several independent groups have found that the miR-140 level is significantly reduced in human OA cartilage,^22,24,41 and that IL-1β suppresses miR-140 expression in chondrocytes in culture.²²

MiR-140 (−/−) mice were generated by two independent groups and showed a mild skeletal phenotype with a short stature as well as craniofacial bone defects.^23,52 Although the structure of the articular joint cartilage appeared grossly normal, miR-140 (−/−) mice showed age-related OA-like changes, such as proteoglycan loss and fibrillation of articular cartilage.²³ Transgenic mice overexpressing miR-140 in cartilage did not show any apparent abnormalities in skeletal development, but importantly, these transgenic mice were resistant to antigen-induced arthritis.²³

Eight target genes have been identified for miR-140 in chondrocytes. Histone deacetylase 4 (HDAC4) was downregulated by direct interaction of miR-140 to its 3′-UTR.²⁵ HDAC4 has been known to negatively regulate hypertrophic differentiation of chondrocytes,⁵³ but it has not been demonstrated yet whether miR-140 plays a role in hypertrophy through suppression of HDAC4. Chemokine ligand 12 (CXCL12), which stimulates chondrocyte hypertrophy,⁵⁴ was also shown to be downregulated by direct interaction of miR-140 to its 3′-UTR, but its physiological relevance was not tested at all.⁵⁵ ADAMTS-5, a critical enzyme for OA pathogenesis, was identified as a direct target and its expression was significantly increased in chondrocytes from miR-140 (−/−) mice and introduction of ds-miR-140 into those chondrocytes reduced ADAMTS-5 expression, suggesting that miR-140 contributes to the cartilage homeostasis by inhibiting OA-like changes through in part, suppressing ADAMTS-5.²³ Smad3 was identified as a direct target, and the transforming growth factor (TGF)-β pathway was shown to be inhibited by miR-140 through suppression of Smad3.⁵⁶ Sp1 transcription factor, which inhibits the cell cycle by activating p15INK4b and p21Waf1/Cip1 promoter, was reported as a direct target, suggesting that suppression of the Sp1 activity by miR-140 is implicated in maintenance of chondrocyte proliferation.²⁶ The insulin-like growth factor binding protein 5 (IGFBP-5),²⁴ BMP2,⁵⁷ and aspartyl aminopeptidase (Dnpep)⁵² were also suggested as target genes of miR-140.

MiR-145

The function of miR-145 has been associated with smooth muscle cells fate decision and various oncogenic pathways.^58,59 Yang et al. showed that the miR-145 level was gradually decreased during TGF-β3-induced chondrogenic differentiation of mouse MSCs.¹⁹ The same group further identified the Sox9 gene as miR-145 target through the 3′-UTR-reporter assay and gain- or loss-of-function experiments.²⁰ Very recently, Martinez-Sanchez et al. also found Sox9 as a direct target of miR-145.⁶⁰

MiR-146

MiR-146 has been reported to be a negative regulator in the inflammatory responses.⁶¹ Altered upregulation of miR-146 has been reported in RA synovial fibroblasts and synovial tissues,⁶² and in the macrophages and T and B cell subsets in RA synovial tissues.⁶³ Additionally, miR-146 was higher in the synovium from RA patients compared to OA patients.⁶³ In OA cartilage, expression pattern of miR-146 appears to be dependent on the severity of OA. Yamasaki et al. found that miR-146 was expressed intensely in low-grade OA cartilage and decreased with increasing cartilage degeneration,⁶⁴ while Jones et al. reported miR-146 as one of the downregulated miRNAs in late-stage OA cartilage.⁴² A direct target of miR-146 is not known yet in chondrocytes. However, IL-1β-induced production of TNF-α was significantly reduced by miR-146 overexpression, indicating that miR-146 is involved in the inflammatory OA state.⁴²

MiR-199a*

An early in situ study of zebrafish embryo has implicated the specific expression of miR-199a* with skeletal systems.¹⁵ miR-199a* was suggested to repress the early chondrogenesis process.²⁷ In the course of BMP2-triggered chondrogenesis of C3H10T1/2 mouse stem cells, the miR-199a* level was immediately decreased dramatically and then increased and remained higher. Overexpression of miR-199a* inhibited the expression of early marker genes for chondrogenesis, such as Sox9, COMP, and Col2A1, whereas anti-miR-199a* increased the expression of these genes. The 3′-UTR-reporter assay led to the identification of Smad1 as a direct target gene for miR-199a*. Expression of p204, a direct target gene of Smad1 and Smad4, was greatly reduced by miR-199a*, indicating that miR-199a* represses Smad1-dependent transactivation via direct targeting to Smad1.

MiR-221

MiR-221 was shown to be involved in the chondrogenesis process.⁶⁵ MiR-221 was strongly increased during the inhibition of c-Jun N-terminal kinase (JNK) signaling, one of the important signaling pathways in the regulation of cartilage formation. Inhibition of JNK signaling suppressed chondrogenic differentiation of chick limb mesenchymal cells possibly by suppression of cell migration and stimulation of cell apoptosis. Anti-miR-221 reversed the chondro-inhibitory actions of a JNK inhibitor on the proliferation and migration of chondroprogenitor cells. The Mdm2 oncoprotein was identified as a direct target gene for miR-221. Additionally, in articular cartilage, miR-221 was suggested as a potential regulator of the mechanotransduction pathway.³⁰

MiR-365

MiR-365 was identified as a mechanoresponsive miRNA in primary chicken chondrocytes cultured in 3-dimensional collagen scaffoldings under cyclic loading conditions,³¹ in which the Indian hedgehog (Ihh) was also induced in this loading conditions and was necessary for mechanostimulation of chondrocyte proliferation.⁶⁶ The expression level of miR-365 was higher in the prehypertrophic chondrocytes among all three zones (proliferating, prehypertrophic, and hypertrophic) of tibia growth plate, coinciding with the Ihh expression pattern. MiR-365 overexpression significantly enhanced chondrocyte proliferation and stimulated differentiation markers, including type X collagen (ColX) and Ihh. HDAC4 was identified as a direct target of miR-365 by the 3′-UTR reporter assay and gain-of- and loss-of-function experiments. Overexpression of HDAC4 reversed miR-365 stimulation of differentiation markers. Therefore, the authors suggest that miR-365, as a mechanoresponsive miRNA, regulates chondrocyte proliferation and differentiation by directly targeting HDAC4 and inducing Runx2 and Ihh expression.³¹

MiR-675

From genome-wide profiling experiments using healthy human chondrocytes, expression of H19 (a primary miRNA that is processed to the mature form, which is miR-675) was found to be as high as the most abundant cartilage matrix genes, Col2A1 and aggrecan.²⁸ Expression levels of both H19 and miR-675 were greater in articular cartilage and much lower in other tissues of mouse. Expression of H9 was dependent on Sox9, possibly through direct interaction of Sox9 to the H9 promoter, because the human H9 gene has two putative Sox9 binding sites in its proximal promoter region. A direct target gene of miR-675 has not been identified, but it was shown that both H19 and miR-675 positively regulated Col2A1 expression by using the gain-of-function and loss-of-function experiment.

miRNAs and Implications for Tissue Engineering

The goal of cartilage tissue engineering is to produce an ECM with the composition, biomechanical and frictional properties that mimic that of the native cartilage. Tissue engineering has adopted new strategies to improve the function of the engineered tissues, and recent cartilage tissue engineering strategies have combined cell-based therapy (e.g., chondrocytes and stem cells) with scaffolds as well as various morphogens, growth and differentiation signals. There still is an unmet need to develop methods to induce complete regeneration and repair of articular cartilage. The recent discovery of miRNAs and their ability to regulate global gene expression patterns in a variety of tissues, including articular cartilage, may revolutionize current tissue engineering strategies.

The advancements in miRNA biology, including miRNAs' functions in cells in health and disease states, have provided the motivation to introduce miRNAs to the disciplines of tissue engineering. From a tissue engineering perspective, miRNAs that have a key function in the differentiation and homeostasis would aid to the regeneration of tissue.^67,68 Tissue engineering with the use of stem cells in combination with miRNA gene therapy may bring considerable progress in the treatment of various diseases. MiRNAs may be integrated in stem cells to induce them to differentiate to repair or regenerate damaged cartilage tissue. Real application of these miRNAs in tissue engineering to enhance differentiation just began to bring promising outcomes. Overexpression or inhibition of specific miRNAs resulted in promising outcomes in tissue engineering purposes. For example, skeletal muscle cell differentiation was enhanced by the overexpression of miR-1 and miR-206⁶⁹ and by the inhibition of miR-133⁷⁰ in 3-D bioartificial muscle constructs. Osteogenic differentiation of human MSCs in a 3-D scaffold was enhanced by the miR-148b mimic and the miR-489 inhibitor.⁷¹ Besides tissue engineering purposes, miRNAs can be used as therapeutic targets and diagnostic/prognostic markers and these utilities are better known in cancer field. Abnormal expression of miRNAs is identified to disrupt signaling networks in cells and leads to pathological changes. These miRNAs are potential therapeutic targets for various diseases. In addition, aberrant miRNA expression profiles are able to be diagnostic and prognostic markers of cancer states.⁷²

Despite the potential for miRNAs in tissue engineering, there are some hurdles to the clinical utilization of miRNAs in tissue engineering. First problem is how to make miRNA therapy efficient, specific, and inducible. Viral vectors are often used for overexpression of a miRNA. For eventual clinical usage, viral vectors should be avoided due to the safety concerns. A delicately controlled induction system is also needed as continuous overexpression or inhibition of a miRNA may be unable to fine tune the regulation of differentiation that is required. Potential tumorigenic aspects of the human embryonic stem cells must be always kept in mind. Regardless, it is obvious that the accumulated understanding and new discoveries of miRNA functions in cartilage and OA will usher the new approaches to tissue engineering.

Conclusions

Functional cartilage regeneration has been a critical aspect in cartilage tissue engineering. More and more miRNAs are revealed to play roles during chondrogenesis, cartilage homeostasis, and OA pathogenesis. As recent studies in the cancer field have highlighted the potential therapeutic aspects of miRNA delivery to certain types of tumors,⁷³ a better understanding of the miRNAs that can promote the differentiated phenotype, or maintain the phenotype has implications for tissue engineering of cartilage.

Footnotes

Acknowledgments

This work was supported by funds from the Lawrence J Ellison Endowed Chair.

Disclosure Statement

No competing financial interests exist.

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