Degenerative alterations to the rotator cuff are generally regarded as the consequence of mechanical abutment, undefined 'rheumatoid' inflammation, or previous accident. Although these causes could describe possible etiologies, they do not completely explain the cellular and molecular alterations seen in and around the rotator cuff, such as chondrogenic transformation and ectopic mineralization of the tendon tissue. With the present set of data, an additional perspective towards a causative explanation is given: chronic activation of morphogenetic factors (BMP-2, BMP-7, TGF-β, VEGF, and FGF) that might actively contribute to the rise of mechanically incompetent and (because of the mineral deposits) chronically irritating tissue components. The differential expression of BMP-2 and BMP-7, with BMP-7 expressed in a decreasing gradient from acromion to tendon, suggests a distinct contribution of BMP-7 to the disease process. Additional expression of inflammatory cytokines (IL-1β and TNF-α) might serve in propagating local inflammation and tissue destruction.
In bursa samples from patients with ruptures of the rotator cuff, expression of collagen types I and III was enhanced compared with normal controls [16
]. In addition, enhanced expression of IL-1β, both secreted and cell-bound IL-1 receptor antagonists, and VEGF have been demonstrated [4
]. Participation of the bursa in the disease progression of the rotator cuff has also been shown in animal experiments with rabbits [19
] and chickens [20
]. Specifically, chemical induction of a bursitis-induced chondrogenic metaplasia of the supraspinatus insertion site [21
]. These reports are not only in line with our findings, but also strongly suggest significant influence of molecular events within the subacromial bursa on the fate of the underlying supraspinatus tissue.
Unfortunately, there is no recent literature on the histology of the normal bursa. Organ material from tumor-related and joint-replacement surgery was available but always showed signs of degeneration, depending on the primary disease, and was thus unrepresentative of a normal situation. Nonetheless, the presence of bioactive BMP in itself supports the hypothesis of its role in induced chondrogenesis, irrespective of whether and, if so, how much it is expressed in normal tissue.
The amounts of BMP we detected are quite significant. Although direct estimates are hard to compare, the mid-ng quantities/mg of bursa tissue represent an overwhelmingly strong potential for morphogenetic signaling. In particular, the in-situ transformation that was achieved by placing the detector cell line C2C12 onto tissue slices reveals the effectiveness of the deposited BMP within the bursa. BMP detected within the cell and extracellular matrix of blood vessels might have been transported there through the bloodstream. However, the microanatomic distribution of immunohistological signals and the results from the PCR analysis strongly suggest local production rather than introduction from outside the bursa. Because we could block the differentiation signal by incubating the extracts with anti-BMP antibodies or soluble BMP receptors, a dominant role of BMPs in activating tissue transdifferentiation can be assumed, although a contribution of other growth factors cannot be excluded.
Because of the normal function of the bursa, the BMP might reach the tendon tissue through anatomic secretion pathways from the glandular elements within the bursa. Unfortunately, too little is reported in the literature about the secretary activity of this normally rather inconspicuous layer of tissue underneath the acromion. It is obvious, however, that there was significant cartilage differentiation, with RNA levels for cartilage collagen types II and X at quite significant levels in parts of the patients' tissue. Collagen to GAPDH ratios of >0.1, as detected here, are usually found in normal articular, mineralizing, and osteoarthritic tissue [22
]. In addition, studies performed in the rat support our observation of long-term preservation of cartilage-related gene expression in the supraspinatus [24
There is some information on the production and role of BMP in adult soft tissue. BMP is produced by gingival and periodontal fibroblasts [25
], megakaryocytes and platelets [26
], cells supporting egg maturation [27
], the kidney [29
], and also connective tissue tumor cells. More importantly, arthritic synovial membranes have been shown to express BMP-2 and BMP-6 and can influence cell turnover [30
]. Early studies showed that BMP induces tissue transdifferentiation of tenocytes into chondrocytes in vitro
] and, more recently, studies showed BMP-induced transdifferentiation of kidney fibroblasts into epithelial cells [32
]. Our finding in itself is not unexpected, retrospectively, but so far, to our knowledge, no attempts have been made to directly link the 'shoulder syndrome' to ectopic overexpression of BMP. Our approach using AP differentiation within the C2C12 cell line gave us a tool to explore primary features of the BMP deposited within the bursa. However, to describe the entire pathologic pathway from soft tissue to mineral deposits and fiber cartilage, more experiments are needed using chondrocytes and primary mesenchymal precursor cells in vitro
or with exogenous deposits of defined BMP in animals.