Time-dependently Appeared Microenvironmental Changes and Mechanism after Cartilage or Joint Damage and the Influences on Cartilage Regeneration

ABSTRACT

Cartilage and joint damage easily degenerates cartilage and turns into osteoarthritis (OA), which seriously affects human life and work, and has no cure currently. The temporal and spatial changes of multiple microenvironments upon the damage of cartilage and joint are noticed, including the emergences of inflammation, bone remodeling, blood vessels, and nerves, as well as alterations of extracellular and pericellular matrix, oxygen tension, biomechanics, underneath articular cartilage tissues, and pH value. This review summarizes the existing literatures on microenvironmental changes, mechanisms, and their negative effects on cartilage regeneration following cartilage and joint damage. We conclude that time-dependently rebuilding the multiple normal microenvironments of damaged cartilage is the key for cartilage regeneration after systematic studies for the timing and correlations of various microenvironment changes.

KEYWORDS:

• Joint damage
• osteoarthritis (OA)
• pH value
• oxygen tension
• biomechanics

Introduction

The biocomposite formed by articular cartilage, calcified cartilage (their combination is cartilage) and subchondral bone is called osteochondral unit,¹ in which cartilage damage can be caused by several conditions including accidents such as a tear to the anterior cruciate ligament, injury, slow degeneration over time (aging), excessive activity (overuse), excessive weight (obesity), poor alignment of joint, necrosis, and other diseases of osteoarthritis (OA) and rheumatoid arthritis etc. Cartilage has a minimal ability to repair itself in terms of structure, function, and strength for its avascular nature and poor capability for adult chondrocytes to secret extracellular matrix. To be worse, the appeared mesenchymal stromal cells (MSCs) show a changed phenotype, which is susceptible to hypertrophy, matrix metalloproteinase-13 (MMP-13) release and osteogenesis.² Generally, cartilage damage is healed by fibrocartilage different from normal cartilage, which is difficult to integrate with surrounding tissues, and ultimately degenerated and disintegrated over time. Cartilage damage usually leads to serious medical consequences, in which constant and severe pain, inflammation, and some degree of disability are typical.

A “seed and soil theory” is raised by Stephen Paget in 1880, which implies that the initiation and progression of disease such as cancer focus not only on the cell itself, “the seed,” but also on “the soil,” in which it derives its nutrients, oxygen, and signals for growth and development.³ The importance of microenvironment is postulated thereafter. This review combs the time-dependently appeared microenvironmental changes and their mechanism following cartilage or joint damages, and the influences of one microenvironmental change on relative tissues, cells, cytokines, and other microenvironments as well as their interplays, focusing on MSCs for their promise in cartilage regeneration, which is summarized in Figure 1. We conclude that systematic studies for the timing and correlations of various microenvironmental changes and time-dependently rebuilding the multiple normal microenvironments of damaged joint may be the key to cartilage regeneration.

Figure 1. The time-dependently (in the gray arrow) altered microenvironments (the bold italic letters) and their influences on relative tissues, cells, cytokines, and other microenvironments (normal black letters) as well as their interplays (as shown in Inflammation, positive effects are gray line and dot lines, the negative effects are dot lines) after cartilage or joint damage.

Extracellular and pericellular matrixes microenvironment

Time-dependently altered extracellular and pericellular matrixes after cartilage and joint damage

After cartilage damage, the concentrations of matrix degradation enzymes, such as MMP-3,⁴ MMP-13, and dsRNA-dependent protein kinase PKR increase,⁵ resulting in loss of proteoglycan and type II collagen on the fifth day, loss of fixed charged density,⁶ change of collagen fiber orientation and increase of cartilage thickness in two weeks.⁷ In addition, the type I collagen, fiberonectin, and their degradation fragments increase in 20 days.^8,9 Significant proteoglycan loss and increase of type II collagen orientation angle and content exist in the middle and deep zones of cartilage tissue in four weeks.¹⁰ The ratio of collagen degradation to synthesis increases after 6 months.⁴ The fibrillation expands to the radial zone at eight months.¹¹ With the development of OA, the staining for type VI collagen and perlecan changes progressively from compact to weakened and less focalized.¹² Laminin is depleted from the pericellular matrix before collagen type IV.¹³ In fact, the microscale spatial pericellular matrix and local extracellular matrix in middle and superficial zones of cartilage are softened before the structural changes in macroscale tissue properties appear.¹⁴ On the other hand, a small leucine-rich proteoglycan of decorin is found to slightly increased ten weeks after OA initiation.¹⁵

The negative effects of altered extracellular and pericellular matrixes on cartilage maintenance and regeneration

The normal components of extracellular and pericellular matrixes are important for maintenance of structure and function in osteochondral tissues and cells, as well as for their self-repair capability. For example, collagen provides an important microenvironment for human OA chondrocytes in response to mechanical stimulation.¹⁶ Type III collagen is a key regulator of collagen fibrillar structure and biomechanics of articular cartilage. Reduction of type III collagen leads to increased heterogeneity and mean thickness of collagen fibril diameter, as well as reduced modulus, and these effects become more pronounced with skeletal maturation. Type III collagen also has a marked contribution to the micromechanics of the pericellular matrix.¹⁷ The proteoglycan-4 secreted by synovial fibroblasts and superficial zone chondrocytes plays important autocrine role in modulating OA synoviocyte proliferation and expression of matrix degrading enzymes.¹⁸ Perlecan is required for the chondrogenic differentiation of synovial MSCs.¹⁹ Laminins and nidogen-2 in pericellular matrix drive chondrogenic progenitor cells toward chondrogenesis.²⁰ Retention of the native chondrocyte pericellular matrix improves its matrix production.²¹ Decorin could increase the adhesion between aggrecan and aggrecan molecules, and between aggrecan molecules and collagen II fibrils, as well as increase the retention of aggrecan in the neo-matrix of chondrocytes. Decorin delays cartilage matrix degeneration and fibrillation,²² but decorin-null cartilage substantially reduces aggrecan content.²³

The altered extracellular and pericellular matrixes lead to changes in mechanical properties and organization in themselves. Decrease of equilibrium modulus, dynamic modulus and phase angle in cartilage two weeks after OA induction,⁷ as well as impair of stiffness in collagen network and increase of permeability after four weeks are found.²⁴ The elastic moduli in extracellular and pericellular matrixes are decreased for 45% and 30% on the medial condyle after OA.²⁵ The stiffness²⁶ and mean Young’s modulus¹² of pericellular matrix are also decreased. However, chondrocyte shear strain is increased for 30% in early-OA model.¹⁴ Loss of organization in pericellular matrix, including perforation, decrease and destruction of local micro-fibrillar collagen intensity, regional collagen type VI signal weakness or absence are observed in early OA.²⁷

The altered extracellular and pericellular matrixes in cartilage affect joint cells. Cartilage wear particles result in a significant increase in DNA and collagen content as well as nitric oxide (NO) and prostaglandins E2 synthesis of synoviocytes.²⁸ The fragments from degraded extracellular matrix may have catabolic properties, such as fibronectin fragments (Fn-fs) induce expression of proinflammatory cytokines, NO, and other inflammatory mediators as well as proteinases in joint, acting as proinflammatory mediators in a positive feedback loop and therefore promoting inflammation and cartilage destruction in OA.²⁹ However, its effect on MSCs is unclear.

Mechanical microenvironment

Time-dependent altered mechanical microenvironment after cartilage and joint damage

Mechanical loading to the articular joint induces a series of mechanical forces, including tension, hydrostatic pressure, shear and compression on cartilage. Due to the damage, the mechanical properties of cartilage are changed. The viscoelastic properties of articular cartilage, especially around the tidemark are decreased in two weeks after OA initiation and degraded as cartilage degeneration progressed.³⁰ The equilibrium and dynamic moduli of cartilage are lowered for up to three times four weeks after OA initiation.¹⁰ The cartilage stiffness³¹ and storage modulus³² of OA patients are reduced. But the exercise-induced cartilage strain is increased.³³ OA cartilage has a higher capacity to hold water.³²

As to subchondral bone, the stiffness is reduced four weeks after OA initiation³⁴ and the modulus is decreased in mild OA patients.³⁵ The shear modulus and radial strain ratio are reduced. But the energy dissipation increases.³⁶ The hyperelastic material constants, the structural parameters,³⁷ and the reduction in cartilage stiffness correlate with OA grade.³¹ The detailed time-dependent alterations in mechanical microenvironment after cartilage and joint damage need further studies.

The negative effects of altered biomechanical microenvironment on chondrocytes and MSCs, as well as effects on OA

It is widely accepted that mechanical forces of a relevant magnitude are essential for maintaining cartilage homeostasis within a healthy joint and playing a vital role in cartilage development. However, excess mechanical stress and strain are experienced by the damaged chondrocytes, which may promote apoptosis³⁸ and anabolic function, increase reactive oxygen species (ROS) production, and decrease uncoupling protein 2 (UCP2) in chondrocytes.³⁹ High fluid shear stress of 20 dyn/cm² experienced after cartilage damage induces the mRNA expression of MMP-12. In fact, the micromechanical environment changes harm chondrocyte activities are before the macro-scale degradations at the tissue level become apparent.⁴⁰

The external mechanical environment has potent capability in regulation of MSC differentiation compared with other two known powerful means of biological factors and substrate stiffness.⁴¹ Suitable biomechanical stimulation promotes the proliferation and chondrogenesis of MSCs.⁴² However, the higher or lower mechanical environment could induce MSCs to other cell types, i.e. osteogenic or adipogenic cells.⁴³

Abnormal mechanical loading also lead to onset and progression of OA.⁴⁴ Increased matrix stiffness disrupts the homeostatic balance between chondrocyte catabolism and anabolism, consequently eliciting OA pathogenesis. Enhancement of matrix cross-linking also augments surgically induced OA pathogenesis. While suppressing these events effectively inhibits OA.⁴⁰

Oxygen tension microenvironment

Altered oxygen tension microenvironment after cartilage and joint damage

Oxygen tensions within cartilage are strongly affected by many factors, including oxygen concentrations in synovial fluid, cartilage thickness, cell density, and cellular oxygen consumption rates, and supply of oxygen from the subchondral bone.⁴⁵ Normally, the oxygen tension of cartilage is estimated to be an average of ~5%, with levels slightly higher (around 6%) at the surface and lower (2 ~ 3%) in the depth,⁴⁶ whilst that of bone marrow is at 7%.^47,48 However, the oxygen tension of cartilage would be changed after damage. The deep and low oxygen tension survival chondrocytes have possibility to contact with oxygen rich synovial fluid due to cartilage lesion. And the blood vessels evolved in subchondral bone or even tidemark with the progression of OA⁴⁹ increase mass transport from bone to cartilage,⁴⁹ which provides opportunity for high oxygen tension in damaged cartilage. On the other hand, the inflammation could induce hypoxia.⁵⁰ The increased mineralization of calcified cartilage may reduce its oxygen tension.⁴⁹

The negative effects of altered oxygen tension microenvironment on chondrocytes, MSCs, and inflammation reactions

Physiological oxygen tension profits, however, the lower or higher oxygen tension is harmful for chondrocyte chondrogenesis. For example, compared with physiological oxygen tension, 21% O₂ promotes dedifferentiation of chondrocytes toward an MSC phenotype,⁵¹ and inhibits the dynamic compression stimulated glycosaminoglycan synthesis.⁵² 1% O₂ reduces hyaluronan synthesis in chondrocytes compared with 21% O₂.⁵³ Oxygen affects energy metabolism and the redox system in normal and OA chondrocytes. In detail, compared with 20% O₂, 1% O₂ significantly reduces, but 5% O₂ significantly increases ATP levels.⁵⁴ Less than 1% O₂ leads to reduction in cell viability, glutathione (GSH): oxidized glutathione (GSSG) ratio and superoxide dismutase 1 (SOD1) and SOD2 protein expressions, increase in glycosaminoglycan release, and alteration in mitochondrial membrane potential and ROS levels in 48 h compared with condition of 5% O₂.^55,56

Physiological oxygen tension profits, however, the higher oxygen tension is harmful for MSC chondrogenesis. For example, compared with physiological oxygen tension, MSCs from 20% O₂ responsive donors have decreased glycosaminoglycan and type II collagen content, and induced hypertrophic markers.⁵⁷ Similarly, human MSCs express typical articular cartilage biomarkers and established inhibitors of hypertrophic differentiation under 2.5% O₂. In contrast, 21% O₂ prevents the expression of these inhibitors and is associated with increased hypertrophic differentiation.⁵⁸ Furthermore, 21% O₂-mediated isolation and expansion of MSCs reduce the number of colonies, the chondrogenic capacity, the mRNA expression of hypoxia inducible factor-2ɑ, but increase the mRNA expression of collagen X.⁵⁹ 20% O₂ enhances the inhibitory effect of inflammation factors on the chondrogenic differentiation of MSCs.⁶⁰

The oxygen tension sensitivity of MSCs is dependent on their differentiative potentials. MSC preparations and articular cartilage progenitors clones of high intrinsic chondrogenicity produce abundant matrix both at 20% and 2% O₂, while poorly chondrogenic cells demonstrate a significant fold-change matrix increase only at 2% but not 20% O₂. Both high and low intrinsic chondrogenicity groups upregulate chondrogenic genes; however, only high intrinsic chondrogenicity groups have a concomitant decrease in hypertrophy-related genes and increase in many common hypoxia-responsive genes at 2% O₂, while low intrinsic chondrogenicity groups downregulate most of these genes. High intrinsic chondrogenicity groups produce comparable type II collagen but less type I collagen at 2% O₂ than at 20% O₂. Type X collagen is detectable in some articular cartilage progenitors pellets at 20% O₂ but reduced or absent at 2% O₂. In contrast, type X collagen is detectable in all MSC preparations at 2% and 20% O₂.⁶¹

Oxygen affects inflammation reactions in articular chondrocytes, where 5% O₂ is optimal compared with 1% and 20% O₂. IL-1 induced reduction in protein translation, increase in autophagy and no effects on senescence are only found at 20% O₂ but not at 1% and 5% O₂. The NO donor induced reduction in ATP levels is observed at all oxygen tensions, but increase of phospho-adenosine monophosphate-activated protein kinase (pAMPK) and senescence is only shown at 5% O₂, and decrease of pAMPK, protein translation and autophagy is only appeared at 1% O₂.⁵⁴

Inflammatory microenvironment

Appearance of and time-dependently altered inflammatory microenvironment after cartilage and joint damage

Following joint damage, macrophage phenotypes accumulate and localize.⁶² The synovial macrophages, fibroblasts and chondrocytes secret inflammatory factors,⁶³ which increase their concentrations in synovial fluid, including interleukin-1ß,6,8, (IL-1ß,6,8), tumor necrosis factor-ɑ (TNF-ɑ), and C-reactive protein in one day⁶⁴ and IL-10 in 2 weeks.⁶³ After acute phase, their concentrations generally drop down, but some are reported to maintain elevated levels than normal conditions for months to years, especially under OA progression.

The negative effects of the inflammatory microenvironment on chondrocytes and MSCs, as well as effects on other cytokines, angiogenesis, and OA

The inflammatory factors elicit catabolism in joint. Inflammation inhibits the synthesis of proteoglycans and type II collagen,⁶⁵ and results in apoptosis of chondrocytes.⁶⁶ In detail, IL-1ß decreases mRNA of type II collagen, aggrecan core protein and MMP-9, while increases MMP-1, 2, 3 and tissue inhibitors of matrix metalloproteinases (TIMPs) in chondrocytes.⁶⁷ TNF-α increases MMP activity, induces MMP-13 and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-5) gene expression, and reduces dynamic compression induced glycosaminoglycan synthesis in chondrocytes dose-dependently in 48 h.⁵² While NO generated in joint induces apoptosis of human articular chondrocytes within 24 h.⁶⁸ NO also inhibits respiration and synthesis of adenosine triphosphate (ATP) and proteoglycans without affecting cell viability, but increases cellular levels of heat-shock protein 70 (hsp70) and heme oxygenase 1 (HO-1)⁶⁹ with cartilage protective effects.^70,71 Prostaglandin E2 and F2 generate synergistic effects on the expression of MMP-12.⁷² While the inflammation induced MMPs, ADAMTS, and other cytokines are known to cause cartilage matrix degradation and angiogenesis.^29,73,74

Inflammation affects proliferation, differentiation, and angiogenesis of MSCs. Exposure of MSCs to proinflammatory cytokines reduces the viability and differentiation abilities to osteogenic, adipogenic and chondrogenic cells,⁷⁵ in addition to their expression levels of SOX-9, TGF-β1, aggrecan, and type II collagen.⁷⁶ The expression of VEGF in MSCs is upregulated by OA synovial fluid.⁷⁷ TNF-α activated MSCs promote endothelial progenitor cell homing and angiogenesis.⁷⁸

Inflammation affects immunomodulation and inflammation modulation of MSCs. Inflammatory cytokines authorize the immunomodulatory capabilities of MSCs, which can orchestrate local and systemic innate and adaptive immune responses through the release⁷⁹ and intervention⁸⁰ of various mediators. Even nonviable MSCs can exert beneficial immunomodulatory effects, but apoptotic MSCs show immunosuppressive functions.⁷⁹ For example, inflammatory environment impairs galectin expression in MSCs, which may limit MSC tissue repair properties following intra-articular administration, for galectins are a family of β-galactoside binding proteins regulating inflammation, adhesion and cell migration in diverse cell types.⁸⁰ Lipopolysaccharide (LPS) and IL-1β increase the expression of pro-inflammatory cytokines, i.e. TNF-α and IL-1β, 6, 8 in MSCs.⁷⁷ On the other hand, MSCs secreted factors under inflammatory environment cause multiple anti-inflammatory effects.⁸¹ In special, MSC exosomes suppress IL-1β-induced NO and MMP13 production in chondrocytes.⁸²

The net outcomes of MSC activation might vary depending on the levels and the types of inflammation⁸³ and MSC sources. Factors secreted by pro-inflammatory macrophages substantially increase MSC attachment and migration, whereas those released by anti-inflammatory macrophages enhance MSC osteogenic activity as well as cell migration.⁸³ Inflammatory synovial environment may not be enough to activate MSC immunomodulatory potential when they are intraarticular administered, yet inflammatory priming enhances MSC immunomodulatory potential,⁸⁴ where inflammatory cytokine primed-MSCs produce less cytotoxic allo-antibodies directed against donor’s leukocyte antigen than naïve-MSCs in secondary intraarticular administration.⁸⁵ IL-1β stimulates more IL-8, oncostatin M (OSM), the angiogenic proteins of vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, VEGF-A, VEGF-C, the proteases of MMP-1 and 3, and the protease inhibitor TIMP3 in naïve MSCs than in MSCs undergoing chondrogenic differentiation in 48 h.⁸⁶

The inflammatory factors promote angiogenesis in joint. Chondrocytes in fracture model play a key role in angiogenesis, which is transcription factor forkhead box-O 1 (FOXO1) dependent and that FOXO1 in chondrocytes regulates a potent angiogenic factor, VEGF-A.⁸⁷ Inflammation drives synovial angiogenesis.⁸⁸ Fibroblast changes under chronic synovial inflammation condition induce angiogenesis under hypoxic conditions.⁸⁹ TNF-α induces the secretion of leucine-rich-alpha-2-glycoprotein 1 in human umbilical vein endothelial cells, which could increase angiogenesis and recruit MSCs in the subchondral bone of OA joints.⁶⁶ NO and prostaglandins also contribute to angiogenesis.^90,91

The inflammatory factors induce the production of other cytokines that aggravate the inflammation condition in joint. For instance, IL-1ß induces IL-1ß, 6, 8, 11, and TNF-α expression,⁹² in addition to NO and prostaglandins E2 production in chondrocytes.^54,67 TNF-α increases NO and prostaglandins E2 dose-dependently.⁵²

Inflammation may involve in the obesity and aging induction of OA. Obese people and animals show a higher systematic level of serum TNF-α, IL-1β, and 6.⁶⁵ Similarly, aged people are characterized by the presence of a low-grade systematic proinflammatory status.⁹³ Highly vascularized synovial tissue, cartilage surface and subchondral bone could expose the obese and aged cartilage to the systemic influences of low grade inflammation and suffer from the negative effects.⁹³

Underneath articular cartilage tissues microenvironment

Time-dependently altered underneath articular cartilage tissues and appearance of bone remodeling microenvironment after cartilage and joint damage

Cartilage damage induces changes in tissues underneath articular cartilage. The thickness of human calcified cartilage decreases with age and the number of tidemarks increases particularly over age of sixty, and these processes are accelerated with increasing age.⁹⁴ The calcified cartilage is extremely hypermineralized, especially in superior regions, which is translocated into subchondral bone and articular cartilage in human end-stage OA.⁹⁵

The subchondral bone changes in damaged cartilage are widely studied for its hypothesized key role in OA progression. The bone remodeling and loss are increased in early-stage, but the remodeling is slowed and subchondral bone is densified in late stage, which are important spatial and temporal pathogenetic process of OA subchondral bone.⁹⁶ In addition, the fibril pattern has changed from a regular periodic banding pattern with apatite crystals parallel to the long axis of the fibrils in grade I OA, to a random and undulated arrangement with nonhierarchical intrafibrillar mineralization and higher calcium to phosphorus ratios accompanied by a circular oriented pattern of apatite crystals in grade IV OA.⁹⁷

The osteophyte bone is emerged in OA patients.⁹⁸ The number and volume of subchondral bone cysts are associated with lateral OA severity, lateral joint space narrowing, alignment, and sex. Greater cyst number and volume are associated with higher bone mineral density.⁹⁹ However, lateral-compartment osteophytes are not associated with biomechanically weaker cartilage or with more-advanced histological signs of degeneration of lateral-compartment cartilage in knees with varus arthritis.¹⁰⁰

The negative effects of altered underneath articular cartilage tissues microenvironment on cartilage and joint cells

Normal tissues and cells underneath articular cartilage profit cartilage maintenance. Normal healthy osteoblasts stimulate an initial increase in the number and later osteogenic differentiation of MSCs, and osteocytes are more influential than osteoblasts in osteogenesis of MSCs.¹⁰¹ Normal healthy osteocytes enhance the IL-17-induced osteogenesis in MSCs through critically orchestrating the activation and differentiation of both osteoblasts and osteoclasts.¹⁰²

The altered tissues underneath articular cartilage promote cartilage damage. Abnormalities in subchondral bone of OA promote joint pain generation and articular cartilage degeneration.¹⁰³ Increased subchondral bone remodeling-induced microstructural damage aggravates OA, which is preceded by osteoporosis.¹⁰⁴ Osteoporosis may also accelerate OA progression because its aberrant bone remodeling leads to an inferior microstructure and worsening biomechanical properties, which potentially affect transmission of loading stress from the cartilage to the subchondral bone.¹⁰⁵

The altered tissues underneath articular cartilage disadvantage joint cells. The pathology of primary OA is accompanied by bone MSC exhaustion, a hallmark of aging.¹⁰⁶ The MLOY4 osteocytic cells cultured on extracellular matrix secreted from primary human subchondral bone osteoblasts of OA patients show a decrease of IL-β1 expression and deactivation of focal adhesion kinase (FAK) cell signaling pathway, which subsequently affect the initial osteocytic cells’ attachment and functions, including morphological abnormalities of cytoskeletal structures, focal adhesions, increased apoptosis, altered osteocyte specific gene expression and increased MMP-2 and -9 expression compared with MLOY4 osteocytic cells cultured on normal extracellular matrix.¹⁰⁷ However, the effect of altered tissues underneath articular cartilage on MSCs is unclear.

Blood vessels and nerves microenvironment

Appearance of blood vessels and nerves microenvironment after cartilage and joint damage

The cartilage is avascular without nerve. However, H-type vessels are increased in subchondral bone of six months OA model and aged mice.¹⁰⁸ The chondrocytes expressing vascular endothelial growth factor (VEGF), the proliferation of endothelial cell and vascular density in articular cartilage are increased, which are associated with subchondral bone marrow replacement by fibrovascular tissue expressing VEGF, and increased nerve growth factor expression within vascular channels.¹⁰⁹ The tidemark is breached by vascular channels along with sympathetic and sensory nerves present within in samples from patients received total knee joint replacement surgery. Perivascular and free nerve fibers and nerve trunks are observed within the subchondral bone marrow and the marrow cavities of osteophytes.¹¹⁰ Osteochondral vascularity is associated with the severity of OA cartilage changes and clinical disease activity.¹¹¹

The negative effects of the appeared blood vessels and nerves microenvironment on cartilage maintenance and regeneration

The invaded blood vessels and nerves to subchondral bone, calcified cartilage or even to tidemark increase the mass transport into cartilage⁴⁹ and contribute to tibiofemoral pain in OA across a wide range of structural disease severity,¹¹⁰ in addition to cartilage degeneration, osteophyte formation, subchondral bone cysts and sclerosis, and synovitis.¹¹² For instance, VEGF destructs condylar cartilage, profits the proliferation of their hypertrophic cells, decreases levels of proteoglycan, and increases expression of VEGF receptor 2 (VEGFR2), MMP-9, MMP-13, and TNF-related activation-induced cytokine.¹¹³ On the contrary, the specific angiogenesis inhibitor PPI-2458 reduces synovial and osteochondral angiogenesis, synovial inflammation, joint damage, and pain behavior of induced OA animals.¹¹⁴ Research shows that sole blockade of angiogenesis in MSCs is sufficient for inducing MSC chondrogenic differentiation.¹¹⁵ However, its effect on MSCs is unclear.

pH value microenvironment

Altered pH value microenvironment after cartilage and joint damage

Normally the pH value in joint is 7.4. However, the pH value of synovial fluid is lowered after inflammation⁵⁰ and obesity.¹¹⁶ The pH value of OA synovial fluid is age- and gender-dependent ranging from 6.80 to 7.68 with a mean of 7.78,¹¹⁷ and lower pH value in female,¹¹⁸ which is changed to 7.60 after revision aseptic operation, and to 7.55 after revision septic operation.¹¹⁷ However, the detailed time-dependent pH change after cartilage and joint damage is unclear.

The negative effects of the altered pH microenvironment on joint cells

Small but not large alkaline pH may be beneficial for MSCs and osteoblasts. pH values of 7.4 and 8.0 upregulate MSC proliferation,¹¹⁹ mineralization.¹²⁰ Weak alkaline conditions (<8.0) stimulate osteogenic differentiation, but inhibit the osteoclasts formation of osteoporotic rat MSCs.¹²¹ While pH value of 8.5 inhibits proliferation and promotes senescence of MSCs.¹¹⁹ Small alkaline pH (<8.4) is beneficial for the proliferation, differentiation and ATP content in developing osteoblasts.¹²² The influence of alkaline pH on chondrocytes is not clear.

Small acidic pH may be harmful for MSCs, chondrocytes and osteoblasts. pH values of 6.3 and 6.7 inhibit proliferation and promote senescence of MSCs.¹¹⁹ 48 h of exposure to pH of 6.6–7.5 decreases the alkaline phosphatase activity and collagen I synthesis of MSCs.¹²³ pH affects elements of the redox system in normal articular chondrocytes. pH 6.2 leads to reduction in cell viability, GSH:GSSG ratio and SOD1 and SOD2 protein expressions, but increase in glycosaminoglycan release, alteration in mitochondrial membrane potential and ROS levels in 48 h compared with condition of pH 7.2.⁵⁵ pH also affects elements of the redox system in human osteoarthritic chondrocytes.⁵⁶ An acidic pH environment (>6.4) increases cell death and pro-inflammatory cytokine release in osteoblasts.¹²⁴ However, irrigation fluid with pH of 6.4 but not 7.6 facilitates intra-articular fibrous cartilage formation and cartilage healing after microfracture procedure.¹²⁵

Future directions

As discussed above, the sequential and related microenvironment changes, i.e. extracellular and pericellular matrix, biomechanics, oxygen tension, inflammation, underneath tissues, blood vessels, and nerves, as well as pH value emerge after cartilage damage, and are not fit for the normal growth and differentiation of chondrocytes, as well as the recruitment, growth, and chondrogenic differentiation of stem cells, which in turn lead to negative feedback loops and contribute to the eventual failure of cartilage repair as shown in Inflammation of Figure 1. Accordingly, we suggest that time-dependently rebuilding the normal multiple microenvironments of damaged cartilage is the key for cartilage regeneration, though one specific microenvironment modification such as inhibition of inflammation or bone remodeling already shows benefit for cartilage regeneration through enzymatic disruption and removal or disruption of function of the factors induced microenvironment changes,^126–128 which is not the focus of our review here.

Considering the microenvironment changes and their negative feedbacks, in order to successfully regenerate cartilage, several questions below exist and are needed to study. (i) The time-dependent microenvironment changes are not understood clearly and systematically, especially the pH and oxygen tension. (ii) The correlations among various microenvironment changes are deficient. The pivotal microenvironmental factors affecting cartilage regeneration are needed to disclose. In particular, the previous appeared microenvironment change affects the subsequent microenvironment change, so as to reveal the influence of the control for previous appeared microenvironment change on the subsequent microenvironment change during cartilage regeneration. For example, whether the newly formed osteophyte is resulted from the hyperoxia, overload and vascularization microenvironments in damaged cartilage for these microenvironments profit osteogenic but not chondrocyte differentiation of MSCs? Furthermore, whether revision of hyperoxia, overload, and vascularization microenvironments could inhibit the osteophyte formation?

Abbreviations

Table

OA	osteoarthritis
MSCs	mesenchymal stromal cells
IL	interleukin
TNF	tumor necrosis factor
ROS	reactive oxygen species
IGF-1	insulin-like growth factor-1
TGF-β1	transforming growth factor beta 1
VEGF	vascular endothelial growth factor
MMP	metalloproteinases
GSH	glutathione
GSSG	oxidized glutathione
SOD	superoxide dismutase
ADAMTS	a disintegrin and metalloproteinase with thrombospondin motifs
AMPK	adenosine monophosphate-activated protein kinase
pAMPK	phospho-AMPK
NO	nitric oxide
OARSI	Osteoarthritis Research Society International

Author contributions

Danyang Yue, Lin Du, Bingbing Zhang, Huan Wu, and Qiong Yang help collecting reference papers and write draft. Min Wang helps collecting information on clinics and discussing on cartilage regeneration. Jun Pan contributes and summarizes the ideas and writes the manuscript.

Acknowledgments

This study was supported by grants from NSF of China (No. 31470902; 11272366; 10972243), the Fundamental Research Funds for the Central Universities of China (2020CDCGSW051, 2018CDPTCG0001/46, 106112015CDJZR238803), Innovation Team in University of Chongqing Municipal Government (CXTDX201601002) and the Chongqing Graduate Student Research Innovation Project (CYS19055).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the Chongqing Graduate Student Research Innovation Project [CYS19055]; Innovation Team in University of Chongqing Municipal Government [CXTDX201601002]; NSF of China [No. 31470902; 11272366; 10972243]; Fundamental Research Funds for the Central Universities of China [2020CDCGSW051, 2018CDPTCG0001/46, 106112015CDJZR238803].