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.

Tissue Engineers Use New Biomaterial to Repair Knee Cartilage


Tissue engineers from Johns Hopkins University School of Medicine’s Translational Tissue Engineering Center (TTEC) have used a biomaterial to stimulate and facilitate the growth of new cartilage in human patients.

An illustration of the cartilage repair surgical procedure. A mini-incision exposes the cartilage defect (top left-hand panel), and any dead tissue is removed from the edges. (B) The adhesive is then applied to the base and walls of the defect, followed by microfracture. (C) Lastly, the hydrogel solution is injected into the defect. (D) Bleeding from the microfracture holes is trapped in and around the hydrogel.Science Translational Medicine/AAAS
An illustration of the cartilage repair surgical procedure. A mini-incision exposes the cartilage defect (top left-hand panel), and any dead tissue is removed from the edges. (B) The adhesive is then applied to the base and walls of the defect, followed by microfracture. (C) Lastly, the hydrogel solution is injected into the defect. (D) Bleeding from the microfracture holes is trapped in and around the hydrogel.
Science Translational Medicine/AAAS

This was a rather small study that only examined 15 patients. All 15 patients had cartilage defects and were scheduled to undergo “microfracture surgery.” Microfracture surgery uses a drill to bore tiny holes in the bone. These small holes allow bone marrow stem cells to leak into the joint space and make new bone and cartilage. In this study, hydrogel scaffolding was troweled into the wound to in order to support and nourish the healing process. The results from this study were published in the Jan. 9 issue of Science Translational Medicine. According to the authors, this study is a proof of concept trial that paves the way for larger trials to test the hydrogel’s safety and effectiveness.

“Our pilot study indicates that the new implant works as well in patients as it does in the lab, so we hope it will become a routine part of care and improve healing,” says Jennifer Elisseeff, the Jules Stein Professor of Ophthalmology and director of the Johns Hopkins University School of Medicine’s TTEC. Cartilage damage results from overuse, injury, disease or faulty genes. Microfracture surgery is a standard of care for cartilage repair, but when holes in cartilage are caused by joint injuries, microfracture surgery often either fails to stimulate new cartilage growth or grows cartilage that is less hardy than the original tissue

To address this problem, tissue engineers, such as Elisseeff, have postulated that the bone marrow mesenchymal stem cells need a nourishing scaffold on which to grow in order to make the right type of cartilage and enough of it. Unfortunately, demonstrating the clinical value of hydrogels has been slow, difficult, and expensive. By experimenting with various materials, Elisseeff and her colleagues have developed a promising hydrogel, and an adhesive that sticks the hydrogel to the bone.

After testing the combination for several years in the lab and in goats, the hydrogel seemed ready for human trials. Elisseeff and her group collaborated with orthopedic surgeons to conduct their first clinical study. 15 patients with holes in the cartilage of their knees received a hydrogel and adhesive implant along in combination with microfracture surgery. In order to compare the efficacy of their hydrogel, another three patients were treated with microfracture surgery alone. After six months, it was clear that the hydrogel implants had caused no major problems. Furthermore, magnetic resonance imaging of these patient’s knees showed that patients with implants had new cartilage filling an average 86% of their defects in their knees, and patients that had received only microfracture surgery had an average of 64% of their tissue replaced. Patients with the implant also reported a greater decrease in knee pain in the six months following surgery, according to the investigators.

As the trial continues, more patients have enrolled. This clinical trial is presently managed by a company called Biomet. These data from this trial is part of an effort to earn European regulatory approval for the device.

Elisseeff and her team have begun developing a next-generation implant in which the hydrogel and adhesive will be combined in a single material. Elisseeff and others are also interested in technologies for joint lubrication that reduce joint pain and inflammation

Repairing Cartilage With Fat-Based Stem Cells May Be Feasible With New Technology


Head-to-head comparisons between bone marrow and fat stem cells have shown that bone marrow stem cells consistently outperform fat stem cells. As I have written in past posts, the present protocols for inducing cartilage from mesenchymal stem cells were developed using bone marrow stem cells. Therefore, the fact that bone marrow stem cells outperform fat stem cells with it comes to cartilage formation is no surprise.

In a study in New Zealand White rabbits, bone marrow stem cells outperformed fat stem cells when it came to the repair the cartilage defects in the knee joint. See Li Q, Tang J, Wang R, Bei C, Xin L, Zeng Y, Tang X. “Comparing the chondrogenic potential in vivo of autogeneic mesenchymal stem cells derived from different tissues.” Artif Cells Blood Substit Immobil Biotechnol. 2011 Feb;39(1):31-8. Here again, the system for chondrocyte differentiation system used was developed with, by, and for bone marrow mesenchymal stem cells. Thus the ability of these cells to outperform fat stem cells says nothing about the ability of fat-based mesenchymal stem cells to form cartilage in alternative culture systems.

Because fat-based stem cells are highly accessible and unlikely to be rejected by the immune system, there is a deep desire to efficiently convert fat-based stem cells into cartilage. Unfortunately, this task is not as straightforward as previously believed. As it turns out, fat-derived stem cells secrete molecules that actually inhibit cartilage formation. However, new research has found that if fat-based stem cells are pre-treated with antibodies that neutralize Vascular Endothelial Growth Factor (VEGF) and growing them in nutrients that are specifically designed to promote cartilage formation can counteract the effects of these molecules.

Chondrocytes make and maintain healthy cartilage. However, damage and diseases, such as osteoarthritis, can destroy cartilage and this can result in pain, compromising the patient’s mobility.

Professor Barbara Boyan, and her colleagues from the Georgia Institute of Technology showed that adipose (fat) stem cells (ASCs) secrete large amounts of factors. Some of these factors, especially the growth factor VEG, prevents cartilage regeneration and actually causes the death (apoptosis) of chondrocytes.

However, by treating ASCs with a media designed to encourage the differentiation of fat-based stem cells into cartilage cells reduced the amount of these secreted factors and prevented the growth of blood vessels. Also, the fat-based stem cells were treated with an antibody that neutralizes VEGF, and this pretreatment prevented chondrocyte death.

Professor Boyan said: “Non-treated ASCs actually impeded healing of hyaline cartilage defects, and although treating ASCs improved the situation they added no benefit compared to cartilage allowed to heal on its own. However we only looked at cartilage repair for a week after treatment. Other people have shown that two to six weeks is required before the positive effect of ASCs on cartilage regeneration is seen.”

Therefore, fat-based stem cells might be able to help repair damaged cartilage, and careful handling plus pre-treatment can help ensure a positive result.

See: “Adipose stem cells can secrete angiogenic factors that inhibit hyaline cartilage regeneration,” Christopher SD Lee et al.; Stem Cell Research & Therapy, 24 August 2012, 3:35, DOI:10.1186/scrt126