Synovium-Derived Stem Cells: A Tissue-Specific Stem Cell for Cartilage Engineering and Regeneration

Abstract

Articular cartilage is difficult to heal once injury or disease occurs. Autologous chondrocyte transplantation is a biological treatment with good prognosis, but donor site morbidity and limited cell source are disadvantages. Currently, mesenchymal stem cells (MSCs) are a promising approach for cartilage regeneration. Despite there being various sources, the best candidate for cartilage regeneration is the one with the greatest chondrogenic potential and the least hypertrophic differentiation. These properties are able to insure that the regenerated tissue is hyaline cartilage of high quality. This review article will summarize relevant literature to justify synovium-derived stem cells (SDSCs) as a tissue-specific stem cell for chondrogenesis by comparing synovium and cartilage with respect to anatomical location and functional structure, comparing the growth characterization and chondrogenic capacity of SDSCs and MSCs, evaluating the application of SDSCs in regenerative medicine and diseases, and discussing potential future directions.

Introduction

The occurrence of disease and injury to joint connective tissues, such as articular cartilage, meniscus, and nucleus pulposus, is increasing due to the aging of the population and greater athletic involvement. While a number of disease processes may lead to cartilage damage, osteoarthritis (OA) and rheumatoid arthritis (RA) are two debilitating diseases that account for a large proportion of musculoskeletal disability among older adults.¹ Another significant source of disability among adults is back pain. Nearly 80% of adults suffer from low back pain at some point in their lifetime and while the cause is often idiopathic, it is thought to be a result of disc degeneration.² Due to the avascular and aneural nature of cartilage tissue, there is poor regeneration and the repair process results in fibrous tissue with biomechanical properties that are significantly inferior to healthy tissue.³ Strategies such as autologous chondrocyte transplantation (ACT), microfracture, and disectomy ease the discomfort of these injuries and restore mobility, but these treatments do not regenerate tissue that resembles its native form. Mesenchymal stem cell (MSC) use in regenerative medicine can reduce the invasiveness and donor site morbidity associated with the current practices and may result in superior regenerative capacity.

Isolated from tissues, such as bone marrow, adipose, periosteum, muscle, perichondrium, and recently synovium, MSCs have become an attractive option for the regeneration of these damaged tissues.^4–13 MSCs are a culture-adherent, quiescent population of slow-cycling cells that respond to physiological turnover and stress stimuli through intense proliferation and differentiation.^4,14 In addition, these cells are positive for the surface markers CD73, CD90, and CD105, and negative for hematopoietic stem cell markers.¹⁵ The proper management of MSC differentiation capacities is one of the keys to regenerative medicine.

Several studies have been performed with bone marrow stem cells (BMSCs) attempting to manipulate their chondrogenic potential to produce constructs mimicking native tissue, but so far these attempts have fallen short. In 2001, MSCs derived from the synovial membrane surrounding joints, known as synovium-derived stem cells (SDSCs), were isolated.¹¹ Increasing attention has been placed on these cells because of their superior chondrogenic potential both in vitro and certain in vivo states. Along with their intimate relationship with the connective tissue of joints, it has been suggested that SDSCs may be “tissue-specific” for connective tissue regeneration. A tissue-specific stem cell is derived from a known tissue type and can respond to organ-specific signals when recruited to that organ; in other words, the differentiation potential reflects the local cell population.^13,16 If SDSCs are tissue specific for connective tissue repair, they will respond most appropriately to signaling in the joint cavity to facilitate cartilage tissue regeneration.

In this article, we seek the evidence to support the tissue specificity of SDSCs for articular cartilage regeneration by providing an overview of the research that has been performed on these cells. We will discuss the similarities and differences of synovium and articular cartilage, compare the growth kinetics and differentiation capabilities of SDSCs and other MSCs, discuss the role of SDSCs in regenerative medicine and disease, and suggest directions for future research.

Synovium and Cartilage: Anatomical Location and Functional Structure

Embryologically, the synovium develops at joints from locally derived mesenchymal cells and does so in a way that supports the biomechanical and biochemical needs of the joint.^17–20 The synovium is a fluid-filled cavity that surrounds joint cartilage and tendons and facilitates smooth movement of joints. The membrane that forms this cavity is visible in blunt dissection and, histologically, two synoviocyte cell types can be observed: macrophage-like cells (type A) and fibroblast-like cells (type B).²¹ Type A cells function in innate and adaptive immunity while type B cells function in the formation of synovial fluid and are believed to be the source from which SDSCs are derived.^14,22

Type B cells are largely responsible for the proper functioning of the synovium as a unit and these cells possess several unique properties to accomplish this goal. UDP-glucose dehydrogenase (UDPGD) is a necessary enzyme in hyaluronan synthesis and type B cells differ from other cell types by showing high UDPGD activity.^21,23 Type B cells can also be recognized by prominent CD44 and vascular cell adhesion molecule 1 (VCAM-1) expression. CD44 attaches hyaluronan to the synoviocytes and the shear forces generated by movement causes more hyaluronan to be produced.²¹ Hyaluronan serves to promote the migration of cells, facilitate extracellular matrix (ECM) remodeling, promote inflammatory response, and inhibit cell adhesion—all important processes in wound healing.^24–27 CD44 has also been suggested as a marker of chondrogenic potential.^28–30 A large percentage of SDSCs express CD44; this number is >80% as shown by Sakaguchi et al. and ∼90% by Bilgen et al.^31,32 Interestingly, UDGPD activity and CD44 expression are lower in RA.²³

Although a sample of synovium contains both type A and type B cells, a method for enriching the sample in type B cells through CD14 negative isolation has been demonstrated.^32,33 The enriched type B sample undergoes more extensive chondrogenesis than the mixed sample, thus adding support to the claim that SDSCs are derived from type B cells.³³ The type A cells do not proliferate as rapidly as the type B cells, and after three passages an enriched population of type B cells is obtained.³³ While the use of CD14 negative isolation is not a requirement for SDSC use, it provides a time and cost benefit.

Working in unison with the synovium, articular cartilage helps ensure proper function of joints. Articular cartilage is an avascular, aneural tissue composed of a high water content matrix of type II collagen and other proteoglycans. The matrix is secreted and maintained by chondrocytes, but has limited regeneration capabilities. Articular cartilage can be divided into three structurally and biologically distinct zones: a superficial zone, a middle zone, and a deep zone with each zone possessing a distinct set of proteins and a unique collagen arrangement. The arrangement of the matrix allows articular cartilage to trap water, imparting tensile strength and allowing it to distribute loads on the joint and facilitate movement.

The proper collagen matrix arrangement is necessary for cartilage formation, growth, repair, and regeneration, but poses a major challenge in the regeneration of native cartilage.³⁴ ACT has been a somewhat effective means of repairing cartilage defects, but Rieppo et al. demonstrated that the repaired tissue lacks the complex collagen arrangement of native tissue.³⁵ Work still continues on elucidating the complete arrangement of native collagen. Interestingly, through transfection with an adenovirus carrying histone deacetylase 4, a zonal structure resembling hyaline cartilage was achieved in pellet culture.³⁶ The mechanism behind this effect has not been completely elucidated, but it is believed to be through histone deacetylase 4 inhibition of the collagen II transcription repressors, runt-related transcription factor 2 (runx-2), Wnt-5a, and myocyte enhancer factor 2c (MEF2C).³⁷

In addition to matrix arrangement, proper function of the joint is maintained by the secretion of superficial zone protein (SZP) (homologous to lubricin, megakaryocyte-stimulating factor precursor, camptodactyly-arthropathy-coxavara-pericarditis protein, and proteoglycan 4) by the superficial zone chondrocytes and synovium.³⁸ A thin layer of SZP on normal articular cartilage reduces the coefficient of friction in joints and provides protection for chondroprogenitor cells at the surface of articular cartilage.³⁸ Abnormal protein deposits, disappearance of the superficial zone, synovial hyperplasia, and premature joint failure are seen in mice lacking the SZP gene.³⁹ As with the synovium, mechanotransduction is important for the secretion of proteins and maintenance of properly functioning tissue. In the knee joint, locations on the femoral condyle that experience increased load show a high expression of SZP. This response is mediated through the transforming growth factor beta (TGF-β) pathway, a pathway that is often manipulated in chondrogenesis.³ Interestingly, the different members of the bone morphogenetic protein (BMP)/TGF-β superfamily elicit different responses in chondrocytes and synoviocytes, showing that while these two cells work together to form a functional unit, they are in fact quite different.³

Chondrocytes and SDSCs share a similar gene expression profile.⁴⁰ SDSCs, like chondrocytes, have been shown to accumulate type II collagen and express the gene for proteoglycan 4, but do not accumulate large amounts of type X collagen.^41,42 Although type X collagen is a marker of hypertrophy, it is sometimes expressed by deep-zone chondrocytes. These properties could make SDSCs readily useful in regenerating superficial zone cartilage and, with the proper growth environment, useful in regenerating middle- and deep-zone cartilage.

Further, the cells of the synovium and articular cartilage develop from the same pool of precursor cells and remain in close relationship into adulthood.^43–45 Additionally, synovial cells and chondrocytes produce cartilage oligomeric matrix protein, link protein, and glycosaminoglycans (GAGs).^46–50 Synovium is the only tissue that can produce hyaline cartilage in synovial chondromatosis and rheumatoid pannus; synovial cells are recruited to articular cartilage defects.^51–55 It was for these reasons that the synovium was hypothesized to contain MSC-like cells.⁵⁶

Synovium Serves as a Stem Cell Niche

What is a stem cell niche and why is it important? A niche is the microenvironment in which a stem cell exists and functions in conjunction with nurturing cells to retain pluripotency, prevent apoptosis, and inhibit excessive cell replication.⁵⁷ The niche effectively houses the stem cells and provides the correct biochemical signals to the cells when an environmental change occurs.⁵⁸ This is much different than embryonic stem cells, which form teratomas when injected into joints.⁵⁹

It was recently demonstrated that there is an in vivo presence of a slow-cycling, MSC-like cell population in murine knee joints.¹⁴ Through iododeoxyuridine labeling and surface staining, cells were shown to spread out through synovium and into both the intimal layer and the subintimal layer. Additional surface markers were used to show that these cell populations were phenotypically distinct. Upon injury, cartilage metaplasia was noted throughout the synovium, but occurred intensely at the synovium–cartilage boundary. The niche was provided with the correct signals to mobilize the dormant SDSCs. This result shows that synovium and cartilage function together in the absence of medical intervention, further demonstrating the tissue-specific capacity of SDSCs. Kurth et al. also demonstrated that SDSCs were not derived from pericytes, further supporting the exclusivity and localization of the synovial niche.¹⁴ It should be noted that Kurth et al. did not rule out the possibility that the synovial cells could be in part derived from bone marrow, articular cartilage, and blood.¹⁴ The synovium is a complex mixture of cells, but no matter where they are derived from, the end phenotypic and genotypic profile of the synovial cells is different from their site of origin.

Further support for the existence of a niche is found in recent studies with an SDSC-derived ECM in which in vitro niches were hypothesized. The largely type I collagen nanostructured ECM deposited by SDSCs not only provides a three-dimensional environment similar to the in vivo environment, but also contains components that affect cell signaling pathways.^60–66 The tissue-specific nature of SDSCs is supported by increased cell proliferation and enhanced chondrogenesis without a concomitant increase in adipogenesis and osteogenesis.⁶⁰ Through binding interactions with ECM, the local concentration of growth factors and morphogens is increased, thus contributing to improved cell capabilities; an ECM containing type I collagen and decorin can retain stem cell phenotype [CD44, CD73, CD90, CD105, and stage-specific embryonic antigen 4 (SSEA-4) expression] and prevent spontaneous differentiation.^64,65,67,68 When compared with growth on plastic flasks, ECM-grown cells exhibit a smaller and more spindle-like morphology that has been associated with increased differentiation capabilities; the proliferation rate through passages on ECM increased in contrast to passaging on plastic flasks.^64,66

In addition, ECM-expanded SDSCs exhibit decreased expression of senescence-associated β-galactosidase and reactive oxygen species and increased expression of the stem cell marker SSEA-4.⁶⁹ When expanded on ECM, SDSCs express higher levels of chondrogenic genes and surface markers than cells similarly expanded on plastic flasks. The mean fluorescence intensity of CD90, a chondrogenic marker, was higher among ECM-expanded cells than plastic-flask-expanded cells.⁶⁰ The upregulation of phospho-TGF-β receptor II on ECM-expanded cells also supports the increased chondrogenic potential through the upregulation of Sox9 and collagen II.⁶⁹ Low levels of types I and X collagen expression suggest that ECM may prevent SDSC hypertrophic differentiation.⁶⁰ The use of SDSC-deposited ECM, an in vitro niche, may help overcome some of the hurdles of in vitro expansion and retain the tissue-specific nature of these cells.

Molecular and Growth Properties Differentiating SDSCs from Other Adult Stem Cells

SDSCs have a demonstrable ability to differentiate into cartilage, bone, adipose, and muscle.¹¹ The fact that these cells are derived from synovium, a tissue that is in intimate contact with articular cartilage, suggests that SDSCs may already possess a strong bias toward the production of intra-articular tissue making them an attractive option for tissue-specific engineering of cartilage tissue. Multiple studies have compared the growth properties, surface characteristics, and chondrogenic potential of SDSCs to MSCs of other origins.⁷⁰ In the studies described throughout this article, the term “synovial-derived stem cell” is given to cells that are derived from different parts of the synovial membrane. This undoubtedly includes a complex mixture of cells, but as a whole, the results of these studies support the notion that SDSCs have increased chondrogenic ability and are thus a better candidate cell source for cartilage regeneration.³¹

In terms of growth, SDSCs possess a greater proliferative rate than other stem cell sources, including bone marrow, periosteum, muscle, and fat.⁷¹ A 100-fold expansion was achieved in 14 days by plating SDSCs at 50 cells per cm² and thus SDSCs should be plated at a low density to achieve maximum proliferation.⁵³ It has also been shown with other MSCs that low-density expansion allows for retention of multilineage differentiation capacity.^72–75 The high proliferation rate necessitates only a small sample of synovium to produce a clinically useful quantity of cells. SDSCs possess a greater colony-forming potential than BMSCs.^31,54 Jo et al. report the colony-forming potential per nucleated cell in SDSCs to be 1 in 12.5–80 compared with 1 in 10³–10⁴ in BMSCs.⁵⁴ Sakaguchi et al. demonstrated that plating at optimal density for 14 days resulted in an average of 21,000 cells per milligram of synovium.³¹ SDSCs retain the proliferation rate and colony-forming potential regardless of patient age, unlike BMSCs.^11,76 SDSCs also retain their chondrogenic potential over four passages.³¹ Other cell types, such as muscle MSCs and adipose MSCs, lose their proliferative capacity after four and seven passages, respectively.³¹

Several studies have been performed to distinguish MSCs from one another through gene profiling and mRNA expression. The SDSC gene profile more closely matches the chondrocyte and meniscal cell gene profile than BMSCs.^16,77 SDSCs showed a 5- to 10-fold reduction in osteocalcin and alkaline phosphatase (ALP) compared with BMSCs.⁷⁸ Higher levels of Activin A, a molecule that may play a role in proliferation and maintenance of pluripotency depending on the cellular environment, were detected in BMSC supernatant than in SDSC supernatant. This could be problematic, as BMSCs with higher Activin A expression may retain some ability to revert to other differentiation states even after induction to chondrogenesis.⁷⁸ Activin A may also be representative of chondrogenic/osteogenic potential, suggesting the hypertrophic nature of BMSCs.⁷⁸ Recently, SDSCs were differentiated from BMSCs and ASCs through proteomic analysis.⁷⁹ The results of the proteomic analysis, based on proteins from a number of biological pathways, showed a close relationship between BMSCs and ASCs, but a distinct difference in the SDSCs.⁷⁹

Although SDSCs are distinct from other MSCs, they are similar in their surface epitope expression and immunosuppressive abilities. Surface epitopes for SDSCs differ very little from those of BMSCs and to date no known epitope has been found to positively identify SDSCs.⁵³ Both cell types are negative for CD14, CD34, and CD45 and positive for CD44, CD73, CD90, and CD105 among others. No significant differences in mean fluorescent intensity were found, except for CD90, which was higher in SDSCs, a finding which may explain the increased chondrogenic potential of SDSCs.^78,80 SDSCs are also positive for CD9, CD10, CD13, CD54, CD55, CD166, and D7-FIB, but these markers are not unique to SDSCs.⁷⁸ Additionally, the ability of SDSCs to suppress T cell proliferation is clinically important and may allow allogenic MSCs from another donor to be used in therapy. MSCs can also exert similar effects on B cells, natural killer cells, and dendritic cells by inhibiting proliferation, maturation, and expression of presentation molecules.⁸¹ The ability of MSCs to decrease dendritic cell secretion of cytokines, such as tumor necrosis factor alpha (TNF-α), interferon gamma, and interleukin 12 (IL-12), and to increase secretion of IL-10 may be useful in the treatment of inflammatory arthritic diseases. Allogenic MSCs were shown to inhibit type II collagen–stimulated T cell proliferation in an RA model.⁸¹

A Comparison of Differentiation Potentials

The ability to differentiate into chondrocytes, osteocytes, and adipocytes is a requirement for a cell population to be considered an MSC.¹⁵ However, the differentiation potentials of MSCs vary greatly. The superiority of SDSCs for cartilage formation has been demonstrated in multiple studies, but the ability of SDSCs to undergo osteogenesis and adipogenesis warrants consideration as this could have clinical relevance.

Many studies performed have used pellet culture when inducing chondrogenesis because cell–cell contact may predispose cells to undergo chondrogenesis and inhibit them from undergoing osteogenesis.⁵³ In a comparison of multiple rat MSCs, pellets larger than 1 mm were obtained from SDSCs, BMSCs, and periosteum MSC cultures, while pellets smaller than 1 mm were obtained from muscle and adipose MSCs.³¹ Additionally, the pellets from SDSCs and periosteum MSCs were heavier than those from BMSCs, muscle, and adipose MSCs.³¹ Synthesis of chondroitin sulfate and hyaluronan was highest in SDSCs and pellet growth was determined to be due to ECM production as the amount of DNA decreases over the culture period.^31,71

Numerous in vitro studies have been performed to assess the chondrogenic potential of SDSCs, while relatively few in vivo studies have examined this ability. Studies in rabbit osteochondral defects have shown that SDSCs have an enhanced chondrogenic capacity in a comparison of MSCs seeded in collagen gel.⁸² Defects with SDSCs and BMSCs contained significant cartilage matrix and scored well histologically, while defects with adipose and muscle MSCs were poorly healed. Additionally, SDSC-treated defects reached native height at 12 weeks and, in a chondrogenic assay, SDSCs produced more cartilage than BMSCs.^31,71 It was noted by Wakitani et al. that although in vivo repair by BMSCs has resulted in clinically acceptable outcomes, the repaired tissue has a cartilaginous-to-fibrous appearance, which may indicate inferior mechanical properties of the tissue compared with native tissue.⁸³

During repair, several outcomes are of concern in the use of MSCs, in particular, dedifferentiation, degeneration, and ossification of hyaline cartilage.⁸⁴ SDSCs can retain metachromatic staining in repaired cartilage up to 24 weeks and do not show signs of ossification.⁸⁵ Although SDSCs are capable of undergoing osteogenesis, this ability seems to be reduced compared with other MSCs. Endogenous BMPs and BMP receptors are either not detected or decrease as the culture time increases and expression of the osteogenic marker runx-2 does not change throughout culture. Some osteogenic proteins are detected, but the pellet does not show signs of hypertrophy or bone formation.⁵³ Importantly, the pellets formed from SDSCs expressed only a small amount of bone sialoprotein mRNA, and no osteocalcin mRNA or signs of calcification were seen after 6 weeks in culture.⁵³ BMSCs and periosteum MSCs display a much greater potential for chondrogenic hypertrophy and osteogenesis.^86–88 ALP staining and activity is noted in SDSCs, BMSCs, and adipose MSCs, but staining appears to be donor dependent and ALP activity is significantly reduced in SDSCs.⁸⁹

It is important to note that two subsets of SDSCs can be obtained from humans: fibrous SDSCs and adipose SDSCs. These two subsets of SDSCs are differentiated by the underlying subsynovial connective tissue layer. Fibrous SDSCs are isolated from areas with a fibrous subsynovial layer, while adipose SDSCs are isolated from areas with an adipose subsynovial layer.⁹⁰ Fibrous SDSCs can be harvested from such places as the lateral part of the knee joint capsule that overlays the noncartilaginous area of the lateral condyle of the femur. Adipose SDSCs are located in the infrapatellar fat pad overlying the patellar tendon. Both types of SDSCs can be obtained from their respective tissues after digestion of a synovectomy sample, followed by mincing, digestion, and filtration.^33,61 Most work appears to be done with fibrous SDSCs, but many authors do not explicitly specify the type of SDSCs used in their experiments, they only document the harvest procedure and location. There are only slight differences between the two SDSC types, but fibrous SDSCs appear to have more desirable properties for cartilage formation and, when compared with adipose MSCs of a nonsynovial origin, both SDSCs were superior in their chondrogenic potential.⁹⁰ Fibrous SDSCs showed greater proliferative and colony-forming potentials and secreted significantly more SZP into the medium.⁹¹ It should be noted that the SZP was secreted into the medium instead of being retained on the surface as it is in vivo. Macroscopically, fibrous SDSC pellets were heavier than adipose SDSC pellets and, histologically, adipose SDSC pellets were fibrous in the center upon TGF-β1 induction, staining for proteoglycans and type II collagen only around the edges, while fibrous SDSCs had proteoglycan and type II collagen staining throughout. Recent findings in our group demonstrate that chondrogenic differentiation could be improved throughout the pellets from adipose SDSCs with pretreatment on ECM deposited by SDSCs.⁶¹

Applications of SDSCs in Joint and Cartilage Tissue Engineering

Currently several options exist for repairing cartilage defects, including ACT, microfracture, and mosaicplasty.⁹² All are relatively successful in relieving pain for the patients, but these procedures do not result in regeneration of native tissue. In ACT, chondrocytes must be harvested from healthy donor tissue and do not have the proliferation capacity of SDSCs, which makes large defects hard to repair.⁹⁰ ACT results in donor site morbidity, lack of adhesion between native cartilage and the implant, inappropriate biomechanical properties, and often chondrocyte hypertrophy.⁹² Patient age is also a concern for ACT and microfracture. In patients over 50, the aforementioned techniques show little effect, most likely due to an age-related decline in the cells' potential to proliferate and differentiate.^93–98 However, this ability is not lost in SDSCs.¹¹

Clinical benefits abound for SDSC use in cartilage tissue engineering. Many of the properties that set SDSCs apart from other MSCs are particularly appealing, but ease of access and removal of synovium for cartilage defect repair results in less morbidity for the patient. Synovium can be obtained arthroscopically with minimal invasiveness and few complications. A small-punch biopsy would be sufficient for growth of a clinically useful quantity of cells.³¹ Additionally, the regenerative capacity of synovium has been demonstrated after surgical and chemical synovectomy.^99,100 In contrast, the harvesting of BMSCs is invasive, painful, and can potentially contribute to morbidity.¹⁰¹ The periosteum is also a source of MSCs; however, surgical invasiveness and complications, such as hypertrophy or ossification of the periosteum, are a concern.^82,102

Articular cartilage repair with SDSCs has been demonstrated in vivo in several animal studies.^{82,85,103,104} Currently SDSCs have the ability to repair cartilage defects in vivo to a limited extent. Using a local adherent technique, a substantial number of cells can attach to the site of injury and facilitate the production of cartilage matrix in rabbits.¹⁰³ SDSCs have demonstrated the ability to form a cartilage matrix with an abundance of type II collagen and sulfated GAGs and variable cell morphology in accordance to local microenvironment. Integration with native tissue was noted over 24 weeks; however, the thickness of the repair tissue decreased at 24 weeks.⁸⁵ SDSCs seeded in polyglycolic acid mesh with fibrin glue produced an abundance of type II collagen and sulfated GAGs, although the equilibrium modulus of the tissue was low compared with native tissue.¹⁰⁴ ECM-expanded SDSCs demonstrated superior tissue regeneration capabilities over plastic-flask-expanded SDSCs after intra-articular injection into articular cartilage defects (unpublished data). New tissue generated by SDSCs showed no type I or X collagen, thus indicating a stable hyaline cartilage phenotype.⁸⁵ Interestingly, the use of a periosteal patch improved the surface regularity and thickness of the regenerated tissue.⁸²

The differentiation ability of SDSCs allows for their use in more than just articular cartilage regeneration. Joints are complex biomechanical structures that depend on tissues other than cartilage to function properly. Ligaments, menisci, and nucleus pulposus are other joint components that are critical to producing smooth movement. While these structures differ in nature, the hypovascularity of the tissues allows for little, if any, regeneration. The use of SDSCs has been demonstrated to help repair meniscal and anterior cruciate ligament defects and produce nucleus pulposus cells.^16,105

Meniscal repair with SDSCs has been demonstrated in an animal model, in which rats with SDSCs injected in the area of injury experienced larger regrowth of meniscal tissue with type II collagen preponderance than did injured rats without an injection of SDSCs.¹⁶ SDSC proliferation occurred after injection, most likely in response to chemokines and cytokines produced as a result of injury.¹⁶ While overproliferation and pannus formation can be a concern, seeding the injury site with more cells results in a better outcome because some cells are resorbed or fail to differentiate.¹⁰³ When SDSCs are compared with BMSCs in meniscal repair, gene profiles show that SDSCs are more similar to meniscal cells, even though the SDSC- and BMSC-repaired meniscal tissue appears the same macroscopically.¹⁶ It has been shown that cells of fibroblastic origin from the synovium and joint capsule mediate meniscal repair through migration, proliferation, and differentiation.^106–110

The origin of low back pain is a complicated process, but degeneration of the intervertebral disks (IVDs) appears to be an underlying cause. Degeneration appears to begin in the nucleus pulposus of the IVD through a progressive loss of the aggrecan-rich matrix. Chondrocytes and nucleus pulposus cells share matrix-producing properties, but phenotypic differences exist.¹¹¹ Relatively few nucleus pulposus cells can be obtained from a disk biopsy and, in monolayer expansion, nucleus pulposus cells lost their proliferative capacity.⁶² SDSCs were cocultured with nucleus pulposus cells in an attempt to replicate this tissue.^62,105 In coculture, SDSCs and nucleus pulposus cells were able to undergo chondrogenesis and produce a matrix similar to nucleus pulposus cells alone.¹⁰⁵ In addition, type X collagen expression was downregulated in coculture, indicating that hypertrophic differentiation may be preventable with this method.¹⁰⁵ Expansion on an SDSC-secreted ECM resulted in increased proliferation; greater type II collagen, aggrecan, and Sox9 mRNA; and maintenance of redifferentiation capacity over an increased number of passages.⁶² The aforementioned results illustrate the ability of a three-dimensional ECM niche to revert terminally differentiated cells to a more stem-like state.

SDSCs in Disease

Ideally, work with tissue-specific SDSCs will lead to better treatment for arthritic disease and joint trauma. Few options exist for patients with these diseases and the treatments that are available serve to reduce or manage pain, but do not treat the underlying problem. The effects of disease, such as OA and RA, on joints and the component cells must be considered, as the distinct properties of the disease will play a critical role in treating patients with their own stem cells.

In disease, the joint is a harsh environment for the proliferation and differentiation of cells, but ECM expansion can increase the proliferation and chondrogenic differentiation potential of SDSCs over plastic flask expansion under the influence of oxidative stress (unpublished data). MSCs have been detected in synovial fluid of OA patients and their involvement in inflammatory arthritis was recently suggested.¹¹² It has been demonstrated that BMSCs accumulate in the synovium prior to clinical onset of arthritis and it is postulated that SDSCs may be shed from diseased joint structures.^29,78 The ability of BMSCs to undergo adipogenesis and chondrogenesis is diminished in advanced OA, but SDSCs retain the capacity to undergo chondrogenesis.^56,113 Surprisingly, the number of MSCs in the synovial fluid of OA patients is increased, an occurrence that is associated with an increased proliferative capacity of OA SDSCs.²⁹ Although adipose SDSCs may not have all the qualities of fibrous SDSCs, these cells may be a better choice for use in patients with OA since fewer inflammatory changes are associated with the infrapatellar fat pad.¹¹⁴ The ability of SDSCs to produce SZP is important for OA treatment as the SZP layer may be absent in OA. Therefore, the ability to produce and implant a layer of cells producing SZP could provide a way to alleviate symptoms.⁹¹

The autoimmune disease RA results in or is manifested through hyperplastic synovial fibroblasts. The synovial intimal layer, normally 1- to 3-cells thick, can increase to 10- to 15-cells thick in patients with RA. In addition, the fibroblasts that were once immunoregulatory adopt an immune function (expression of major histocompatibility, type II, encoded by human leukocyte antigen (HLA-DR) molecules and production of inflammatory cytokines) in RA due to the presence of TNF-α and an associated increase in major histocompatibility (MHC) class II expression contributes to further immune response.¹¹⁵ The chondrogenic potential of SDSCs in rheumatoid synovium is inversely related to the amount of synovial inflammation and proportion of infiltrating monocytes; the absolute number of SDSCs is reduced with increasing inflammation.³⁰ The reduction in cell number could be an effect of synovial hyperplasia seen in RA, a process that may be initiated by migration of BMSCs into the joint.^30,112 For the clinical use of SDSCs in RA, the lowest level of inflammation should be obtained before collecting or applying SDSCs; even in the worst cases, a sufficient number of cells can be isolated for clinical use.^81,116 Importantly, the phenotype of RA-SDSCs is not changed from the typical SDSC expression profile and the same is true for OA-SDSCs.³⁰ However, it was shown that CD44 expression is inversely related to the inflammation state and UDPGD activity is lowered, while VCAM-1 expression is maintained in RA patients.^23,30

The composition of the synovial fluid is also changed due to disease and it has been shown to have an effect on the differentiation of MSCs.¹¹⁷ Interestingly, synovial fluid from OA joints and acute traumatic injuries stimulates chondrogenesis and proteoglycan synthesis in contrast to synovial fluid from RA joints and chronic traumatic injuries.¹¹⁷ By stimulating RA-MSCs with OA synovial fluid and TGF-β3, the matrix formation was increased, suggesting that RA synovial fluid inhibits the TGF-β3 chondrogenesis pathway.¹¹⁷ It is suggested that MSCs retain their epigenetic profiles after expansion and thus studies should be conducted to determine whether RA-SDSCs fit this model. By comparing RA-SDSCs before and after expansion it could be determined whether the epigenetic profile of in vivo RA-SDSCs is maintained or whether they can be reverted back to a healthy SDSC epigenetic profile.³⁰

It is conceivable that SDSCs could be used for clinical treatment of inflammatory conditions not by producing a tissue structure or differentiating along a specific lineage, but by harnessing the SDSCs for the production of anti-inflammatory and immunoregulatory factors.¹¹⁶

Future Directions

SDSCs offer a tissue-specific advantage over other MSC sources in terms of chondrogenic potential, but the cartilage product is far from perfect. Several key areas must be addressed if full regeneration is to be achieved. These areas include, but are not limited to, unique surface markers to allow isolation of the most stem-like or chondroprogenitor-like cells, growth factors that mimic the body's natural growth factor response during development, and biotribology of the regenerated surface to ensure a proper and integrated functioning with native tissue.

As of yet, no specific surface or cellular marker has been described to positively identify MSCs.⁸¹ The International Society for Cellular Therapy has set markers that MSCs must be positive and negative for, but this set certainly does not select for cells that are most multipotent or chondrogenic. Recent work has been done with subpopulations of MSCs expressing different surface markers, such as CD73, CD105, CD106, and CD271.^118,119 Interestingly, some subpopulations seem to be more differentiated than others and possess a stronger chondrogenic potential.

Numerous growth factors and cell signaling molecules have been experimented with to evaluate their effect on cartilage production. Varying concentrations, combinations, and application times have led to an incredible amount of data, but there is still no cartilage construct that has the complexity or organization of native tissue. Reproducing native tissue in vitro may not be possible yet, but elucidating the pathways and the timing that occur in fetal development and growth into adulthood may bring us one step closer. It is only reasonable to assume that, since the physiological formation of articular cartilage occurs due to the action of a sequence of growth factors, hyaline cartilage formation by SDSCs must also occur as a result of this phenomenon.^120–122 Underlying many of these issues is the need for a complete understanding of the biochemical and molecular pathways that moderate chondrogenesis.⁹² A better understanding of these pathways would allow researchers to systematically alter the environments of in vitro cultures and, in vivo, would help engraft the transplanted cells and lock them into a differentiated state.^81,89

Finally, a better understanding of the nature of native and regenerated cartilage will ensure proper function at the site of injury. Biotribology is the study of the interactions between biological surfaces in terms of friction, wear, and lubrication. By understanding the biotribology of the tissue, work can be done to ensure that the surface of a regenerated cartilage implant is as similar as possible to the native cartilage. This is critical in a joint because surface contact between cells can affect apoptosis, growth, communication, sensing, morphology, and remodeling.^123–126 Knowledge of the biotribology of native tissue will allow the regenerated tissue to contribute to joint homeostasis and lubrication through the production and secretion of specific proteins, lubricants, and autocrine and paracrine factors.¹²⁷ A regenerated or implanted tissue with these properties will ultimately increase the longevity of the procedure.¹²⁷

Conclusion

The results of work done with SDSCs support the use of these cells as a tissue-specific cell for the production of joint connective tissue constructs. Further support for the utility of SDSCs in cartilage regeneration lies in the genetic and physical relationships between SDSCs and chondrocytes. The proper combination of inductive signals, responding cells, and three-dimensional microenvironment will hopefully lead to a suitable regenerative process using SDSCs that will not only ease the pain of millions of people, but also regenerate healthy, normal tissue.

Footnotes

Acknowledgments

The authors thank Suzanne Smith for her help in editing the article. This project was supported by Research Grants from the Musculoskeletal Transplant Foundation and the AO Foundation.

Author Contributions

M.P.: Planning; B.A.J. and M.P.: Data collection, data analysis, and article preparation and editing.

Disclosure Statement

The authors declare that they have no conflict of interest.

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