Mesenchymal Stem Cells Repair Cartilage Defects in Cynomolgus Monkeys


Repairing cartilage defects in the knee represents one of the primary goals of orthopedic regenerative medicine. Cartilage that covers the joints, otherwise known as articular cartilage, has a limited capacity for repair, which leads to further degeneration of the cartilage when it is damaged if it remains untreated. A number of surgical options for treating cartilage defects include microfracture, osteochondral grafting, and cell-based techniques such as autologous chondrocyte implantation (ACI). Each of these procedures have been used in clinical settings. Unfortunately cartilage injuries treated with microfracturing deteriorate with time, since the cartilage made by microfracturing has a high proportion of softer. less durable fibrocartilage.  Also osteochondral grafting suffers from a lack of lateral integration between host and donor cartilage.

Alternatively, tissue engineering has shown some promise when it comes to the healing of cartilage defects.  Mesenchymal stem cells (MSCs) are multipotent progenitor cells that have the ability to differentiate into several different cell lineages including cartilage-making chondrocytes.  MSCs have theoretical advantages over implanted chondrocytes when it come to healing potential.  MSCs have the ability to proliferate without losing their ability to differentiate into mature chondrocytes and produce collagen II and aggrecan. In the short-term, bone marrow-derived MSCs combined with scaffolds have been successful in cartilage repair using animal models such as rabbits (Dashtdar H, et al., J Orthop Res 2011; 29: 1336-42) sheep (Zscharnack M, et al., Am J Sports Med 2010; 38: 185769) and horses (Wilke MM, et al.,l J Orthop Res 2007; 25: 9132).  

In a recent study, Kazumasa Ogasawara and Yoshitaka Matsusue and their colleagues from Shiga University of Medical Science in Shiga, Japan, tested the ability of expanded bone marrow-derived MSCs that had been placed in a collagen scaffold to improve healing of cartilage defects in cynomolgus macaques (type of monkey).  Before this study, there were no previous studies using MSCs from primates for cartilage repair.  The monkey MSCs were shown to properly differentiate into fat, bone, or cartilage in culture, and then were transplanted into the injured cartilage in the cynomolgus macaque.  The efficacy of these cells were ascertained at 6, 12, and 24 weeks after transplantation.

In culture, the cynomolgus MSCs were able to differentiate into fat, bone, and cartilage.

Characteristics of bone marrow-derived MSCs. Panel (a) demonstrates the colony-forming properties of MSCs isolated from bone marrow of cynomolgus macaques using the present protocol (arrows). Bar: 1 cm. Panel (b) shows the adipogenetic properties of MSC-derived cells from staining of lipid droplets with oil red O (arrowheads). Bar: 20 μm. Panel (c) confirms the osteoblastic properties of MSC-derived cells with alkaline phosphatase staining (arrowheads). Bar: 30 μm. Panel (d) confirms the chondrogenetic properties from immunostaining of type-II collagen. Type-II collagen-positive matrix is stained red. Bar: 0.5 mm. Read More: http://informahealthcare.com/doi/full/10.3109/17453674.2014.958807.
Characteristics of bone marrow-derived MSCs. Panel (a) demonstrates the colony-forming properties of MSCs isolated from bone marrow of cynomolgus macaques using the present protocol (arrows). Bar: 1 cm. Panel (b) shows the adipogenetic properties of MSC-derived cells from staining of lipid droplets with oil red O (arrowheads). Bar: 20 μm. Panel (c) confirms the osteoblastic properties of MSC-derived cells with alkaline phosphatase staining (arrowheads). Bar: 30 μm. Panel (d) confirms the chondrogenetic properties from immunostaining of type-II collagen. Type-II collagen-positive matrix is stained red. Bar: 0.5 mm.
Read More: http://informahealthcare.com/doi/full/10.3109/17453674.2014.958807.

Upon transplantation into cartilage defects in the knee cartilage of cynomolgus monkeys, MSCs were compared with collagen gel devoid of MSCs.  The knees that received the transplantations did not show any signs of irritation, bone spurs or infection.  All of the animals had so-called “full-thickness cartilage defects,” and those in the non-treated group showed cartilage defects that did not change all that much.  The cartilage defects of the gel group had sharp edges at 6 weeks that were thinly covered with reparative tissue by 12 weeks, and at 24 weeks, the defect was covered with thick tissue, but the central region of the defects often remained uncovered, with a hollow-like deformity.  In the cartilage defects of those animals treated with MSCs plus the collagen gel, the sharp edges of the defects were visible at 6 weeks after the operation, but at 12 weeks, the defects were evenly covered with yellowish reparative tissue.  At 24 weeks, the defects were covered with watery hyaline cartilage-like tissue that was very similar to the neighboring naïve cartilage.

Macroscopic observations of the repaired defects in the 3 groups at 6 weeks (a, d, g), 12 weeks (b, e, h), and 24 weeks (c, f, i) after implantation. Scale bar: 5 mm. Arrow in (d): the sharp edge of the defect is visible at 6 weeks in the gel group. Arrow in (f): a hollow-like deformity remains in the central region of the defect, despite thick coverage by the reparative tissue. Arrow in (g): the sharp edge of the defect is also visible in the MSC group at 6 weeks. Read More: http://informahealthcare.com/doi/full/10.3109/17453674.2014.958807.
Macroscopic observations of the repaired defects in the 3 groups at 6 weeks (a, d, g), 12 weeks (b, e, h), and 24 weeks (c, f, i) after implantation. Scale bar: 5 mm. Arrow in (d): the sharp edge of the defect is visible at 6 weeks in the gel group. Arrow in (f): a hollow-like deformity remains in the central region of the defect, despite thick coverage by the reparative tissue. Arrow in (g): the sharp edge of the defect is also visible in the MSC group at 6 weeks.
Read More: http://informahealthcare.com/doi/full/10.3109/17453674.2014.958807.

When evaluated at the tissue level, Ogasawara and Matsusue and others used a stain called toluidine blue to visualize the amount of cartilage made by each treatment.  As you can see in the picture below, the non-treated group didn’t do so well.  In the full-thickness defect the region below the cartilage was filled with amorphous stuff 6 weeks after the procedure, and at 12 weeks, amorphous stuff faintly stained with toluidine blue, which reflects the conversion of the amorphous stuff into bone.  At 24 weeks, bone tissue reappeared below the cartilage zone, even though the bone did not look all that normal (no trabecular structure but woven bone-like structure).

In the gel group, cartilage-like tissue is seen at 6 weeks, and at 12 weeks, the faintly stained layer covered the cartilage defect. At 24 weeks, the defect was covered with the cartilage-like stuff, even though the central region had only a little cartilage, as ascertained by toluidine blue staining.  The bone underneath the cartilage looked crummy and there was excessive growth of cartilage into the region underneath the cartilage layer.

In the MSC group, the bone underneath the cartilage healed normally, and at 12 weeks, the boundary between the articular cartilage and the bone layer beneath it had reappeared.  At 24 weeks, the thickness of the toluidine blue-stained cartilage layer was comparable to that of the neighboring naïve cartilage.

Even though the gel group showed most cartilage-rich tissue covering the defect, this was due to the formation of excessive cartilage extruding through the abnormal lower bone layer.  Despite the lower amount of new cartilage produced, the MSC group showed better-quality cartilage with a regular surface, seamless integration with neighboring naïve cartilage, and reconstruction of the bone underneath the cartilage layer.

Histological findings after toluidine blue staining in the 3 groups at 6 weeks (a, d, g), 12 weeks (b, e, h), and 24 weeks (c, f, i) after implantation. Scale bar: 2 mm. Dotted line in (a): amorphous reparative tissue filling the subchondral region. Arrowheads in (b): faint toluidine blue staining that reflects involvement of endochondral ossification. Arrowhead in (c): toluidine blue-negative reparative tissue covering the defect. Dotted line in (c): reconstructed subchondral bone consisting of woven bone-like structure. Arrowhead in (d): toluidine blue-positive cartilaginous tissue. Arrowhead in (e): thin faintly toluidine blue-positive layer covering the defect. Arrowhead in (f): the unstained central region of the cartilaginous layer covering the defect. Arrow in (f): excessive cartilage extruding through the deficient tidemark. Dotted line in (g): woven bone-like subchondral bone already re-appearing at 6 weeks. Arrowhead in (h): reconstructed tidemark distinctly discriminating the articular cartilage from the subchondral bone.
Histological findings after toluidine blue staining in the 3 groups at 6 weeks (a, d, g), 12 weeks (b, e, h), and 24 weeks (c, f, i) after implantation. Scale bar: 2 mm. Dotted line in (a): amorphous reparative tissue filling the subchondral region. Arrowheads in (b): faint toluidine blue staining that reflects involvement of endochondral ossification. Arrowhead in (c): toluidine blue-negative reparative tissue covering the defect. Dotted line in (c): reconstructed subchondral bone consisting of woven bone-like structure. Arrowhead in (d): toluidine blue-positive cartilaginous tissue. Arrowhead in (e): thin faintly toluidine blue-positive layer covering the defect. Arrowhead in (f): the unstained central region of the cartilaginous layer covering the defect. Arrow in (f): excessive cartilage extruding through the deficient tidemark. Dotted line in (g): woven bone-like subchondral bone already re-appearing at 6 weeks. Arrowhead in (h): reconstructed tidemark distinctly discriminating the articular cartilage from the subchondral bone.

This protocol has been nicely optimized by Ogasawara and Matsusue and their research team.  From these data, they conclude:  “Application in larger defects is certainly in line with future clinical use. If MSCs—under optimized conditions—turn out to be superior to chondrocyte implantation in experimental cartilage repair, the procedure should be introduced to clinical practice after well-controlled randomized clinical trials.”  Hopefully, clinical trials will commence before long.  This procedure uses a patient’s own MSCs, and if such a procedure could reduce or delay the number of knee replacements, then it would surely be a godsend to clinicians and patients alike.

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Published by

mburatov

Professor of Biochemistry at Spring Arbor University (SAU) in Spring Arbor, MI. Have been at SAU since 1999. Author of The Stem Cell Epistles. Before that I was a postdoctoral research fellow at the University of Pennsylvania in Philadelphia, PA (1997-1999), and Sussex University, Falmer, UK (1994-1997). I studied Cell and Developmental Biology at UC Irvine (PhD 1994), and Microbiology at UC Davis (MA 1986, BS 1984).

4 thoughts on “Mesenchymal Stem Cells Repair Cartilage Defects in Cynomolgus Monkeys”

    1. The cells were loaded into a collagen scaffold and “troweled” into the defect. This procedure was done arthroscopically and under live fluoroscopy. The HiQ Cells are from fat, whereas the cells in this experiment were harvested from bone marrow. Also the HiQ Cells are injected into the joint, but not blindly. Instead the cells are injected into the precise region of the cartilage defects and laid onto the affected site. The HiQ Cells are currently in use in Australia whereas this procedure is experimental.

  1. I understand, it just seemed like this cartilage at the picture, with this new experimental procedure was overall better at the whole joint. Maybe I was just fooled by the pictures. Nice if this could be done arthroscopically at the whole joint. Will this university get the clearance from the Japanese regulators to perfom this in humans in the near future? Do you know anything about the Regeneus outcome? If I understand correctly they have to “remove” an antibody with MSC’s from the fat so the injected cells will not kill the chondrocytes?

  2. Regereus is presently collecting data on a few hundred patients to assessment the clinical efficacy of their HiQ Cell procedure. They have published a small pilot study, but according to the information on their web site, the results of the pilot study are largely being recapitulated in the larger study: HiQ cell treatment relief pain as well as placebo, but they also decrease the degradation of the articular cartilage of the knee (as ascertained by the decreased excretion of cartilage breakdown products in urine) and also improve the mobility of the knee. Here’s what is said at the HiQ Cell website:

    “Professor Jegan Krishnan, chair of Orthopaedic Surgery at the Flinders Medical Centre, Flinders University, Adelaide, an independent expert in orthopaedics, developed the Joint Registry in conjunction with Regeneus.

    It is a voluntary Registry and all patients treated who have had HiQCell treatment are asked if they would like to be included. Patients are tracked from a pre-treatment baseline, at two weeks, at six months and then annually. 305 patients had consented to be included in the registry as of 23 January 2014, representing 77% of all patients treated.” See http://www.imaginelessjointpain.com.au/about-hiqcell-treatment/hiqcell-patient-results.

    As to removing an antibody from fat-based mesenchymal stem cells, I am unsure what you mean. Converting adipose tissue-based MSCs into chondrocytes required the addition of transforming growth factor-b (TGF-b),ascorbate, and dexamethasone. Under these conditions, fat-based MSCs will secrete the extracellular matrix proteins of cartilage (collagen Type II, collagen Type VI, and aggrecan) when maintained in an appropriate 3D matrix for 1 – 2 weeks in culture. In a 3-D culture, they form cartilage even sooner. As to isolating these MSCs from fat, MSCs are much denser than fat and can be cheaply and easily isolated from the mature fat cells. Finally as to MSCs killing off chondrocytes, that contradicts a report by Danièle Noëla’s group that fat-based MSCs promote chondrocyte survival (see Stem Cell Research 2013; 11(2):834–844).

    As to your question, “Will this university get the clearance from the Japanese regulators to perform this in humans in the near future?” I have no idea. I suppose that Ogasawara and Matsusue will apply for a clinical trial with their treatment, but that is all conjecture on my part. I agree that the cartilage in this picture does look quite nice, but that is probably one of their best pictures.

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