Treatment of Full-Thickness Rotator Cuff Tendon Tear Using Umbilical Cord Blood-Derived Mesenchymal Stem Cells and Polydeoxyribonucleotides in a Rabbit Model

Abstract

Objective: The aim of this study was to investigate regenerative effects of ultrasound- (US-) guided injection with human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) and/or polydeoxyribonucleotide (PDRN) injection in a chronic traumatic full-thickness rotator cuff tendon tear (FTRCTT) in a rabbit model.

Methods: Rabbits (n = 32) were allocated into 4 groups. After a 5 mm sized FTRCTT just proximal to the insertion site on the subscapularis tendon was created by excision, the wound was immediately covered by a silicone tube to prevent natural healing. After 6 weeks, 4 injectants (0.2 mL normal saline, G1-SAL; 0.2 mL PDRN, G2-PDRN; 0.2 mL UCB-MSCs, G3-MSC; and 0.2 mL UCB-MSCs with 0.2 ml PDRN, G4-MSC + PDRN) were injected into the FTRCTT under US guidance. We evaluated gross morphologic changes on all rabbits after sacrifice. Masson's trichrome, anti-type 1 collagen antibody, bromodeoxyuridine, proliferating cell nuclear antigen, vascular endothelial growth factor, and platelet endothelial cell adhesion molecule stain were performed to evaluate histological changes. Motion analysis was also performed.

Results: The gross morphologic mean tendon tear size in G3-MSC and G4-MSC + PDRN was significantly smaller than that in G1-SAL and G2-PDRN (p < 0.05). However, there were no significant differences in the tendon tear size between G3-MSC and G4-MSC + PDRN. In G4-MSC + PDRN, newly regenerated collagen type 1 fibers, proliferating cell activity, angiogenesis, walking distance, fast walking time, and mean walking speed were greater than those in the other three groups on histological examination and motion analysis.

Conclusions: Coinjection of UCB-MSCs and PDRN was more effective than UCB-MSC injection alone in histological and motion analysis in a rabbit model of chronic traumatic FTRCTT. However, there was no significant difference in gross morphologic change of tendon tear between UCB-MSCs with/without PDRN injection. The results of this study regarding the combination of UCB-MSCs and PDRN are worth additional investigations.

Figure 1

Human umbilical cord blood-derived mesenchymal stem cells (MSCs), polydeoxyribonucleotide (PDRN), and ultrasound images. (a) PDRN. (b) MSCs. (c) Injection was made in the left shoulder subscapularis full-thickness tears under ultrasound guidance. (d) Longitudinal ultrasound image shows the needle (arrow) in the left shoulder subscapularis of the rabbit. T: tendon; I: injectant.

Figure 2

Timeline of saline, PDRN, MSC, and PDRN with MSC injection. Normal saline (0.2 mL; group 1: SAL), PDRN (0.2 mL; group 2: PDRN), MSC (0.2 mL; group 3: MSC), and MSCs with PDRN (both 0.2 mL; group 4: MSC + PDRN) were injected under ultrasound guidance into the left shoulder subscapularis full-thickness tears 6 weeks after the tears were created. The analysis including gross morphology of tear site, histologic examination, and motion analysis was performed 4 weeks after injection of four different solutions. All rabbits were euthanized by carbon monoxide inhalation 4 weeks after injection of the different solutions. PDRN: polydeoxyribonucleotide; MSC: human umbilical cord blood-derived mesenchymal stem cell.

Figure 3

Gross morphological (A1–B4) findings of the subscapularis tendons in groups 1, 2, 3, and 4. (A1–A4) Pretreatment images. FTT is observed in all four groups. (B1–B4) Posttreatment images. FTT is shown and no gross morphologic changes between pretreatment and four weeks posttreatment in G1-SAL and G2-PDRN. There are significant differences in gross morphologic changes between pretreatment and four weeks posttreatment in G3-MSC and G4-MSC + PDRN. Normal saline (0.2 mL; group 1: SAL), PDRN (0.2 mL; group 2: PDRN), MSC (0.2 mL; group 3: MSC), and MSCs with PDRN (both 0.2 mL; group 4: MSC + PDRN). MSC: human umbilical cord blood-derived mesenchymal stem cell; PDRN: polydeoxyribonucleotide; FTT: full-thickness tendon tear.

Figure 4

Gross morphology of tear site at 4 weeks postinjection. FTT was observed in all eight rabbits in G1-SAL. In G2-PDRN, a PTT was observed in three rabbits and FTT in five rabbits. In G3-MSC, a PTT was observed in three rabbits, FTT in two rabbits, and CH in three rabbits. In G4-MSC + PDRN, a PTT was observed in five rabbits, FTT in one rabbit, and CH in two rabbits. SAL (normal saline 0.2 mL); PDRN (PDRN 0.2 mL); MSC (MSC 0.2 mL); and MSC + PDRN (MSC 0.2 mL with PDRN 0.2 mL). PDRN: polydeoxyribonucleotide; MSC: human umbilical cord blood-derived mesenchymal stem cell; FTT: full-thickness tendon tear; PTT: partial-thickness tendon tear; CH: nearly complete healing.

Figure 5

Subscapularis tendon tear size at 4 weeks postinjection. ^∗P < 0.05, one-way ANOVA, Tukey's post hoc test between two groups. SAL (group 1, normal saline 0.2 mL); PDRN (group 2, PDRN 0.2 mL); MSC (group 3, MSC 0.2 mL); and MSC + PDRN (group 4, MSC 0.2 mL with PDRN 0.2 mL). PDRN: polydeoxyribonucleotide; MSC: human umbilical cord blood-derived mesenchymal stem cell.

Figure 6

Histological findings of the subscapularis tendons in group 1 (SAL), group 2 (PDRN), group 3 (MSC), and group 4 (MSC + PDRN). (A1–A4) Newly regenerated tendons are shown in the blue-stained fibers (black arrow; Masson's trichrome stain; X200) in groups 2, 3, and 4. Few regenerative collagen fibers were seen in group 1. (B1–B4) Regenerated tendon fibers (black arrow; X200) were stained with anti-type 1 collagen antibody in groups 2, 3, and 4. Few regenerated tendon fibers were seen in group 1. (C1–D4) Numerous BrdU- and PCNA-stained cells (black arrow, X400, X200) were observed in regenerated tendon fibers in groups 2, 3, and 4. Few BrdU- and PCNA-stained cells were observed in group 1. (E1-F4) Numerous VEGF-positive cells and PECAM-1 positive microvascular densities (black arrows, X200) were observed in groups 2, 3, and 4. Few VEGF-positive cells and PECAM-1-positive microvascular densities were observed in group 1. MTS: Masson's trichrome stain; COL-1: collagen type 1; BrdU: 5-bromo-2′-deoxyuridine; PCNA: proliferating cell nuclear antigen; MSC: human umbilical cord blood-derived mesenchymal stem cell; PDRN: polydeoxyribonucleotide; VEGF: vascular endothelial growth factor; PECAM: platelet endothelial cell adhesion molecule.

Figure 7

Semiquantitative score of histological findings and immunoreactivity of stain. The immunoreactivity of MTS and anti-type 1 collagen antibody stain and proportion of BrdU-, PCNA-, VEGF-, and PECAM-1-positive cells were scored as detailed in Materials and Methods. ^∗P < 0.05 one-way ANOVA, Tukey's post hoc test among groups. SAL (group 1, normal saline 0.2 mL); PDRN (group 2, PDRN 0.2 mL); MSC (group 3, MSC 0.2 mL); and MSC+PDRN (group 4, MSC 0.2 mL with PDRN 0.2 mL). MSC: human umbilical cord blood-derived mesenchymal stem cell; PDRN: polydeoxyribonucleotide; VEGF: vascular endothelial growth factor; PECAM: platelet endothelial cell adhesion molecule.

Figure 8

Motion analysis of the rabbits at 4 weeks postinjection. In G4-MSC + PDRN, walking distance, fast walking time, and mean walking speed are greater than those in the other three groups on motion analysis. ^∗P < 0.05 one-way ANOVA, Tukey's post hoc test among groups. Group 1 (normal saline 0.2 mL). Group 2 (PDRN 0.2 mL). Group 3 (MSC 0.2 mL). Group 4 (MSC 0.2 mL with PDRN 0.2 mL). MSC: human umbilical cord blood-derived mesenchymal stem cell; PDRN: polydeoxyribonucleotide.