CIMB, Vol. 44, Pages 5827-5838: Effect of Adipose-Derived Mesenchymal Stem Cells (ADMSCs) Application in Achilles-Tendon Injury in an Animal Model

Figure 1. Workflow scheme. Eight female New Zealand white rabbits in which tenotomy was performed, were divided into two groups (four animals per group). The ADMSC group received male ADMSCs after tenotomy, whereas the lesion group remained untreated (no intervention). At Week 12, tissue biopsies of both groups were taken in order to perform histological and molecular analysis. An additional healthy group of four male rabbits was simultaneously analyzed as a control.

Figure 1. Workflow scheme. Eight female New Zealand white rabbits in which tenotomy was performed, were divided into two groups (four animals per group). The ADMSC group received male ADMSCs after tenotomy, whereas the lesion group remained untreated (no intervention). At Week 12, tissue biopsies of both groups were taken in order to perform histological and molecular analysis. An additional healthy group of four male rabbits was simultaneously analyzed as a control.

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Figure 2. Flow cytometry analysis. Adherent cells that showed MSC morphology were characterized using specific markers for stem cells according to the International Society for Cellular Therapy (ISCT). Negative controls of each antibody’s own isotype were included to rule out nonspecific signals and unlabeled cells (dotted line curves). CD73, CD44, and CD105 stained datasets were found positive after overlay, whereas CD34 and CD14 stained datasets were negative, thus discarding a hematopoietic origin of the sampled cells and suggesting an MSC phenotype.

Figure 2. Flow cytometry analysis. Adherent cells that showed MSC morphology were characterized using specific markers for stem cells according to the International Society for Cellular Therapy (ISCT). Negative controls of each antibody’s own isotype were included to rule out nonspecific signals and unlabeled cells (dotted line curves). CD73, CD44, and CD105 stained datasets were found positive after overlay, whereas CD34 and CD14 stained datasets were negative, thus discarding a hematopoietic origin of the sampled cells and suggesting an MSC phenotype.

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Figure 3. Identification of SRY. The sex-determining region Y (SRY) gene, which has an amplified product of 241 bp was identified in 3 out of 4 female rabbits of the ADMSC group and absent in all the members of the lesion group. GAPDH (287 bp) was used as an internal control and was identified in all members as expected. Male and female controls are also shown.

Figure 3. Identification of SRY. The sex-determining region Y (SRY) gene, which has an amplified product of 241 bp was identified in 3 out of 4 female rabbits of the ADMSC group and absent in all the members of the lesion group. GAPDH (287 bp) was used as an internal control and was identified in all members as expected. Male and female controls are also shown.

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Figure 4. Mean fold gene expression % for collagen I relative to GAPDH in healthy, ADMSC, and lesion groups determined by quantitative RT-PCR. Standard error of the mean (SEM) is also depicted. * p < 0.05.

Figure 4. Mean fold gene expression % for collagen I relative to GAPDH in healthy, ADMSC, and lesion groups determined by quantitative RT-PCR. Standard error of the mean (SEM) is also depicted. * p < 0.05.

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Figure 5. Mean fold gene expression % for collagen III relative to GAPDH in healthy, ADMSC, and lesion groups determined by quantitative RT-PCR. Standard error of the mean (SEM) is also depicted. * p < 0.05.

Figure 5. Mean fold gene expression % for collagen III relative to GAPDH in healthy, ADMSC, and lesion groups determined by quantitative RT-PCR. Standard error of the mean (SEM) is also depicted. * p < 0.05.

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Figure 6. Histopathology. (A) H&E staining of ADMSC group. Vascular neoformation is observed along with moderately disorganized fibers and formation of blood vessels with cellular aggregates. (B) Masson’s trichrome of ADMSC group. Vascular neoformation is observed along with moderately disorganized fibers. (C) H&E staining of lesion group. Areas of inflammatory infiltrate are observed along with abundant areas of adipose infiltration between the collagen fibers, few blood vessels, and necrotic foci. (D) Masson’s trichrome of lesion group. Areas of inflammatory infiltrate are observed along with abundant areas of adipose infiltration between the collagen fibers, few blood vessels, and necrotic foci.

Figure 6. Histopathology. (A) H&E staining of ADMSC group. Vascular neoformation is observed along with moderately disorganized fibers and formation of blood vessels with cellular aggregates. (B) Masson’s trichrome of ADMSC group. Vascular neoformation is observed along with moderately disorganized fibers. (C) H&E staining of lesion group. Areas of inflammatory infiltrate are observed along with abundant areas of adipose infiltration between the collagen fibers, few blood vessels, and necrotic foci. (D) Masson’s trichrome of lesion group. Areas of inflammatory infiltrate are observed along with abundant areas of adipose infiltration between the collagen fibers, few blood vessels, and necrotic foci.

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Table 1. Flow cytometry targets and antibodies.

Table 1. Flow cytometry targets and antibodies.

TargetAntibodyFluorophoreBrand Mesenchymal CD105Anti-CD105 IgG1, κFITCSerotecCD73Anti-CD73 IgG1, κPEInvitrogenCD44Anti-CD44 IgG2a, κPEChemicon Hematopoietic CD14Anti-CD14 IgG2a, kPEAbd SerotecCD34Anti-CD34 IgG1, κPEBD BiosciencesControlIgG2a, κ IsotypePEBioLegendControlIgG1, κ IsotypePEBioLegend

Table 2. Collagen I gene expression.

Table 2. Collagen I gene expression.

Collagen I Healthy GroupADMSC GroupLesion GroupMean Fold Gene Expression0.60.30.2Mean Fold Gene Expression %68.5%36.6%24.1%

Table 3. Collagen III gene expression.

Table 3. Collagen III gene expression.

Collagen III Healthy GroupADMSC GroupLesion GroupMean Fold Gene Expression0.50.20.1Mean Fold Gene Expression %54%26.3%11.9%

Table 4. Gene expression ratios.

Table 4. Gene expression ratios.

Collagen ICollagen IIIADMSC Group/Lesion Group Expression Ratio1.52.2

Table 5. Histopathology results with GHBCS.

Table 5. Histopathology results with GHBCS.

ADMSC Group (Mean with IQR) (n = 4)Lesion Group (Mean with IQR) (n = 4)pCollagen1.5 (1–2)2 (2–3)0.13Angiogenesis1 (0–1)0 (0–0.75)0.23Cartilage0 (0–0)0 (0–0)0.99

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