Cartilage Repair Using Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Embedded in Hyaluronic Acid Hydrogel in a Minipig Model


Cartilage shows lousy regenerative capabilities. Fortunately, it is possible to regenerate cartilage with human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) that have been embedded in a hyaluronic acid (HA) hydrogel composite. In fact, such a combination has shown remarkable results in rat and rabbit models.

In this present study, published in Stem Cells Translational Medicine, Yong-Geun Park and his colleagues from SungKyunKwan University School of Medicine, in Seoul, South Korea sought to confirm the efficacy of this protocol in a in a pig model using three different hUCB-MSC cell lines.

Park and his coworkers generated full-thickness cartilage injuries in the trochlear groove of each knee in 6 minipigs. Three weeks later, an even larger cartilage defect, 5 mm wide by 10 mm deep, was created, followed by an 8-mm-wide and 5-mm-deep boring. In short, the knee cartilages of these minipigs were very messed up.

Trochlear-groove

To these knee cartilages, a mixture (1.5 ml) of hUCB-MSCs (0.5 × 107 cells per milliliter) and 4% HA hydrogel composite were troweled into was then cartilage defects of the right knee. The left knee served as an untreated control. Each cell line was used in two minipigs.

Macroscopic findings of the osteochondral defects of the porcine knees. At 12 weeks postoperatively, the defects of both knees had produced regenerated tissues that were pearly white and firm. These new tissues, which resembled articular cartilage, appeared adherent to the adjacent cartilage and had restored the contour of the femoral condyles (smooth articular surfacewithout depression). The regenerated tissue of the control knee (left knee) looked fibrillated. Grossly, no differencewas seen in the quality of the repaired tissue in the transplanted knee (right knee) among the three groups with different cell lines. (A): Group A. (B): Group B. (C): Group C. Abbreviations: HA, hyaluronic acid; hUCB-MSCs, human umbilical cord blood-derived mesenchymal stem cells.
Macroscopic findings of the osteochondral defects of the porcine knees. At 12 weeks postoperatively, the defects of both
knees had produced regenerated tissues that were pearly white and firm. These new tissues, which resembled articular cartilage, appeared adherent to the adjacent cartilage and had restored the contour of the femoral condyles (smooth articular surface without depression). The regenerated tissue of the control knee (left knee) looked fibrillated. Grossly, no difference was seen in the quality of the repaired tissue in the transplanted knee (right knee) among the three groups with different cell lines. (A): Group A. (B): Group B. (C): Group C. Abbreviations: HA, hyaluronic acid; hUCB-MSCs, human umbilical cord blood-derived mesenchymal stem cells.

12 weeks after surgery, the pigs were sacrificed, and the degree of subsequent cartilage regeneration was evaluated by gross and more detailed microscopic analysis of the knee tissue. The transplanted knee showed superior and more complete joint-specific (hyaline) cartilage regeneration compared with the control knee. The microscopic characteristics of the knee cartilage showed that those animals that received the hUCB-MSCs had greater rates of cell proliferation and cells that differentiated into cartilage-making cells.

Microscopic findings of the regenerating osteochondral defects on porcine articular cartilage (safranin O and fast green staining). At 12 weeks postoperatively, the surface of the repairing tissue in the control knee (left knee) was poorly stained for glycosaminoglycan. In the transplanted knee (right knee), both the regenerated tissue and the adjacent cartilage to which it had become adherent exhibited the normal orthochromatic staining properties with safranin O. (A): Group A. (B): Group B. (C): Group C. Scale bars = 2 mm. Abbreviations: HA, hyaluronic acid; hUCB-MSCs, human umbilical cord blood-derived mesenchymal stem cells.
Microscopic findings of the regenerating osteochondral defects on porcine articular cartilage (safranin O and fast green staining). At 12 weeks postoperatively, the surface of the repairing tissue in the control knee (left knee) was poorly stained for glycosaminoglycan. In the transplanted knee (right knee), both the regenerated tissue and the adjacent cartilage to which it had become adherent exhibited the normal orthochromatic staining properties with safranin O. (A): Group A. (B): Group B. (C): Group C. Scale bars = 2 mm. Abbreviations: HA, hyaluronic acid; hUCB-MSCs, human umbilical cord blood-derived mesenchymal stem cells.

These data show consistent cartilage regeneration using composites of hUCB-MSCs and HA hydrogel in a large animal model. These experiments could be a stepping stone to a human clinical trial in the future that treats osteoarthritis of the knees with hUCB-MSCs embedded in HA hydrogel.

Improving Cartilage Production By Stem Cells


To repair cartilage, surgeons typically take a piece of cartilage from another part of the injured joint and patch the damaged area, this procedure depends on damaging otherwise healthy cartilage. Also, such autotransplantation procedures are little protection against age-dependent cartilage degeneration.

There must be a better way. Bioengineers want to discover more innovative ways to grow cartilage from patient’s own stem cells. A new study from the University of Pennsylvania might make such a wish come true.

This research, comes from the laboratories of Associate professors Jason Burdick and Robert Mauck.

“The broad picture is trying to develop new therapies to replace cartilage tissue, starting with focal defects – things like sports injuries – and then hopefully moving toward surface replacement for cartilage degradation that comes with aging. Here, we’re trying to figure the right environment for adult stem cells to produce the best cartilage,” said Burdick.

Why use stem cells to make cartilage? Mauck explained, “As we age, the health and vitality of cartilage cells declines so the efficacy of any repair with adult chondrocytes is actually quite low. Stem cells, which retain this vital capacity, are therefore ideal.”

Burdick and his colleagues have long studied mesenchymal stem cells (MSCs), a type of adult stem cell found in bone marrow and many other tissues as well that can differentiate into bone, cartilage and fat. Burdick’s laboratory has been investigating the microenvironmental signals that direct MSCs to differentiate into chondrocytes (cartilage-making cells).

chondrocytes
chondrocytes

A recent paper from Burdick’s group investigated the right conditions for inducing fat cell or bone cell differentiation of MSCs while encapsulated in hydrogels, which are polymer networks that simulate some of the environmental conditions as which stem cells naturally grow (see Guvendiren M, Burdick JA. Curr Opin Biotechnol. 2013 Mar 29. pii: S0958-1669(13)00066-9. doi: 10.1016/j.copbio.2013.03.009). The first step in growing new cartilage is initiating cartilage production or chondrogenesis. To do this, you must convince the MSCs to differentiate into chondrocytes, the cells that make cartilage. Chondrocytes secrete the spongy matrix of collagen and acidic sugars that cushion joints. One challenge in promoting MSC differentiation into chondrocytes is that chondrocyte density in adult tissue is rather low. However, cartilage production requires that the chondrocytes be in rather close proximity.

Burdick explained: “In typical hydrogels used in cartilage tissue engineering, we’re spacing cells apart so they’re losing that initial signal and interaction. That’s when we started thinking about cadherins, which are molecules that these cells used to interact with each other, particularly at the point they first become chondrocytes.”

Desmosomes can be visualized as rivets through the plasma membrane of adjacent cells. Intermediate filaments composed of keratin or desmin are attached to membrane-associated attachment proteins that form a dense plaque on the cytoplasmic face of the membrane. Cadherin molecules form the actual anchor by attaching to the cytoplasmic plaque, extending through the membrane and binding strongly to cadherins coming through the membrane of the adjacent cell.
Desmosomes can be visualized as rivets through the plasma membrane of adjacent cells. Intermediate filaments composed of keratin or desmin are attached to membrane-associated attachment proteins that form a dense plaque on the cytoplasmic face of the membrane. Cadherin molecules form the actual anchor by attaching to the cytoplasmic plaque, extending through the membrane and binding strongly to cadherins coming through the membrane of the adjacent cell.

In order to simulate this microenvironment, Burdick and his collaborators and colleagues used a peptide sequence that mimics these cadherin interactions and bound them to the hydrogels that were then used to encapsulate the MSCs.

According to Mauck, “While the direct link between cadherins and chondrogenesis is not completely understood, what’s known is that if you enhance these interactions early during tissue formation, you can make more cartilage, and, if you block them, you get very poor cartilage formation. What this gel does is trick the cells into think it’s got friends nearby.”

See L Bian, et al., PNAS 2013; DOI:10.1073/pnas.1214100110.