Multipotent mesenchymal stem cells from human subacromial bursa: potential for cell based tendon tissue engineering

Abstract

Rotator cuff injuries are a common clinical problem either as a result of overuse or aging. Biological approaches to tendon repair that involve use of scaffolding materials or cell-based approaches are currently being investigated. The cell-based approaches are focused on applying multipotent mesenchymal stem cells (MSCs) mostly harvested from bone marrow. In the present study, we focused on characterizing cells harvested from tissues associated with rotator cuff tendons based on an assumption that these cells would be more appropriate for tendon repair. We isolated MSCs from bursa tissue associated with rotator cuff tendons and characterized them for multilineage differentiation in vitro and in vivo. Human bursa was obtained from patients undergoing rotator cuff surgery and cells within were isolated using collagenase and dispase digestion. The cells isolated from the tissues were characterized for osteoblastic, adipogenic, chondrogenic, and tenogenic differentiation in vitro and in vivo. The results showed that the cells isolated from bursa tissue exhibited MSCs characteristics as evidenced by the expression of putative cell surface markers attributed to MSCs. The cells exhibited high proliferative capacity and differentiated toward cells of mesenchymal lineages with high efficiency. Bursa-derived cells expressed markers of tenocytes when treated with bone morphogenetic protein-12 (BMP-12) and assumed aligned morphology in culture. Bursa cells pretreated with BMP-12 and seeded in ceramic scaffolds formed extensive bone, as well as tendon-like tissue in vivo. Bone formation was demonstrated by histological analysis and immunofluorescence for DMP-1 in tissue sections made from the scaffolds seeded with the cells. Tendon-like tissue formed in vivo consisted of parallel collagen fibres typical of tendon tissues. Bursa-derived cells also formed a fibrocartilagenous tissue in the ceramic scaffolds. Taken together, the results demonstrate a new source of MSCs with a high potential for application in tendon repair.

FIG. 1.

Morphological appearance, proliferation, and surface marker expression of Bursa-derived MSCs. (A) Bursa MSCs display spindle shaped morphology. (B) The cells were efficient in forming colonies suggesting stem cell properties. (C) Bursa MSCs displayed rapid growth in culture. (D) Flow cytometry analysis for the expression of putative MSCs surface markers demonstrated expression of CD44, CD73, and CD105 with absence of CD45; thus, the cells are devoid of hematopoetic stem cells. The cells were also negative for CD146 expression, indicating uniqueness of the cell population. MSCs, mesenchymal stem cells. Color images available online at www.liebertpub.com/tea

FIG. 2.

Multilineage differentiation of Bursa MSCs in vitro. (A) Osteogenic differentiation: The cells exhibited ALP activity when cultured in osteogenic medium and this expression was highest at day 14 compared to other time points. Alizarin Red staining of osteogenic cultures at 21 days demonstrated extensive mineral deposition. Analysis of osteoblasts related genes of differentiating bursa MSCs demonstrated expression of OCN, OSX, OPN, and Runx2 indicating that the cells possess ability to differentiate into osteoblasts. (B) Bursa MSCs differentiated into adipocytes as demonstrated by extensive Oil Red O staining; analysis of adipogenic related genes showed expression of LPL, PPAR-γ2, and adipsin. (C) Chondrogenic differentiation indicated by H&E staining of pellet cultures and expression of type II and aggrecan genes. The results demonstrate multilineage differentiation of bursa cells. Con, cells cultured in maintenance medium; OM, osteogenic medium; AM, adipogenic medium; CM, chondrogenic medium; H&E, haematoxylin and eosin; ALP, alkaline phosphatase activity; OCN, osteocalcin; OPN, osteopontin; OSX, osterix. Color images available online at www.liebertpub.com/tea

FIG. 3.

Tenocyte differentiation in vitro. (A) Morphological appearance of bursa MSCs that were not incubated with BMP-12 (CON) and incubated with BMP-12 for 7 days. Bursa MSCs incubated with BMP-12 assumed an aligned and elongated morphology. (B) Expression of tendon markers by Bursa MSCs incubated in a medium supplemented with BMP-12. Tendon markers expressed by the cells were SCX, TNMD, Tenacin C, and Col1a1 at 3 and 7 days following exposure to BMP-12. (C). Tendon marker expression was assessed by RT-PCR. BMP-12, bone morphogenetic protein-12; SCX, Scleraxis; TNMD, tenomodulin. ±SD, *p<0.05, **p<0.01, ***p<0.001. N=4, triplicate experiments.

FIG. 4.

Bone formation in vivo by Bursa MSCs. (A) Bursa MSCs that were not pretreated with BMP-12 showed bone formation in the ceramic scaffolds. (B) Scaffold implanted in mice without cells demonstrated absence of bone formation. (C) Scaffold seeded with bursa MSCs pretreated with BMP-12 showed extensive bone formation in vivo. (D) Empty scaffold implanted in mice did not show any bone formation. (E, G) Immunofluorescence showed DMP-1 staining within the newly made bone in scaffolds seeded with either of the bursa cell populations. (F, H) Immunofluorescence staining for DMP-1 in scaffolds in which primary antibodies were omitted. (I–L) DAPI staining of respective scaffolds. (M–P) Overlay images of DMP-1 immunofluorescence staining and DAPI staining of respective tissue sections. The data demonstrate that bursa MSCs differentiate into osteoblasts in vivo and deposit bone. Magnification 100×. Color images available online at www.liebertpub.com/tea

FIG. 5.

Fibrocartilage formation by the bursa MSCs. (A) H&E staining of the fibrocartilagenous area within the tissue section from the scaffold that was seeded with bursa MSCs not pretreated with BMP-12. (B) Fibrocartilagenous area stained with toluidine blue showed staining of cartilage matrix. (C) Immunofluorescence staining for type II collagen in the fibrocartilagenous tissue confirmed that bursa MSCs form cartilage in vivo. Magnification 100×. Color images available online at www.liebertpub.com/tea

FIG. 6.

Formation of tendon-like tissue in vivo by the bursa MSCs. Bursa MSCs formed tendon-like tissues in vivo. (A) Bursa MSCs not pretreated with BMP-12 formed tendon-like tissue in vivo. (B) Bursa MSCs pretreated with BMP-12 appeared to form more tendon-like tissues. (C) Sirus red staining for collagen in a scaffold seeded with bursa MSCs not pretreated with BMP-12. (D) Sirus red staining in a scaffold seeded with bursa MSCs pretreated with BMP-12. Sirus Red staining demonstrated bundles of parallel collagen fibres typical of tendon tissues for both cell populations. Because there are no specific markers available for tendon cells, the tissues were identified based on their morphological appearance. The data are consistent with data reported previously on tendon-like tissue formation by tendon stem cells in vivo. Magnification 100×. Color images available online at www.liebertpub.com/tea