Researchers at the Stanford University School of Medicine have discovered a way to regenerate, in mice and human tissue, the cushion of cartilage found in joints.
Loss of this slippery and shock-absorbing tissue layer, called articular cartilage, is responsible for many cases of joint pain and arthritis, which afflicts more than 55 million Americans.
Nearly one in four adult Americans suffer from arthritis, and far more are burdened by joint pain and inflammation generally.
The Stanford researchers figured out how to regrow articular cartilage by first causing slight injury to the joint tissue, then using chemical signals to steer the growth of skeletal stem cells as the injuries heal.
The work was published Aug. 17 in the journal Nature Medicine.
“Cartilage has practically zero regenerative potential in adulthood, so once it’s injured or gone, what we can do for patients has been very limited,” said assistant professor of surgery Charles K.F. Chan, Ph.D.
“It’s extremely gratifying to find a way to help the body regrow this important tissue.”
The work builds on previous research at Stanford that resulted in isolation of the skeletal stem cell, a self-renewing cell that is also responsible for the production of bone, cartilage and a special type of cell that helps blood cells develop in bone marrow.
The new research, like previous discoveries of mouse and human skeletal stem cells, were mostly carried out in the laboratories of Chan and professor of surgery Michael Longaker, MD.
Articular cartilage is a complex and specialized tissue that provides a slick and bouncy cushion between bones at the joints.
When this cartilage is damaged by trauma, disease or simply thins with age, bones can rub directly against each other, causing pain and inflammation, which can eventually result in arthritis.
Damaged cartilage can be treated through a technique called microfracture, in which tiny holes are drilled in the surface of a joint.
The microfracture technique prompts the body to create new tissue in the joint, but the new tissue is not much like cartilage.
“Microfracture results in what is called fibrocartilage, which is really more like scar tissue than natural cartilage,” said Chan.
“It covers the bone and is better than nothing, but it doesn’t have the bounce and elasticity of natural cartilage, and it tends to degrade relatively quickly.”
The most recent research arose, in part, through the work of surgeon Matthew Murphy, Ph.D., a visiting researcher at Stanford who is now at the University of Manchester.
“I never felt anyone really understood how microfracture really worked,” Murphy said.
“I realized the only way to understand the process was to look at what stem cells are doing after microfracture.” Murphy is the lead author on the paper. Chan and Longaker are co-senior authors.
For a long time, Chan said, people assumed that adult cartilage did not regenerate after injury because the tissue did not have many skeletal stem cells that could be activated.
Working in a mouse model, the team documented that microfracture did activate skeletal stem cells.
Left to their own devices, however, those activated skeletal stem cells regenerated fibrocartilage in the joint.
But what if the healing process after microfracture could be steered toward development of cartilage and away from fibrocartilage?
The researchers knew that as bone develops, cells must first go through a cartilage stage before turning into bone. They had the idea that they might encourage the skeletal stem cells in the joint to start along a path toward becoming bone, but stop the process at the cartilage stage.
The researchers used a powerful molecule called bone morphogenetic protein 2 (BMP2) to initiate bone formation after microfracture, but then stopped the process midway with a molecule that blocked another signaling molecule important in bone formation, called vascular endothelial growth factor (VEGF).
“What we ended up with was cartilage that is made of the same sort of cells as natural cartilage with comparable mechanical properties, unlike the fibrocartilage that we usually get,” Chan said. “It also restored mobility to osteoarthritic mice and significantly reduced their pain.”
As a proof of principle that this might also work in humans, the researchers transferred human tissue into mice that were bred to not reject the tissue, and were able to show that human skeletal stem cells could be steered toward bone development but stopped at the cartilage stage.
The next stage of research is to conduct similar experiments in larger animals before starting human clinical trials.
Murphy points out that because of the difficulty in working with very small mouse joints, there might be some improvements to the system they could make as they move into relatively larger joints.
The first human clinical trials might be for people who have arthritis in their fingers and toes.
“We might start with small joints, and if that works we would move up to larger joints like knees,” Murphy says. “Right now, one of the most common surgeries for arthritis in the fingers is to have the bone at the base of the thumb taken out.
In such cases we might try this to save the joint, and if it doesn’t work we just take out the bone as we would have anyway. There’s a big potential for improvement, and the downside is that we would be back to where we were before.”
Longaker points out that one advantage of their discovery is that the main components of a potential therapy are approved as safe and effective by the FDA.
“BMP2 has already been approved for helping bone heal, and VEGF inhibitors are already used as anti-cancer therapies,” Longaker said. “This would help speed the approval of any therapy we develop.”
Joint replacement surgery has revolutionized how doctors treat arthritis and is very common: By age 80, one in 10 people will have a hip replacement and one in 20 will have a knee replaced.
But such joint replacement is extremely invasive, has a limited lifespan and is performed only after arthritis hits and patients endure lasting pain.
The researchers say they can envision a time when people are able to avoid getting arthritis in the first place by rejuvenating their cartilage in their joints before it is badly degraded.
“One idea is to follow a ‘Jiffy Lube’ model of cartilage replenishment,” Longaker said. “You don’t wait for damage to accumulate *- you go in periodically and use this technique to boost your articular cartilage before you have a problem.”
BMP Signaling in Tissue-Derived Osteoblasts
The Calvarial Bones Have Two Tissue-Lineages
Using genetic mouse model, the murine calvaria has been demonstrated originated from with dual-tissue lineages (Jiang et al., 2002; Kuratani, 2005), namely, the cranial neural-crest cells (CNC) and paraxial mesoderm mesenchymal stem cells.
The CNC cells that originate from the dorsal neural tube appear early during embryogenesis, and can diversify into multiple cell types, and contribute to most cranial bones, including the nasal-frontal bones, maxillary, frontal bone, and mandible (Chai and Maxson, 2006).
Paraxial mesoderm-derived cells contribute to the formation of parietal bone (Jiang et al., 2002; Kuratani, 2005). Both CNC-derived and paraxial mesoderm derived osteoprogenitor cells undergo intramembranous ossification to produce cranial bones.
Some bones in the cranial base are also from CNC, but they are formed via endochondral ossification, where mesenchymal cells first differentiate into the chondrocytes to form the cartilage primordial.
The intramembranous ossification happens with a direct differentiation into osteoblasts progenitors from the mesenchymal cells (Mishina and Snider, 2014).
Different bones are connected by different sutures. Nasal and metopic sutures are derived from CNC, and coronal sutures are derived from mesoderm, which connect CNC-derived frontal bone and mesoderm-derived parietal bone, and the sagittal suture is derived from CNC, which separate two mesoderm-derived parietal bone (Mishina and Snider, 2014).
However, CNC-derived and paraxial mesoderm derived osteoblasts show distinct differences in osteogenic potential, the regenerative capacities and ossification (Reichert et al., 2013).
The main difference between CNC-derived osteoblasts and mesoderm-derived osteoblasts has been demonstrated in vitro (Xu et al., 2007), namely, CNC-derived osteoblasts display robust proliferation, and the extent of the cell differentiation is much less, and the extent of bone formation is faster compared to mesoderm-derived osteoblasts, exhibiting minimal capacities of bone nodules formation in vitro (Xu et al., 2007).
When mesoderm-derived osteoblasts are cultured with the addition of CNC-derived osteoblasts, the inferior performance of ossification in mesoderm-derived osteoblasts have been improved (Doro et al., 2019), suggesting that CNC input can favor the osteogenic capacities and the extent of ossification (Doro et al., 2019) (Figure 1).
The Levels of BMP Signaling in Tissue-Derived Osteoblasts
Bone morphogenetic protein signaling in bone has been reviewed previously (Nie et al., 2006; Chen et al., 2012; Graf et al., 2016; Wu et al., 2016).
Briefly, BMP ligands bind to their receptors in the membrane, triggering phosphorylation of R-Smads (Smad1, Smad5, and Smad9) that complex with co-Smad (Smad4) and translocate into the nucleus to drive target gene expressions. B
MP-Smad signaling is well-known to be regulated by extracellular antagonists (e.g., Noggin) and intracellular inhibitors (e.g., Smad6 and Smad7).
In a previous study, BMPRs were found with higher expressions in CNC-derived osteoblasts, while the expressions of the Noggin were higher in mesoderm-derived osteoblasts compared to that in CNC-derived osteoblasts from 2 to 5-day-old mice (Xu et al., 2007).
Based on our high-through sequencing data, the level of BMPRs in embryonic frontal bone tissues were higher than that in embryonic parietal bone tissues (Hu et al., 2017).
The inhibition of BMP signaling using Noggin results in increased apoptosis and osteogenesis in CNC-derived osteoblasts, and similarly, the exogenous stimulation of BMP signaling using BMP2 results in reduced apoptosis and osteogenesis in mesoderm-derived osteoblasts (Senarath-Yapa et al., 2013), suggesting that the modulation of BMP signaling in vitro is able to influence the extent of osteogenic potentials in CNC- and mesoderm-derived osteoblasts (Figure 1).
Functions of BMP Signaling in the Development of Cranial Bones
There are 15 BMPs in humans and rodents. Among them, BMP2, BMP4, and BMP7, as well as growth differentiation factor 5 (GDF5) are essential for embryonic skeletal development, while BMP6, BMP7, and GDF6 are essential for late stages of skeletal development (Graf et al., 2016; Wu et al., 2016).
A number of BMPs are expressing in craniofacial bones in a temporospatial manner, including BMP2, BMP4, BMP3, BMP5, BMP6, and BMP7 as well as GDF1 and GDF6.
Genetic mouse models have been used to verify the functions of BMP signaling in calvarial bones in vivo. In CNC cells, the deletion of BMP2 using Wnt1-Cre leads to craniofacial anomalies that resemble the symptoms of the Pierre Robin sequence (PRS), including smaller craniofacial bones (Chen et al., 2019c).
Mutation of BMP2 in CNC leads to abnormal coordination between the proliferation and differentiation of osteogenic progenitors (Chen et al., 2019c). GDF6 is expressed in the primordia of mouse frontal bones, and GDF6 removal results in coronal suture fusion and defective frontal and parietal bones.
The accelerated differentiation of suture mesenchyme was found earlier than the onset of calvarial ossification (Clendenning and Mortlock, 2012). BMP4 is a major regulator in shaping the craniofacial cartilage (Albertson et al., 2005).
Interestingly, the inactivation of BMP2 and BMP4 using Wnt1-Cre in preosteoblasts and periosteal dura can result in defective skull and cerebral veins. BMP2/BMP4, which can be secreted from CNC or mesoderm-derived preosteoblasts and dura, can function in a paracrine manner to regulate the morphogenesis of the cerebral veins (Tischfield et al., 2017), revealing the unrecognized importance of BMP signaling in the maintenance of tissue–tissue interactions for craniofacial organ growth (Table 1).
Functions of BMP signaling in the development of cranial bones.
|BMP2||Wnt1-Cre||Smaller craniofacial bones||Chen et al., 2019c|
|BMP2/BMP4||Wnt1-Cre||Defective skull and dural cerebral veins||Tischfield et al., 2017|
|BMPRIA||Wnt1-Cre||Defective temporomandibular joint||Gu et al., 2014|
|enhanced BMPRIA||Wnt1-Cre||Inhibitory osteogenesis||Gu et al., 2014|
|BMP7||Wnt1-Cre||Alteration of oral cavity morphology||Kouskoura et al., 2013|
|BMP7/BMP4||Mef2c-Cre||Defective mesenchymal transition||Bai et al., 2013|
|BMPRIA||P0-Cre||Wide-open anterior fontanelles||Saito et al., 2012|
|BMPRIA||Pax3-Cre||Reduction in neural-crest cells||Stottmann and Klingensmith, 2011|
|BMPRIA||Wnt1-Cre||Post-migratory development of a subset of NCC derivative cell types||Stottmann and Klingensmith, 2011|
|Smad1||Col2a1-Cre||Defective calvarial bone||Wang et al., 2011|
|Smad4||Wnt1-Cre||Defective mid-gestation||Nie et al., 2008|
|Smad4||Wnt1-Cre||Underdevelopment of branchial arch||Ko et al., 2007|
|ALK2||Wnt1-Cre||Impaired neural-crest cells||Kaartinen et al., 2004|
|ALK2||Wnt1-Cre||Multiple craniofacial defects||Dudas et al., 2004|
|BMPRII||Prx1-Cre||Normal skeletons||Gamer et al., 2011|
|BMPRIA||Dermo1-Cre||Defective ventral body wall formation||Sun et al., 2007|
Bone morphogenetic protein receptors are heterodimers complex composed of type I receptors and type II receptors. There are also different type I and type II receptors, which create a complex ligand-receptor interaction network and allows for specific outcomes for the skeleton.
Among the three type I receptors, BMPRIA has been best-studied and shown to be indispensable for hindbrain neural tube closure (Stottmann and Klingensmith, 2011). Deletion of BMPRIA in CNC using P0-Cre leads to 100% abnormal phenotype with wide-open anterior fontanelles.
This phenotype in the craniofacial mesenchyme results in an activated p53 apoptosis pathway and a downregulation of c-Myc and Bcl-XL. Therefore, the optimal BMPRIA-mediated signaling is essential for CNC-derived frontal bone development (Saito et al., 2012).
Further exploration of the phenotype of the deletion of BMPRIA in CNC cells using Wnt1-Cre results in a defective temporomandibular joint (Gu et al., 2014). The constitutive activation of BMPRIA in CNC cells leads to the craniosynostosis, which happened through the induction of p53-mediated apoptosis in nasal cartilage (Hayano et al., 2015).
Three type II receptors (BMPRII, ActRIIA, and ActRIIB) were also important for the signaling transduction. Deficiencies of BMPRII, one of the three type II receptors, result in normal skeleton using Prx1-Cre, suggesting that different requirements of BMPRs in transducing the signaling to shape the calvaria development (Gamer et al., 2011) (Table 1).
However, the roles of other two type II receptors, ActRIIA and ActRIIB, in craniofacial bones are still unclear.
The intracellular mediator Smad1 is needed for bone development, and deficient Smad1 results in defective calvarial bone (Wang et al., 2011). The inactivation of Smad4 in CNC cells leads to birth death, accordingly, the defective mid-gestation and increased cell death (Nie et al., 2008).
Additionally, Smad4 deficiency leads to underdevelopment of the first branchial arch (Ko et al., 2007). Improper mutation from a rare transmitted frameshift in inhibitory Smad6 (p. 152 fs∗27) can be inherited from non-syndromic craniosynostosis parents (Timberlake et al., 2018), emphasizing the importance of BMP-Smads signaling in shaping CNC-derived craniofacial development.
The orchestration of the BMP signaling pathway eventually converges at crucial transcriptional factors to regulate the osteogenesis and ossification. For example, Msx2, a bona fide downstream target of BMP signaling, regulates the activities of osteoblast-specific transcriptional factors Runx2 and Osterix (Osx). Mutations of Runx2 (Lee et al., 1997; Mundlos et al., 1997) and Osx (Nakashima et al., 2002) lead to severe defects in bone ossification.
Msx2 can label a special population of mesenchymal precursor cells in the cranial vault (Sakagami et al., 2018). Deficient Msx1/2 using Wnt1-Cre leads to a larger defect in frontal bone (Roybal et al., 2010).
A deficiency of Runx2 in CNC cells results in defective ossification, including the frontal bone, mandible, and nasal bone. Runx2 is required both for mesoderm- and CNC-derived cells to differentiate and ossify.
But CNC-derived frontal bone is more dependent on the activity of Runx2 (Shirai et al., 2019). Neural crest-specific inactivation of Osx resulted in a complete absence of intramembranous skeletal bones that derived from the CNC. Besides, the CNC-derived endochondral skeletal bones were also affected (Baek et al., 2013).
Taken together, the data suggested that a precise responsiveness to BMP signaling in CNC cells is crucial for the proper morphogenesis of the calvaria, and the BMP signaling can be counted on for the superior osteogenic potential in CNC-derived bones.
BMP Signaling in Adult Calvarial Stem-Cell Niche
Bone morphogenetic protein ligands, BMPRs, and intracellular Smads are expressed in suture mesenchyme cells (Opperman, 2000; Ishii et al., 2015). Noggin expression is highly related to patent sutures (Warren et al., 2003), and improper Noggin expression can prevent cranial suture fusion (Warren et al., 2003).
BMPRIA mutations in osteoclasts, osteoblasts, or cartilage result in defective bone remodeling or growth (Mishina et al., 2004; Kamiya et al., 2008; Okamoto et al., 2011; Jing et al., 2013, 2015), and constitutively activated BMPRIA in neural-crest cells results in craniosynostosis (Komatsu et al., 2013). BMP2 is ectopically expressed in Gli3 mutant mesenchyme, which can lead to abnormal osteoblasts differentiation (Tanimoto et al., 2012).
Osteoprogenitors from Gli1+ suture-derived stem cells was found to release Ihh, which is required to maintain the homeostasis of Gli1+ suture-derived stem cells, and this process is fine-tuned by BMPRIA (Guo et al., 2018).
Further, the paracrine BMP2/BMP4, secreted by preosteoblasts, is principally required for the morphogenesis of dural cerebral veins (Tischfield et al., 2017), which then influence the state of the suture-derived stem niche.
This suggested the unrecognized importance of tissue–tissue interactions in suture biology. Therefore, we proposed a BMP diagram where different factors that from osteoprogenitors, osteoclasts, and suture-derived stem cells can coordinate each other at spatial and temporal levels, in either a paracrine or an autocrine manner, to converge together to fine-tune the homeostasis of the suture-derived stem cells through precise communications (Figure 2).
reference link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7075941/
More information: Matthew P. Murphy et al. Articular cartilage regeneration by activated skeletal stem cells, Nature Medicine (2020). DOI: 10.1038/s41591-020-1013-2