Inducible endogenous stem cells within human rotator cuff muscle to promote muscle regeneration after rotator cuff repair

Abstract

Objectives:Rotator cuff (RC) tears are the most common upper extremity injury, with over two million Americans seeking medical attention annually. Secondary muscle degradation following RC tears, including atrophy and fatty infiltration (FI), are critical factors that directly determine the clinical outcome of patients with this injury In mouse studies, the importance of fibro/adipogenic (FAP) cells in the development of FI after RC tears has been shown, determining that these cells are the predominant cell line responsible for the development of FI. FAP cells have garnered considerable interest in mediating both positive and negative effects on muscle cells. In regenerative conditions, FAPs stimulate satellite cell (SC) growth and differentiation, but in degenerative states, these cells differentiate into fat and fibrotic tissue. Additionally, FAPs have the ability to express factors consistent with beige fat, which may have important regenerative potential. In this study, we harvested samples from supraspinatus muscle in patients undergoing RC repair to determine for 1) the presence of FAP cells; 2) the presence of satellite cells; and 3) the ability of FAPs to differentiate into fat and/or fibrotic tissue. We hypothesized that both FAPs and SC would increase, and FAPs would display considerable cell plasticity and have the ability to differentiate into beige fat, adipocytes, and fibroblasts.Methods:The study was approved by the institutional IRB. At the time of surgery, a 3mm × 3 mm Supraspinatus (SS) and Deltoid (DL) biopsy was collected during surgery from 20 different clinical cases (Average age was 65 years, with 15 men and 5 women). Half of the biopsy was used for histology and half for Flow cytometry. For the vitro analysis, FAPs were isolated from human specimens using BD Aria II with PI live and Dead staining, and further isolated using CD31-, CD45-, CD184-, CD29-. CD56-, CD34+, CD140a + markers. SCs were sorted with PI live and dead staining, and further isolated using CD31-, CD45-, CD184+, CD29-, CD56+, CD34-, CD140a + markers. FAPs were cultured in 24 wells cell culture plates in standard media (F10+20%FBS+1% Antibiotics), Fibrogenic media (10ng/ml TGFβ-1) and Adipogenic media (stempro kit) for 2 weeks. For the Amibegron treatment group, FAPs were cultured in adipogenic media for 2 weeks first and switch to Adipogenic media with 10uM Amibegron (Sigma). FAPs were fixed with 4% PFA and stained with collagen I for quantify fibrosis index and perilipin A for Adipogenic index. The Paired ANOVA was used for statistical comparisons between groups, with p<0.05. Data are presented as mean +/- standard deviation.Results:A total of 20 patients were included in the study with different tear sizes. While SC number did not increase when comparing tear sizes, FAP cell number increased with increasing tear sizes. Patients with RC tears all had significantly more FAPs (7.8+/-2% vs 1.3 +/- .4%, p<0.05) compared to patients with partial tears. When divided by tear size, patients with medium and large tears had more FAPs compared to partial and small tears, a difference that was statistically significant. Histological analysis demonstrated that muscle architecture was preserved, but those with full thickness tears had more fat and fibrosis, with increased FAPs (Stained Red, Figure 2). FAPs from patients with full thickness tears had increased ability to differentiate into adipocytes and fibroblasts compared to partial tears (Figure 3), and had increased expression of UCP-1 (P<0.05). FAPs that were isolated from full thickness tears demonstrated increased expression of several promyogenic factors including IGF-1 and follistatin.Conclusions:Patients with full thickness RC tears have increased FAPs compared to control muscle and partial tears. FAPs from full thickness tears increase with tear size and have more differentiation potential compared. This data confirms that these cells are likely the cellular source for FI in humans, but that they also may serve a regenerative potential as they show differentiation plasticity and expression markers consistent with a beige fat phenotype. The fact that FAPS are able to differentiate and express pro-myogenic factors such as IGF-1 and follistatin suggest that they may serve as an endogenous stem cell source for improving muscle quality after RC repair.