Implantation of allogenic synovial stem cells promotes meniscal regeneration in a rabbit meniscal defect model

Abstract

Background: Indications for surgical meniscal repair are limited, and failure rates remain high. Thus, new ways to augment repair and stimulate meniscal regeneration are needed. Mesenchymal stem cells are multipotent cells present in mature individuals and accessible from peripheral connective tissue sites, including synovium. The purpose of this study was to quantitatively evaluate the effect of implantation of synovial tissue-derived mesenchymal stem cells on meniscal regeneration in a rabbit model of partial meniscectomy.

Methods: Synovial mesenchymal stem cells were harvested from the knee of one New Zealand White rabbit, expanded in culture, and labeled with a fluorescent marker. A reproducible 1.5-mm cylindrical defect was created in the avascular portion of the anterior horn of the medial meniscus bilaterally in fifteen additional rabbits. Allogenic synovial mesenchymal stem cells suspended in phosphate-buffered saline solution were implanted into the right knees, and phosphate-buffered saline solution alone was placed in the left knees. Meniscal regeneration was evaluated histologically at four, twelve, and twenty-four weeks for (1) quantity and (2) quality (with use of an established three-component scoring system). A similar procedure was performed in four additional rabbits with use of green fluorescent protein-positive synovial mesenchymal stem cells for the purpose of tracking progeny following implantation.

Results: The quantity of regenerated tissue in the group that had implantation of synovial mesenchymal stem cells was greater at all end points, reaching significance at four and twelve weeks (p < 0.05). Tissue quality scores were also superior in knees treated with mesenchymal stem cells compared with controls at all end points, achieving significance at twelve and twenty-four weeks (3.8 versus 2.8 at four weeks [p = 0.29], 5.7 versus 1.7 at twelve weeks [p = 0.008], and 6.0 versus 3.9 at twenty-four weeks [p = 0.021]). Implanted cells adhered to meniscal defects and were observed in the regenerated tissue, where they differentiated into type-I and II collagen-expressing cells, at up to twenty-four weeks.

Conclusions: Synovial mesenchymal stem cells adhere to sites of meniscal injury, differentiate into cells resembling meniscal fibrochondrocytes, and enhance both quality and quantity of meniscal regeneration.

Figures

Fig. 1

Method of calculating the quantity of tissue regeneration. The area of the entire defect (D; blue dotted line; left) and the regenerated tissue (R; blue dotted line; right) were calculated. The regenerated tissue-to-defect ratio (R/D) was used to quantify the amount of regenerated meniscal tissue. The ideal quantity of regeneration would result in a value of 1, and incomplete regeneration would result in a value of

Fig. 2

Figs. 2-A through 2-F Synovial mesenchymal stem cells have high proliferation capacity and multipotentiality. Synovium refers to synovial mesenchymal stem cells, and bone marrow refers to bone-marrow mesenchymal stem cells. Fig. 2-A Histological appearance of synovial mesenchymal stem cells (left) and bone-marrow mesenchymal stem cells (right) at passage 3. Both groups formed monolayers of spindle-shaped cells that adhered to plastic culture dishes. Scale bar indicates 200 μm. Fig. 2-B Colony formation of synovial mesenchymal stem cells and bone-marrow mesenchymal stem cells at passage 3. Nucleated cells from synovium and bone marrow were plated at 100 and 1000 cells per 60-cm2 dish and cultured for fourteen days (n = 5 cultures each). Culture dishes stained with crystal violet are shown. Fig. 2-C Graph showing the number of colonies (>2 mm) per dish at 100 or 1000 cells per 60 cm2. *P < 0.01. Fig. 2-D Adipogenesis. Adipocyte colonies were stained with oil red O. Scale bar represents 200 μm. Fig. 2-E Calcification. Calcified colonies were stained with alizarin red. Scale bar represents 500 μm. Fig. 2-F Chondrogenesis. Gross photograph of a pellet (left). Scale bar represents 1 mm. Histological section of the pellet stained with safranin O (right). Scale bar represents 100 μm.

Fig. 3

Figs. 3-A and 3-B Meniscal biopsy and implantation of synovial mesenchymal stem cells. Fig. 3-A Intraoperative photograph made following meniscal biopsy. A 1.5-mm-diameter full-thickness cylindrical defect (arrow) was produced in the inner two-thirds of the anterior horn of the medial meniscus. Fig. 3-B Intraoperative photograph demonstrating implantation of the synovial mesenchymal stem cells. Two million synovial mesenchymal stem cells in 50 μL of phosphate-buffered saline solution were placed directly into the meniscal defect of experimental knees with use of a 27-gauge needle. In control knees, the same volume of plain phosphate-buffered saline solution was used.

Fig. 4

Synovial mesenchymal stem cells (MSCs) promote meniscal regeneration (macroscopic observation). Macroscopic findings of the meniscus at four, twelve, and twenty-four weeks after the implantation of synovial mesenchymal stem cells. The specimens in which full-thickness grossly visible defects remained are denoted by an asterisk. Scale bar represents 2 mm.

Fig. 5-A

Low and high-power images of representative sections of regenerated meniscus stained with safranin O at four, twelve, and twenty-four weeks after implantation of synovial mesenchymal stem cells. The inset shows the area seen at higher magnification in the photomicrograph below. Scale bars represent 200 μm.

Fig. 5-B

Low and high-power representative images of the normal meniscus stained with safranin O. Scale bars represent 200 μm.

Fig. 5-C

Regenerated tissue-to-defect ratios are displayed as the mean and the standard deviation for synovial mesenchymal stem cell (MSCs) and control groups at each end point. *The difference between the groups was significant (p

Fig. 5-D

Results of the histological scoring system for regenerated meniscus. The scores are displayed as the mean and the standard deviation. *The difference between the groups was significant (p

Fig. 6

Figs. 6-A and 6-B CM-DiI-labeled synovial mesenchymal stem cells (MSCs) adhere to sites of meniscal injury and remain at twenty-four weeks. Representative macroscopic appearance (Fig. 6-A) and histological sections (Fig. 6-B) of the meniscal defect after implantation of CM-DiI-labeled synovial mesenchymal stem cells under bright light (top) and fluorescence (bottom). In the histological sections (Fig. 6-B), the white solid line indicates the outer edge of the meniscus, and the white dotted line indicates the border between native meniscus and regenerated tissue. Scale bar represents 400 μm.

Fig. 7

Figs. 7-A and 7-B Green fluorescent protein (GFP)-positive synovial mesenchymal stem cells (MSCs) adhere to sites of meniscal injury in experimental knees, where they produce type-I and type-II collagen. Representative macroscopic appearance (Fig. 7-A) and histological sections (Fig. 7-B) of the meniscal defect one day to twelve weeks after the implantation of GFP-positive synovial mesenchymal stem cells under fluorescence. In the histological sections (Fig. 7-B), the white solid line indicates the outer edge of the meniscus. The white dotted line indicates the border between native meniscus (N) and regenerated tissue (R). The inset shows the area seen at higher magnification in the photomicrograph on the right. The scale bars represent 200 μm. Figs. 7-C and 7-D Fluorescent images of the regenerated meniscus at twelve weeks after implantation of GFP-positive synovial mesenchymal stem cells. The sections were immunostained with anti-type-I collagen (Fig. 7-C) and anti-type-II collagen (Fig. 7-D). Nuclei were counterstained with DAPI (4′, 6-diamidino-2-phenylindole dihydrochloride; blue) to demonstrate the cellular background (GFP-positive cells as well as cells not expressing GFP). GFP-positive cells are shown in green (bottom left), and type-I collagen-producing cells (Fig. 7-C) and type-II collagen-producing cells (Fig. 7-D) are shown in red (top right). Arrows in merge (bottom right) indicate double-positive cells of GFP and type-I collagen (Fig. 7-C) or type-II collagen (Fig. 7-D). Scale bar represents 25 μm. Col I = type-I collagen, and Col II = type-II collagen.